|Home | About | Journals | Submit | Contact Us | Français|
Dear colleagues, fellow scientists and guests,
It gives me tremendous pleasure to welcome you to the sixth International Symposium on Myocardial Cytoprotection (ISMC 2010). The venue of the symposium by tradition is the breathtaking and beautiful Pécs, the southern capital of Hungary. Pécs with its rich cultural heritage going back to the ancient Roman times was voted to be the cultural capital city of Europe for 2010.
The first ISMC symposium dates back to 1996 which was organized by the Department of Experimental Surgery of Pécs Medical University and the Experimental Section of the Hungarian Society of Cardiology. The conferences that followed consecutively in 1998 and 2000 had already made Pécs a meeting point for enthusiastic scientists in the field of cardiovascular work from all across Europe.
The fourth and fifth ISMC conference held in 2003 and 2006 innovatively went transatlantic and brought a new society closer to the symposium, the International Academy of Cardiovascular Sciences under the leadership of Professor Naranjan S Dhalla which made scientific contributions reach new heights with fresh and exciting ideas. This symposium also saw the emerging role of leading speakers from the United States and Canada which added flavour to both the scientific and social programs. This year’s symposium along with its previous contributors will be held in collaboration with the Heart Institute of Pécs University bringing together a new and exciting group of scientists presenting their colourful ideas to enrich the symposium.
Conventionally the ISMC has over the five previous symposia granted an atmosphere for international and national researchers of cardiovascular work to come together, discuss and present their latest concepts and ideas from bench to bedside in experimental and clinical work. The ISMC has also added to its credit the opportunity for young scientists to participate and make priceless contributions making room for discussion and debate in cardiovascular research.
I thank all the participants in advance from several countries for their precious efforts. As hosts to this event we will make every effort to make their stay memorably packed with a mixture of science, culture, hospitality and tradition. With a warm welcome, I hope to see you at ISMC 2010 in Pécs.
Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely distributed endogenous neuropeptide, also occurring in the cardiovascular system. Among others, PACAP has been suggested as a cardioprotective factor. It has been shown that PACAP inhibits cardiac fibrosis and protects cardiomyocytes against oxidative stress and in vitro ischemia/reperfusion. The aim of the present study was to investigate whether PACAP is protective in doxorubicin-induced cell death of cardiomyocytes.
Primary culture of neonatal rat cardiomyocytes was prepared from ventricular slices of 2-4-day-old Wistar rats. Non-treated cells served as control (Group I). In Group II 1 μM of doxorubicin was added to the media while in Group III cells were treated with 1 μM of doxorubicin together with 20 nM PACAP1-38. In Group IV to antagonize the effect of PACAP1-38, 250 nM of the PACAP antagonist PACAP6-38 was added simultaneously with 1 μM doxorubicin. Cells were exposed to the mentioned concentration of chemicals for 24 h. Viability of cells were measured by MTT assay, the amount of apoptotic cells was assessed by flow cytometry following annexin V/propidium iodide double staining. The rate of apoptosis was further examined by measuring caspase-3 activity and phospho-Bad using flow cytometry.
In doxorubicin-treated group (II) a lower number of living cells was observed with an increase of apoptotic cells while PACAP administration (Group III) led to a significant increase in the percentage of living cells and reproducible decrease in the rate of apoptosis. This beneficial effect of PACAP1-38 was diminished by PACAP6-38. Furthermore, doxorubicin increased the activation of pro-apoptotic caspase-3 and decreased the phosphorylation of Bad, while simultaneous PACAP treatment reduced the caspase-3 activation and increased the level of phospho-Bad. PACAP antagonist abolished the advantageous effect of PACAP1-38 on caspase-3 and phospho-BAD activation as compared to the control group and the group receiving co-treatment with PACAP1-38 and doxorubicin.
In summary, our present results show that PACAP effectively counteracts the apoptosis-inducing action of the cardiotoxic doxorubicin in vitro, involving caspase-3 and Bad inactivation and it has a clinical importance at the myocardial complications of the doxorubicin-treated patients.
This work was supported by the Hungarian Science Research Fund OTKA K78434, K72592, CNK 78480, ETT278-04/2009, Bolyai Scholarship.
In this study we investigated mechanical and molecular alterations within the left ventricle of laboratory mice with anterior infarction due to ligation of the left anterior descending coronary artery (LAD).
Cardiomyocytes were obtained from hearts of untreated (Control) and infarcted (MCI) female OF-1 laboratory mice 10 weeks after occluding the LAD, from two different areas: the infarcted anterior wall (Ant) and the opposite site (Inf) of the left ventricle. During the mechanical measurements calcium-activated active force (F0), calcium-independent passive force (Fpassive) and calcium sensitivity of force production (pCa50) were measured at 1.9 and 2.3 μm sarcomere lengths (SL). The phosphorylation status of the cardiac troponin I (cTnI) was assessed by Western immunoblotting with specific antibodies against protein kinase A- or protein kinase C-specific phosphorylation sites.
No significant differences could be found in the absolute active forces between the four groups neither at 1.9 nor 2.3 μm SLs [F0 (1.9 μm) ~ 5.6 kN/m2; F0 (2.3 μm) ~ 6.9 kN/m2]. In contrast Fpassive values were significantly higher at the MCI Ant areas at 1.9 μm SL (Control Ant: 0.21±0.03 kN/m2; Control Inf: 0.11±0.03 kN/m2; MCI Ant: 0.33±0.05 kN/m2; MCI Inf: 0.14±0.03 kN/m2; mean±SEM). At identical SL pCa50 was significantly lower at MCI Ant site than Control Ant or MCI Inf sites [pCa50 (1.9 μm): Control Ant: 5.80±0.02; Control Inf: 5.84±0.03; MCI Ant: 5.71±0.03; MCI Inf: 5.80±0.02; pCa50 (2.3 μm): Control Ant: 5.92±0.02; Control Inf: 5.94±0.04; MCI Ant: 5.80±0.03; MCI Inf: 5.89±0.01; pCa50 mean±SEM]. Increased calcium sensitivity at the larger sarcomere length was present in every group (ΔpCa50 ~ 0.1), indicating that the Frank-Starling mechanism was intact. The phosphorylation pattern of the cTnI was similar in the MCI and Control hearts, and at both sites of the infarcted hearts.
In conclusion, myocardial infarction resulted in a decrease in calcium sensitivity of force production. The phosphorylation status of cTnI could not explain this alteration, hence other types of posttranslational protein modifications may be also involved.
Several peptide hormones of cardiac origin exert autocrine/paracrine effects in myocardial ischaemia-reperfusion, in addition to their systemic actions. These include the natriuretic peptides (e.g. ANP and BNP) and adrenomedullin (AM). After acute myocardial ischaemia, release of processed pro-BNP fragments from myocardium is observed to be an early phenomenon and correlates with infarct size. Circulating levels of pro-BNP fragments continue to rise in proportion to the degree of ventricular dysfunction and N-terminal pro-BNP is a strong prognostic factor. In myocardial infarction, BNP exerts multiple systemic and cardiac actions. With regard to the latter, “counter-regulatory” roles in attenuating proliferative responses (myocyte hypertrophy, interstitial fibrosis) may be particularly important. Additionally, ANP and BNP are cytoprotective in the context of acute ischaemia-reperfusion. The protective effects are clearly related to elevation of cGMP concentration but the distal effectors of the survival signalling cascade are unclear. We have observed that BNP administered during early reperfusion limits infarct size, serving as a pharmacological mimetic of ischemic postconditioning. Further work has revealed that the protective action of BNP at reperfusion is dependent on the opening of KATP channels. AM shares many of the cardiac actions of the natriuretic peptides. It too displays cardioprotective effects during reperfusion and this action is clearly mediated via the NO/cGMP pathway. Moreover, AM+/− mice, with half the normal level of endogenous AM, sustain larger infarcts and exhibit increased early mortality compared to wild-type animals, suggesting that AM is a critical factor regulating myocardial tolerance to ischemia-reperfusion injury. Therapeutic activation of the cGMP pathway by manipulating natriuretic peptides and AM may have potential to limit infarct size by salvaging myocardium from the injurious consequences of reperfusion.
Although several studies have shown that erythropoietin (EPO) administered before ischemia may reduce myocardial injury after ischemia/reperfusion, only a few papers examined the effects of EPO on the reperfusion injury with controversial results. In the present study, our aim was to investigate whether EPO or darbopoetin (a modified synthetic EPO-analogue with a longer circulating half life) is able to protect the heart against reperfusion injury and to estimate a dose-response relationship.
Primary cultures of neonatal cardiomyocytes were treated with 1, 10, 100 or 500 U/ml EPO and subjected to 2.5 hrs of hypoxia and 2 hrs of reoxygenation followed by tripane blue viability test to estimate the effective dose range of EPO. In separate experiments, in mechanically ventilated anesthetized male Wistar rats (250–350 g) myocardial infarction was induced by 30 min coronary occlusion followed by 2 hours of reperfusion. Rats (n=8–10/each group) were given either vehicle, B-type natriuretic peptide (10 nmol/kg + 2 nmol/kg/15 min infusion; positive control) or EPO (5000 U/kg) 5 min before the onset of reperfusion. Darbopoetin (2.5, 5, 15 and 25 μg/kg) was administered 15 min before the end of ischemia. Infarct size was determined by the standard Evans blue/TTC method.
EPO in 100 U/ml concentration increased cell viability significantly when compared to control group (from 59.6±1.5% to 66.2±2.3%). In the in vivo rat model of myocardial infarction, both EPO (45.3±4.8%) and darbopoetin (44.6±6.1%) administered in 5000 U/kg and 5 μg/kg, respectively, reduced infarct size significantly when compared to vehicle (65.1±2.5%). The degree of protection was similar to that of the positive control BNP (40.3±7.4%). Neither lower nor higher doses of darbopoetin showed any significant infarct size limiting effect when compared to vehicle.
In the present study we showed that EPO may protect cardiomyocytes against hypoxic damage. Furthermore, both EPO and darbopoetin were able to diminish reperfusion injury administered during ischemia before the onset of reperfusion. It seems that the range of the infarct size reducing effectiveness of EPO or darbopoetin is quite narrow, however, both may be potent drugs, which may decrease acute ischemia/reperfusion injury applied before revascularization in patients with acute myocardial infarction.
Reperfusion of the myocardium is imperative to limit the damage caused by occlusion of an artery following myocardial infarction. However restoration of blood flow to the ischaemic tissue causes damage itself, a phenomenon known as reperfusion injury. Modifying cellular processes at this point can reduce the lethal cell damage caused. Exogenous nitric oxide (NO) reduce this injury at reperfusion, signalling through soluble guanylyl cyclase (sGC), a heterodimeric haem protein that catalyses the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). BAY 41-2272 is one of several NO-independent, haem-dependent stimulators of sGC which stimulates sGC to increase the production of cGMP. Previous studies have shown that the cGMP analogue 8Br-cGMP is cardioprotective at reperfusion. We therefore hypothesised that administration of BAY 41-2272 at reperfusion would be cardioprotective, limiting infarct size.
Hearts from male Sprague-Dawley rats (300–350g) were Langendorff perfused with modified Krebs-Henseleit buffer at constant pressure. After stabilisation hearts were subjected to 35 min regional ischaemia by occluding the left descending coronary artery. At 30 min ischaemia BAY 41-2272 was perfused at 3μM, 1μM, 300nM or 100nM for 15 min (10 min into reperfusion). Controls were perfused with the same buffer in the absence of the drug. To characterise the infarct limiting properties of BAY 41-2272 further, Langendorff experiments were carried out in which the sGC inhibitor ODQ or the NO donor NOC-9 were perfused with or without BAY 41-2272. Hearts were then sectioned and stained with triphenyltetrazolium chloride and infarct was expressed as a percentage of the risk zone. To confirm that BAY 41-2272 elevated cGMP levels, radioimmunoassay studies were performed to quantify cGMP levels.
Administration of BAY 41-2272 at early reperfusion caused a concentration-dependent limitation of infarct size (control 32.5±2.8 % vs. BAY 41-2272 3μM 17.0±2.2 %, mean±SEM, p<0.05). Concomitant perfusion of BAY 41-2272 and NOC-9 treated hearts reduced infarct size to 20.8±1.8 %. Perfusion with the sGC inhibitor ODQ prevented infarct limitation by BAY 41-2272 (29.6±1.7 %) (n≥6 per group, one-way ANOVA). cGMP levels in LV myocardial samples were elevated by 88 % at reperfusion when treated with BAY 41-2272.
These data show that exogenous NO as well as NO-independent activation of sGC at reperfusion is cardioprotective but dependent on the presence of the ferrous haem moiety on the β subunit of sGC. Although further studies need to be performed, the data does suggest that this protection is mediated through cGMP elevation.
It is well known that chronic hypertension can induce apoptosis and fibrosis in arterial wall through oxidative stress. In consequence of oxidative stress the poly(ADP-ribose)polymerase enzyme (PARP) is activated further aggravating the oxidative cell damage. In experimental models with spontaneously hypertensive rats (SHR) L-2286, a quinazoline-type PARP inhibitor could ameliorate these unfavorable alterations.
Male 10-week-old SHR rats were divided into two groups: one received no treatment (SHR-C) and the other received L-2286 treatment 5 mg/kg/day orally for 32 weeks. Male CFY rats were used as age-matched, normotensive controls (CFY). At the end of the experimental period the activation state of signal transduction proteins was monitored by Western blotting in the carotid arteries. To detect the oxidative damage, histologic samples were stained for 3-nitrotyrosine as an indicator of peroxynitrite production. Caspase-independent cell death in arterial wall was investigated by immunohistochemistry and immunfluorescence using antibody against apoptosis inducing factor (AIF).
L-2286 treatment enhanced the phosphorylation of the prosurvival factor Akt-1 (p<0.05) while phosphorylation of JNK and p-38 MAPK were significantly decreased in the SHR-L group. The activation of RhoA and the phosphorylation of 20-kDa myosin light chain (pMLC) also significantly ameliorated among SHR-L (p<0.05). Immunohistochemistry demonstrated strong expression of nitrotyrosine in the vasculature of SHR-C which was decreased in SHR-L. In SHR-C upregulated translocation of AIF was demonstrated which was decreased in SHR-L due to L-2286 treatment.
We demonstrated that PARP inhibition had a beneficial effect in chronic hypertension against vascular remodeling via activation of Akt signaling pathway decreasing vascular reactivity, oxidative stress, and apoptosis.
Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with a diverse physiological profile. Its production in mammalian systems has been attributed to two key enzymes in the cysteine biosynthesis pathway, cystathionine β-synthase and cystathionine γ-lyase. The rate of H2S production in tissue homogenates is in the range of 1–10 pmoles per second per mg protein, resulting in low micromolar extracellular concentrations. It is at these physiological concentrations that H2S is cytoprotective in various models of cellular injury. The reported cytoprotective effects of H2S are partially related to its ability to neutralize reactive oxygen species, to inhibit leukocyte-endothelial cell interactions, to promote vascular smooth muscle relaxation, to reduce apoptotic signaling, and to reversibly modulate mitochondrial respiration. Although the physiological and cardioprotective effects of H2S have previously been documented, the signaling mechanisms that mediate these effects have not been fully evaluated. Therefore, we sought to determine signaling mechanisms induced by H2S in the setting of acute myocardial ischemia and heart failure.
Using an in vivo mouse model of myocardial ischemia and reperfusion injury, we have found that H2S in the form of sodium sulfide (Na2S) is able to provide significant reductions in myocardial infarction and circulating Troponin-I levels when it is administered either before or after ischemia. Additionally, we have recently reported that Na2S provides a clear protection against left ventricular structural and functional impairment in response to ischemic-induced heart failure. In terms of mechanisms of action, H2S induces the nuclear localization of Nrf2, a transcription factor that regulates the gene expression of a number of antioxidants, and increases the phosphorylation of PKC and STAT-3. Activation of these signaling molecules leads to an increase in the expression of several antioxidants (HO-1 and Trx1) and antiapoptogens (HSP90, HSP70, Bcl-2, Bcl-xL, and COX-2), which protect the heart against oxidative stress and cell death.
These findings indicate that the cardioprotective effects of H2S are mediated in large part by a combination of antioxidant and antiapoptotic signaling. Furthermore, these findings suggest that H2S may be of therapeutic benefit in the treatment of myocardial infarction and ischemic-induced heart failure.
The vanilloid receptor 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli; therefore TRPV1 antagonists become a promising therapeutic candidate to target the pain pathway. However, functional TRPV1 expression was observed in the arteriolar smooth muscle cells affecting tissue blood distribution. This latter finding suggests not only a possible side effect of TRPV1 antagonist therapy for analgesia, but also presents a possible new drug target to regulate microvascular diameter. Here we characterized the vascular smooth muscle located TRPV1 mediated vasoconstriction to establish its pharmacological properties. Vascular diameter of isolated, pressurized skeletal (m. gracilis) muscle arterioles (diameter: 207±7 μm, n=56) possessing spontaneous myogenic tone was measured upon treatment by various TRPV1 agonists/partial agonists. Potency, efficacy, kinetics of action, receptor desensitization and in some cases changes in intracellular Ca2+ concentration was determined. Some of the tested TRPV1 agonists evoked substantial constrictions (capsaicin, MSK-195, JYL-79), while others were without effect (resiniferatoxin, JYL-273). A complete desensitization of arteriolar TRPV1 was observed after agonist treatments (with the exception of capsaicin). A partial agonist (JYL-1511) was used to estimate the sufficient level of TRPV1 stimulation which evokes desensitization of arteriolar TRPV1. Finally, a TRPV1 antagonist, AMG9810, suggested that the antagonism is competitive. Our data suggests that the arteriolar smooth muscle located TRPV1 has a different structure-activity relationship than the sensory neuron located receptor. These differences may help to design antagonists devoid of vascular side effects as well as new drug candidates regulating microvascular diameter.
Cardiomyocytes isolated from failing human hearts have elevated resting tension (RT) due to altered spring characteristics of titin. In the present study we tested whether oxidative damage at the 100 nm wide titin-actin overlap region near the Z-discs contributes to this elevated RT by presetting a higher tension on the more distal extensible titin segments.
Left ventricular (LV) myocardium was procured from explanted donor hearts (Control), from surgical biopsies in aortic stenosis patients (AS) and from LV transvascular, endomyocardial biopsies in heart failure (HF) patients. Mechanical parameters of isolated, triton-permeabilized, single cardiomyocytes were obtained during force measurements at a sarcomere length of 2.2 μm. Dithiothreitol (DTT) and gelsolin treatments were applied in vitro. With the reducing agent DTT we tested the hypothetical reversible effect of SH-oxidation in myofilaments. With the actin capping protein gelsolin we removed the thin filament from the sarcomeres to investigate the contribution of titin-actin interaction to the RT of cardiomyocytes. Immunofluorescent images were obtained of cardiomyocytes co-stained for alpha-actinin (Z-disc marker) and nitrotyrosine (oxidative stress marker).
DTT reduced RT of cardiomyocytes in every group (Control n=9; AS n=7; HF n=6). The higher the RT prior to DTT, the larger the fall in RT after DTT (P<0.0001, r=0.79). In HF (n=5), actin removal inhibited the effect of DTT on RT. After gelsolin treatment a fall in RT was present also in every group (Control n=11; AS n=11; HF n=14). The higher the RT prior to gelsolin, the larger the fall in RT after gelsolin (P<0.0001, r=0.63). Control and AS cardiomyocytes stained positively for nitrotyrosine in areas in between the Z-discs. In HF positive nitrotyrosine staining was distributed over the entire cell, including the Z-disc areas.
Titin-actin interaction at the Z-discs contributes to elevated RT of failing human cardiomyocytes. RT can be reduced with antioxidant treatment and/or thin filament removal. Possibly because of oxidative damage to the Z-disc proteins, the effect of titin-actin interaction on RT is especially prominent in HF cardiomyocytes with elevated RT. This could be of relevance to the contractile dysfunction in HF.
The goal of this study was characterization of the importance of the vascular angiotensin converting enzyme (ACE) and ACE inhibitors in coronary bypass graft surgery (CABG) patients.
Vascular tissue (distal saphenous vein (n=33) and/or radial artery (n=34) segments) and blood samples were collected from CABG patients. We detected the potency of angiotensin I (AngI) and angiotensin II (AngII) to evoke vascular contraction with and without ACE inhibitors, and we determined vascular and plasma ACE concentrations.
AngI and AngII treatment evoked significant constriction in both types of vessels. The ratio of the potencies (EC50) of AngII and AngI was significantly lower in the radial artery compared to the saphenous vein. (0.17±0.03 nM vs. 0.51±0.14 nM, p=0.003). These suggested 3-fold more effective AngI conversion in saphenous vein samples. We found significant inhibition of contractions with telmisartan and captopril in both saphenous veins and radial arteries. Vascular ACE expression was significantly higher in saphenous veins compared to radial arteries (9.7±1.0 ng/mg vs. 5.3±0.7 ng/mg, p=0.01). In contrast, serum ACE concentration was similar in blood samples of the same patients (saphenous vein patients: 132±10 ng/ml vs. radial artery patients: 162±20 ng/ml).
ACE-inhibitor therapy after CABG surgery may be an important short as well as long term strategy for preventing saphenous vein graft stenosis and occlusion.
A major hitch in the effectiveness of stem cell therapy is the death of stem cells due to the oxidative environment present in the normal tissue. Reduction of oxidative stress or maintaining a reduced environment in the target tissue can enhance the stem cell survival and can enhance cardiac regeneration after stem cell therapy.
To study the efficiency of maintaining the reduced tissue environment via pretreatment with natural antioxidant resveratrol in stem cell therapy.
We pretreated male Sprague Dawley rats with resveratrol (2.5 mg/kg/day gavaged for 2 weeks). Left anterior descending coronary artery (LAD) occlusion followed by direct injection of adult cardiac stem cells stably expressing EGFP on the border zone of the myocardium through survival surgery. The prevalence of cardiac reduced environment was seen in resveratrol treated rat hearts via significantly enhanced redox signaling observed through the nuclear localization of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1) 7 days after LAD occlusion. Significantly improved cardiac functional parameters (left ventricular ejection fraction and fractional shortening) and enhanced stem cell survival and proliferation (expression of cell proliferation marker Ki67) and differentiation of stem cells towards the regeneration of the myocardium (expression of EGFP) was evident 28 days after LAD occlusion in rats treated with resveratrol compared to control rats.
Maintaining a reduced tissue environment by treatment with resveratrol in rats enhanced the cardiac regeneration by adult cardiac stem cells via improved cell survival, proliferation and differentiation leading to improved cardiac function.
In small arteries and arterioles increases in intraluminal pressure elicit the development of myogenic tone. It has also been shown that in arterial vessels high intraluminal pressure elicits the release of reactive oxygen species eliciting constrictions. We hypothesized that in small veins pressure induces myogenic tone and H2O2 elicits constriction.
Gracilis muscle venules were isolated from Wistar rats, then cannulated and incubated in PSS in the presence of 10 mmHg of intraluminal pressure (Pi) at T=37C° in a special vessel chamber. Changes in diameter were measured in response to increases in intraluminal pressure and hydrogen peroxide (H2O2). Catalase (CAT, which metabolizes hydrogen peroxide (H2O2)) and non-specific inhibitor of cyclooxygenases (COXs) (indomethacin) were used.
Isolated rat gracilis muscle venules developed substantial tone in response to increases in intraluminal pressure (active diameter: ~260 μm, whereas passive diameter: ~370 μm at 10 mmHg). Presence of catalase significantly reduced the development of myogenic tone (control: 65±5 % vs. control+CAT: 80±4 % of PD, respectively at 10 mmHg). Also, indomethacin inhibited – in part the development of myogenic tone (control: 75±4 % vs. control+INDO: 86±3 % of PD, respectively at 10 mmHg). We have also found that H2O2 elicited concentration dependent constrictions (max.: 137±8 to 61±18 μm), which were inhibited by the presence of indomethacin.
Thus in skeletal muscle venules pressure elicits the development of myogenic tone, which is – in part – mediated by H2O2. Also H2O2 elicited indomethacin-inhibitable constrictions. These findings suggest that reactive oxygen species may have an important physiological role in the regulation of venous blood flow.
Supported by: Hungarian Sci. Res. Funds/OTKA-T48376, K67984; Health Sci. Council/ETT 364/2006
Earlier studies have shown that cardiac dysfunction in hearts subjected to ischemia-reperfusion (I/R) injury is associated with depression in sarcolemmal (SL) and sarcoplasmic reticular (SR) activities as well as impairment of endothelial function with respect to NO generation. Although L-Arginine, a NO donor, has been demonstrated to improve cardiac function and prevent subcellular defects in I/R hearts, the mechanisms of these effects are not understood. By employing isolated rat hearts perfused at constant flow (CF) or constant pressure (CP), we have shown that cardiac dysfunction as well as depressions in SL Na+-K+ ATPase and SR Ca2+-transport activities in CF hearts subjected to 30 min of global ischemia followed by 60 min reperfusion were greater than those in CP hearts. The I/R induced decrease in NO formation was more and the increase in calpain activity was greater in CF hearts in comparison to the CP hearts. All these changes in cardiac function as well as subcellular and biochemical activities due to I/R in CF hearts were attenuated by L-Arginine whereas those in CP hearts were augmented by L-NAME, an inhibitor of NO synthase. L-Arginine was found to depress the increased level of calpain activity in I/R hearts by nitrosylation of the enzyme. Furthermore, leupeptin, an inhibitor of calpain, was observed to prevent the I/R induced cardiac dysfunction, defects in SL Na+-K+ ATPase and SR Ca2+-transport as well as calpain activation in the heart. These results suggest that the beneficial effects of L-Arginine on cardiac function and subcellular organelles may be related to attenuation of calpain activation in hearts subjected to I/R injury.
Supported by a grant from the Canadian Institutes of Health Research
ACE inhibitors (ACEi) are important antihypertensive agents. We aimed to set up a method to measure serum ACE activity to monitor the efficacy of ACEi treatment.
Serum ACE activity was determined in blood samples of 150 patients. In addition, their clinical history and medication was also recorded.
Blood pressure was higher in hypertensive patients with prescribed ACEi compared to patients without ACEi medication (139±2.2 n=79; 129±3.2 n=38; p=0.0145). Specific ACE activity increased in parallel with dilution (77.5±7.5 fold increase, n=58) in patients with effective ACEi therapy. In contrast, when this increase in specific activity was lower than 10, the effectivity of ACEi therapy was considered to be insufficient. ACEi treatment was ineffective in 16 cases (20.25%) among patients with ACEi therapy (n=79). Importantly, these patients had higher blood pressure than the patients with effective ACE inhibition (149±7.6 n=15 versus 137±2.1 n=58; p=0.039). To estimate the contribution of insufficient patient’s compliance to the failure of therapy, the efficacy of statin treatment was also assessed in the same patients. Total cholesterol level of patients without statin treatment was not related to the efficacy of the ACEi therapy (in patients without ACEis: 5.63±0.21mmol/l, n=34, in patients with effective ACEi therapy: 5.49±0.25mmol/l, n=22 p=0.3439). In contrast, effectivity of statin therapy was parallelled with the effectivity of ACEi treatment: total cholesterol in the case of patients with effective ACEi was 4.69±0.2, n=27, compared to 6.84±0.4, n=9, in patients with insufficient ACE inhibition (p<0.0001).
In summary, a method was developed to determine the effectivity of ACEi therapy. We found ineffective ACE inhibition (parallelled with ineffective statin therapy) in 20% of patients, suggesting inadequate patient’s compliance. We propose the importance of confirmation of effective ACEi therapy in the evaluation of the prescribed medication.
Heart failure is a serious condition, with a mortality rate greater than 50% over 5 years in severe cases. In spite of extensive research investigating novel therapeutic strategies to treat heart failure for the time being no effective medical therapy exists. The only cure for severe heart failure remains heart transplantation. Apelin-angiotensin receptor-like 1 (APJ) signalling is an important mediator of cardiovascular regulation. APJ mRNA levels are decreased in patients with heart failure (HF) and upregulated in the myocardium from patients treated with LVADs as a bridge to transplantation. The role of APJ gene expression in the clinical recovery observed in HF patients following combined LVAD and pharmacological treatment including the β2-adrenoceptor agonist, clenbuterol (Clen) remains unknown. Real-time PCR was used to measure APJ mRNA expression in left ventricular (LV) samples collected at LVAD implant and explant from 7 patients with functional improvement (time on LVED; 10.3±6.3 months) to allow LVAD removal without requiring transplantation (REC) and from 5 patients who failed to recover function (time on LVED ; 23.7±5.3 months) and were transplanted (TX). Correlation between ejection fraction (EF) and APJ expression was tested using Spearman’s correlation test. APJ gene expression was up-regulated in REC (9.8±4.3-fold at explant vs. implant; mean±SEM, p<0.05; mean±SEM) but unchanged in TX (1.4 ± 3.4-fold p=ns). There was a significant correlation (r = 0.7832, p<0.05) between the change in EF from implant to explant (Δ EF:51.6±8.6 %) during the recovery showing the change in APJ expression in REC. This change with improvement in EF suggests a role for APJ in myocardial recovery. To study whether the observed effects are due to mechanical unloading or Clen treatment, Lewis rats underwent heterotopic abdominal heart transplantation and were treated with either 2 mg/kg/day Clen or saline (Sal). Donor unloaded (UNL) and recipient control (C) hearts were collected after 7 days. APJ gene expression was significantly upregulated in UNL but Clen treatment had no effect (C+sal=1.4±0.1; UNL+sal=2.0±0.1; C+Clen=1.6±0.1; UNL+Clen=2.0±0.2; n=6 per group; p=0.009). We conclude that APJ may play an important role in functional recovery following mechanical unloading in patients treated with LVADs.
Previously, it was suggested that release of nuclearly formed ADP-ribose polymers (PAR) or ADP-ribosylated proteins could be responsible for the cytosolic and mitochondrial effects of poly(ADP-ribose) polymerase (PARP) activation in oxidative stress. In the present report, we provided a novel alternative mechanism. We found that reactive oxygen species (ROS)-activated PARP-1 regulated the activation of JNK and p38 MAP kinases since inhibition of PARP by pharmacons, small interfering RNA (siRNA)-silencing of PARP expression or transdominant expression of enzymatically inactive PARP resulted in inactivation of these mitogen activated protein kinases (MAPKs). This regulation was achieved by increased expression and enlarged cytoplasmic localization of MAPK phosphatase-1 (MKP-1) upon PARP inhibition in oxidative stress since changes in MKP-1 expression were reflected in the phosphorylation state of JNK and p38. Furthermore, we found that in MKP-1-silenced cells, PARP inhibition was unable to exert its protective effect indicating the pivotal role of JNK and p38 in mediating oxidative-stress induced cell death as well as that of increased MKP-1 expression in mediating protective effect of PARP inhibition. We suggested that regulation of a protein that can directly influence cytoplasmic signaling cascades at the expression level represents a novel mechanism for cytoplasmic action of PARP inhibition.
We have shown previously that nitric oxide (NO) results in antiarrhythmic protection during myocardial ischaemia – an effect in part mediated through the modification of gap junction channels. There is some evidence that sodium nitrite (NaNO2), given prior to reperfusion protects against reperfusion injury. Thus, in the present study we aimed to examine whether infusion of NaNO2 prior to and during ischaemia or just prior to reperfusion would provide protection against ischaemia and reperfusion-induced arrhythmias, and whether this effect involves gap junctions.
In dogs anaesthetised with chloralose and urethane myocardial ischaemia was induced by a 25 min occlusion of the left anterior descending coronary artery (LAD) followed by rapid reperfusion. Fifteen dogs served as controls (C), whereas fourteen animals were infused intravenously with NaNO2 in a dose of 0.2 μmol·kg−1·min−1 starting the infusion 10 minutes prior to and maintained over the entire occlusion period (NaNO2-PO). In the other five dogs NaNO2 was administered by the same route 10 minutes prior to reperfusion (NaNO2-PR). The number and incidence of ventricular tachy-cardiac episodes (VT) and the incidence of ventricular fibrillation (VF) as well as the number of ventricular premature beats (VPBs) were assessed in relation to changes in tissue impedance – an indirect measure of gap junction coupling. Ischaemia severity was evaluated by changes in total activation time (TAT) and epicardial ST-segment using a mapping electrode.
Compared to the controls, NaNO2 in both administration forms significantly reduced the number of VPBs (477±96 vs. 156±78 and 84±67), the incidence of VT (87% vs. 29% and 20%) and increased survival (0% vs. 50% and 80%) from the combined occlusion-reperfusion insult. Furthermore, NaNO2 significantly reduced the ischaemia induced increase in TAT and in epicardial ST-segment. These effects of NaNO2 were particularly marked during the later period of the occlusion and were associated with the attenuation of those marked impedance changes that occurred in the controls prior to the occurrence of the Ib phase arrhythmias.
We conclude that the protective effect of NaNO2 can be attributed to the release of NO, and the maintenance of NO availability during myocardial ischaemia. This reduces ischaemia severity and suppresses arrhythmia generation especially during Ib phase, which is, at least in part, mediated through gap junctions.
This work was supported by OTKA (project number: K75381)
Mitochondrial cardiomyopathy is associated with pathological remodeling of cardiomyocyte Ca2+ signalling partly due to decreased expression of the sarcoplasmic reticulum (SR) Ca2+-buffer calsequestrin (CASQ2). This study aimed to determine if CASQ2 downregulation is directly caused by impaired mitochondrial function.
The mitochondrial uncoupler FCCP was used to induce mitochondrial stress in cultured neonatal rat cardiomyocytes. Ca2+ transients and reactive oxygen species (ROS) were measured by confocal microscopy using the indicators fluo-4 and MitoSOX red, respectively. Quantitative PCR and Western blots were used to quantify CASQ2 mRNA and protein expression, respectively. To identify potential regulatory elements in the CASQ2 gene, luciferase reporter constructs were made in which regions of the mouse CASQ2 promoter or intron 1 were ligated to pGL3-Basic.
There was a concentration-dependent downregulation of calsequestrin (CASQ2) in response to mitochondrial stress. Further, there were changes in cardiomyocyte Ca2+ signals involving decreases in SR Ca2+ content and amplitude and duration of Ca2+ sparks. While caspase 3, p38 and p53 inhibitors had no effect on FCCP-induced CASQ2 downregulation, this downregulation was attenuated by the ROS scavenger N-acetylcysteine (NAC). NAC not only decreased FCCP-induced ROS, but it also restored the Ca2+ signals, SR Ca2+ content and Ca2+ spark properties to control levels. Further, the Casq2 intron 1 reporter construct responded similarly as the endogenous gene when exposed to FCCP +/− NAC.
Mitochondrial uncoupling leads to transcriptional changes in CASQ2 expression that manifest as compromised Ca2+ signalling, and these changes can be prevented by ROS scavengers. Further, the similar response of the CASQ2 intron 1 reporter construct to FCCP and NAC suggests that this region may harbour regulatory elements that confer sensitivity to mitochondrial stress. These data not only show a link between mitochondrial dysfunction and cardiomyocyte Ca2+ handling, but they also indicate an important role for ROS in cardiomyocyte function and gene expression. Impaired mitochondrial function has been linked to several cardiac pathologies as well as normal ageing; therefore, the mechanisms described here might be involved in a wide spectrum of cardiac conditions.
Disbalance of reactive types of oxygen (ROS) and antioxidative defence system (ADS) leads to condition known as oxidative stress. Phospodiesterase-5 (PDE5) catalyzes cyclic GMP (cGMP) degradation, as well as its intracellular content. Specific inhibition of that isoenzyme induces an increase of cGMP concentration, relaxation of smooth muscle cells and consequently vasodilation. Regarding unclarified mechanisms of the side-effects, induced by PDE5 inhibition, the aim of our study was to examine the effects of different phosphodiesterase-5 (PDE5) inhibitors on oxidative stress in isolated rat hearts. The hearts were excised from Wistar albino male rats (BM about 200 g, 8 weeks, each group-12 rats) and perfused with buffer at a constant pressure. After the first sequence of coronary perfusion pressure (CPP) changes (basic protocol) the hearts were perfused with novel PDE5 inhibitors, Sildenafil, Tadalafil and Vardenafil, all in different doses (10, 20, 50, 200 nM), alone or in combination with nitric oxide synthase inhibitor (L-NAME, 30mM). The oxidative stress markers (index of lipid peroxidation measured as TBARS and superoxide anion radical (O2−)) were determined in coronary venous effluent. Both Tadalafil and Vardenafil significantly decrease TBARS and O2− in all doses. On the other hand, Sildenafil did not change either parameter significantly. Additional application of L-NAME (with each PDE5 inhibitor) induced further decrease of TBARS and O2− in all applied doses in the Tadalafil and Vardenafil group, while Sildenafil-induced changes weren’t significantly affected. On the basis of these results we can draw a conclusion that these PDE5 inhibitors have different dose dependent effects - decrease of oxidative stress markers (TBARS and O2−), without significant contribution of the L-arginine/NO system.
Atrial fibrillation (AF) is severe arrhythmia, which largely affects the quality of life. The main form of treating AF is still pharmacological. The development of new antiarrhythmic drugs for treating AF would be promoted by a dog AF model. Therefore, the aim of the present study is to investigate the properties of three currents which determine atrial repolarization, the transient outward (Ito), ultra-rapid delayed rectifier (IKur) and acetylcholine sensitive potassium current (IK,ACh) in isolated atrial myocytes originated from normal (SR) and tachypaced atrial fibrillating (PF) dogs by applying the whole cell patch clamp technique at 37°C.
We have identified in all atrial canine myocytes a chromanol 293B (100 μM) sensitive current. The current amplitude was 1300±236 pA (measured at 50 mV) and the current inactivation was best fitted by two exponentials with the following components: τ1=111±18 ms and A1= 349±33 pA; τ2= 12.4±2.6 ms and A2= 866±189 pA. Chromanol 293B accelerated the inactivation in the following manner: τ1=62±28 ms and A1=91±15 pA; τ2=1,62±0,25 ms and A2=256±78 pA. Selective IKur blocker 100 μM aminopyridine (4-AP) was applied as a pharmacological tool for identifying IKur measured as a sustained current activated by depolarizing pulses to positive potentials. 100 μM 4-AP did not significantly change either the peak or the steady state current in dog atrial myocytes. IK,ACh was activated by cholinergic agonist carbachol. In sinus rhythm (SR) carbachol activated a large current either at inward or outward directions (current amplitude at −100 mV, was −78.7±9.4 pA vs −269±39 pA, before and after carbachol, respectively). Selective IK,ACh blocker tertiapin (10 nM) blocked the carbachol induced current by 57 %. In atrial myocytes from PF dogs we could measure the presence of a constitutively active IK,ACh, which could be blocked by 26 % with 10 nM tertiapin (−301±25 pA vs −217±31 pA before and after tertiapin, respectively, p<0.05, n=5).
The presence of the constitutively activated IK,ACh in tachypaced dogs indicates the presence of electrical remodelling, thereby we concluded that the applied chronic tachypaced model induced chronic AF in dogs. It seems that IKur plays a less significant role in the canine atrial repolarization than that reported in human atrial myocytes. However, understanding the cause for these interspecies differences requires further investigations.
We have evidence that peroxynitrite (PN) exerts preconditioning-like protection against ischaemia and reperfusion (I/R)-induced ventricular arrhythmias in vivo. Moreover, both the preconditioning (PC) procedure and the administration of PN significantly increased nitrotyrosine (NT) levels, a marker of endogenous PN generation. We have now examined whether this endogenous PN formation plays a trigger role in the antiarrhythmic effect of PC and PN infusion.
Chloralose-urethane anaesthetised dogs were subjected to a 25 min occlusion of the left anterior descending coronary artery (LAD) followed by rapid reperfusion. PC was initiated by two 5 min occlusions either in the absence (PC, n=9) or in the presence of urate (UA+PC, n=9), a scavenger of PN. Urate was administered in intravenous infusion (0.2 mg/kg/min) 10 min prior to and over the PC procedure. In two other groups, the PC occlusions were replaced by similar durations PN infusion (100 nM) also in the presence (UA+PN, n=8) and in the absence (PN, n = 9) of urate. Two groups served as controls; in one of these saline (C, n=14), in the other one urate (UA+C; n=9) were infused. Severities of ischaemia and of ventricular arrhythmias (VPBs, VT, VF, survival) as well as plasma nitrite/nitrate (NOx) levels, tissue superoxide and NT formations were determined.
Compared to the controls both PC and PN markedly reduced the total number of VPBs (246±75 vs 24±10 and 55±19), the incidence of VF during occlusion (43% vs 0% and 11%) and increased survival (S: 0% vs 66% and 50%) following reperfusion. Both interventions prevented the ischaemia-induced reduction in NOx levels during occlusion and the increase in super-oxide and NT productions after reperfusion. The administration of UA attenuated the antiarrhythmic effect of PN (VPBs: 226±121; VF: 50%; S: 25%) but did not modify this effect of PC (VPBs: 12±6 VF: 0% and S: 66%). In the presence of UA the NOx levels and the production of superoxide and NT remained unchanged in the PC dogs but they were similar to the controls in the PN treated dogs. Interestingly, UA in dogs subjected only to I/R proved to also be antiarrhythmic (VPBs: 50±14; VF: 0% and S: 55%).
These results indicate that the local administration of PN in nanomolar concentrations may result in an antiarrhythmic protection but the formation of endogenous PN is not necessary for the antiarrhythmic effect of PC.
Cardiac tamponade (CT) is a severe clinical syndrome most often caused by high-energy thoracic injuries. Due to the fast circulatory redistribution CT leads to unmanageable peripheral microcirculatory complications, gastrointestinal hypoperfusion and mucosal barrier failure with bacterial translocation. These events are linked to inflammatory activation with increased production of reactive oxygen- and nitrogen intermediates. In our previous in vitro experiments methane gas showed considerable biological activity and proved to be an effective radical scavenger. Our aim was to investigate the comparative anti-inflammatory effects of methane inhalation and selenium (Se), a known cofactor of the enzymes of the redox balance on the hemodynamics and microcirculatory changes in a large animal model of CT.
In anaesthetized, ventilated and thoracotomized minipigs (n=5) CT was induced for 60 min by intrapericardial administration of colloid solution, meanwhile the mean arterial pressure (MAP) was kept between 40–45 mmHg. Macrohemodynamic changes, small intestinal pCO2 gap values (by tonometric probe), blood superoxide and hydrogen peroxide production (chemiluminometric method) were monitored for 180 min. The methane-treated group (PT+Met) was ventilated with 2.5% methane/air mixture, starting at the 5th min before the end of CT, and lasting for 20 min. The PT+Se group was treated with continuous Se infusion (Selenase T; 25 μg/kg/h iv) after CT induction (n=5). The 4th group served as sham-operated control.
After CT cardiac output was normalized, but the increase in the intestinal pCO2 gap (34±8 vs. 53±9 mmHg) and the superoxide and hydrogen peroxide (H2O2) producing capacity of blood referred to the peripheral microcirculatory impairment. Both Se and methane treatments significantly decreased the superoxide and hydrogen-peroxide production and improved the mucosal microcirculation (PT+Met: 26±6 vs. 53±9 mmHg; PT+Se: 36±7 vs. 53±9 mmHg).
Short-term methane inhalation was able to improve the microcirculatory consequences and reactive oxygen intermediate production after CT with the same effectiveness as continuous iv treatment with Se. Both compounds have antioxidant properties and can be used to ameliorate the harmful splanchnic consequences of CT.
Grant support: ETT 442/2009, OTKA K751612008
The expected vascular effect of acetylcholine (ACh) on coronary vessels is endothelium dependent relaxation. However, within different species and experimental conditions constrictor, dilator and biphasic action of ACh was equally seen. During human coronarography ACh-induced coronary constriction was found independent of pathological morphology. The outcome of vascular effects of ACh is based on the balance between endothelium dependent and vascular smooth muscle mediated mechanisms.
In our studies effects of ACh were investigated on isolated perfused rat hearts using constant flow and heart rate. Responses to ACh were characterized by changes in coronary perfusion pressure (CPP). Endothelial function was tested by bradykinin (BK, 30 nmol), while that of vascular smooth muscle by sodium nitroprusside (SNP, 1nmol) bolus injections. First, responses to ACh infusions (0.01-0.1-1μM 5-5 min) were studied at the generally used low perfusion rate (CPP: 41±1 mmHg) providing proper O2 supply. Second, effects of ACh were studied at elevated CPP (via constant vasopressin infusion of 1IU/L; CPP: 91±2 mmHg). During the latter conditions complex responses to ACh were also studied upon denudation of the endothelium (3 μL/min TritonX-100 5 min), and non-specific blockade (pentoxifillin 200μM) of the phosphodiestherase enzyme (PDE).
In low perfusion conditions (n=27) ACh resulted in dose-dependent constrictions (ΔCPPmax: +27±8 mmHg, p<0.01), BK increased (+8±1 mmHg, p<0.001), SNP decreased (−6±1 mmHg, p<0.001) CPP. At elevated CPP (n=28) responses to ACh became tri-phasic. Initial coronary dilatation (ΔCPPmax: −8±1 mmHg, p<0.01) was followed by vasoconstriction (ΔCPPmax: +16±3 mmHg, p<0.01), and upon cessation of the infusion a secondary dilatation was seen (ΔCPPmax: −28±3 mmHg, p<0.01). Moreover, the effect of BK was reversed yielding significant vasodilatation (ΔCPPmax: −33±2 mmHg, p<0.01). The dilatory response to ACh (ΔCPPmax: −25±5 vs. +2±2 mmHg, p<0.05) and BK (−23±5 vs. +3±3 mmHg, p<0.05) was completely blocked by denudation (n=7), while the coronary constriction phase of ACh (ΔCPPmax: +18±10 vs. +3±3 mmHg, p<0.05) was modestly and gradually decreased by PDE inhibition (n=6).
1) The direction (constriction/dilatation) and the extent of the coronary effects of ACh and BK are fundamentally affected by the vascular tone itself. 2) Complex vascular responses to ACh are mediated by the parallel activation of endothelium dependent relaxation and direct vascular smooth muscle contraction. 3) PDE blockade via enhancing endothelial dilation perfectly counteracts the coronary constrictor component of ACh.
This study was supported by the research grant TÁMOP-4.2.2/08/1/KMR
In a previous study we have shown that the maintenance of nitric oxide (NO) availability during ischaemia is important for protection against arrhythmias occurring during ischaemia/reperfusion (I/R). There is also evidence that the administration of sodium nitrite (NaNO2) prior to reperfusion protected against reperfusion injury; an effect which was attributed to the release of NO from nitrite under ischaemic conditions. The aim of the present study was to examine whether NaNO2 is able to modify the ischaemia and reperfusion-induced ventricular arrhythmias in anaesthetised dogs. We also examined the possible mechanism(s) of the potential antiarrhythmic protection.
Control dogs (C; n=15) were infused with saline and subjected to a 25 min occlusion of the left anterior descending coronary artery (LAD) followed by rapid reperfusion. In two other groups, NaNO2 was given in intravenous infusion in a dose of 0.2 μM/min/kg, either prior to and during occlusion (NaNO2-PO; n=14), or 10 min prior to reperfusion (NaNO2-PR; n=5). Arrhythmia severity was assessed as the number of ventricular premature beats (VPBs) and tachycardiac episodes (VT) as well as the incidences of VT and ventricular fibrillation (VF). In blood samples, taken from the coronary sinus at various time intervals during the experiments, plasma nitrate/nitrite (NOx) levels were measured by Griess reaction. Superoxide (O2·) production was determined by DHE staining in myocardial tissue samples taken following reperfusion.
NOx levels were markedly increased in NaNO2 treated groups over the entire occlusion period. This was in contrast to the controls, where the NOx levels were reduced by the end of the occlusion. Ventricular arrhythmias in both groups of NaNO2 treated dogs (NaNO2-PO, NaNO2-PR) were also markedly suppressed. Thus, compared to the controls, the number of VPBs (477±96 vs. 156±78, 84±67) and the incidence of VT (87% vs. 29%, 20%) were markedly reduced and survival from the combined ischaemia and reperfusion was significantly increased (0% vs. 50%, 80%). Superoxide production, determined following reperfusion was also substantially attenuated in dogs infused with NaNO2.
The results show that the administration of NaNO2, no matter whether infused prior to occlusion or just prior to reperfusion, protects against ischaemia and reperfusion-induced arrhythmias and confirm our previous findings that the maintenance of NO availability during ischaemia is important for the antiarrhythmic protection. Furthermore, we suggest that NO is involved in the reduction of superoxide and peroxynitrite production during reperfusion which might explain the high incidence of survival in the NaNO2 treated dogs.
This work was supported by OTKA (project number: K75381)
The inflammatory reaction is characterized by increased generation of reactive oxygen species (ROS), mainly provided by different types of leukocytes. Limitation of oxidative stress is beneficial at least under conditions of chronic inflammation. Since in plants a big variety of antioxidative compounds are present, we are interested to determine the effects of different extracts often used in phytotherapy on cellular models of inflammation.
The following extracts, as prepared for the production of phytotherapeutic drugs, were used: Peppermint, lemon balm, camomile, willow bark. These extracts, as well as the corresponding extracts solvents, were tested in different models of cellular generation of ROS, determined by luminol-enhanced chemiluminescence: buffy coat cells from human blood, stimulated by opsonized zymosan, alveolar macrophages present in an excised specimen from porcine lung, a submucosal preparation from mouse ileum stimulated by histamine. In order to determine general antioxidative properties, the AAPH reaction, for comparison trolox or vitamin c was used.
The results show that all extracts have strong antioxidative properties. Although peppermint is usually the most potent extract, the others show different activity in the various test systems. This finding indicates that the extracts exert their effects probably not only by direct radical quenching reactions, but also by interfering with the pathways responsible for cellular radical production.
Mechanisms of regulation of myocardial oxygen consumption (MVO2), despite their great practical significance, haven’t yet been fully explained. Angiotensin converting enzyme inhibitors (ACE-I) due to their cardioprotective, vasculoprotective, antiatherogenic effects, as well as because of tissue protection, are widely used in therapy of CVD. However, a consensus on the effect of ACE-I on MVO2 under the condition of endothelial dysfunction during the process of atherosclerosis, has not been reached yet. The aims of the study were to determine the influence of captopril, in the animal treated by atherogenic diet on: 1) MVO2 of rabbit heart mitochondria, 2) plasma prekallikrein concentration (PKC), focusing on NO levels, 3) antioxidative protection (AOP), and 4) atherogenic risk of lipid origin.
In the course of twelve-week treatment, the rabbits (n=10/group) were treated daily, in group A: physiologic saline; B: atherogenic (2% hypercholesterolemic) diet; C: atherogenic diet and 2 hours after that captopril (25 mg/day) and D: captopril only (25 mg/day).
Our results showed that captopril (group D) reduced the MVO2 but normal values of ADP/O and RCR indexes, as well as AOP, PKC, nitrite and lipid parameters levels were kept. The treatment with atherogenic diet (group B) also reduced MVO2 but with lower ADP/O index, impaired AOP and PKC, elevated nitrite and lipid parameters levels. The addition of captopril (group C) preserved MVO2, followed by minor decrease of PKC and AOP, the increase in nitrite concentration was not statistically significant (p>0.05) compared with the results obtained in group B.
Taken together, these results showed that due to these beneficial effects, the mechanisms of MVO2 inhibition under the influence of captopril were, most probably, different from the mechanisms of respiration of rabbit heart mitochondria during experimental atherosclerosis. These results may represent a contribution to the research of other authors in clarifying the complex mechanisms of regulation of MVO2 in physiological and pathophysiological conditions.
The mechanisms leading to microvascular dysfunction in diabetes mellitus have still not been clearly elucidated. Our studies and studies by others revealed several mechanisms, which could underline the development of dysfunction. Isolated, pressurized skeletal muscle arterioles (150 microm. at 80 mm Hg) of rats with streptozotocin-induced diabetes mellitus (Type 1 DM) exhibited substantially reduced dilations induced by increases in flow compared to those of normal rats, which was in part due to reduced nitric oxide mediation. The responses could be improved by sepiapterin, precursor of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin. Correspondingly, transient high-glucose treatment (tHG; incubation with 30 mM glucose for 30 min) elicited reduced dilator response, which could be improved by superoxide dismutase or sepiapterin or inhibition of either the glycolysis by 2-deoxyglucose or mitochondrial complex II by 2-thenoyltrifluoroacetone.
In type 2 DM (T2-DM) the reduced arteriolar dilations to ACh, the nitric oxide (NO) donor NONOate, and increases in flow were significantly augmented either by in vitro inhibition of NAD(P)H-oxidase, or by in vivo rosiglitazone (ROSI) treatment (an agonist of peroxisome proliferator-activated receptor-gamma: PPARgamma activation). Also, systolic and mean arterial pressures and total peripheral resistance were significantly elevated in T2-DM mice compared with those of control. Correspondingly, the basal tone of skeletal muscle arterioles of T2-DM mice was increased due to an enhanced COX-2-dependent production of constrictor prostaglandins.
Also, we have found significant contribution of polyol pathway to arteriolar dysfunction in the presence of hyperglycemia, which elicited oxidative stress, reduced NO, and enhanced PGH(2)/TXA(2) mediation of vasomotor responses.
Collectively, these findings suggest that type 1 and type 2 DM leads to vasomotor dysfunction likely due to the transient and chronic elevation of glucose. Increased levels of glucose via its increased metabolism, elicits enhanced production of superoxide, which decreases the bioavailability of NO and the level of the NOS cofactor BH(4), resulting in a reduction of vasomotor responses mediated by NO. Whereas, PPARgamma activation, by reducing oxidative stress, increases NO bioavailability. In addition, high glucose activates COX-2 leading to the enhanced production of constrictor prostaglandins. These alterations in microvascular NO and prostaglandin synthesis may contribute to the increase in peripheral resistance and blood pressure in diabetes mellitus. The revealed mechanisms may facilitate to develop better therapies to treat microvascular dysfunction in various forms of diabetes mellitus.
Supported by the Hungarian National Scientific Research Fund (OTKA) K71591, T67984 and American Heart Association, Founders Aff. 0855910D and National Institutes of Health grant PO1-HL-43023
The concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is increased in patients with endothelial dysfunction. The present study was designed to measure and compare serum ADMA, symmetric dimethylarginine (SDMA) and L-arginine levels in blood samples obtained from coronary sinus and from peripheral vein in patients undergoing coronary revascularization with or without cardiopulmonary bypass.
Two groups of patients with coronary heart disease (CHD) were selected for elective coronary bypass graft surgery (CABG). Patients were subjected to CABG surgery with cardiopulmonary bypass (CPB) (n=20) or with off-pump CABG surgery (OPCAB) (n=21). Blood samples for measurements of ADMA, SDMA and L-arginine were withdrawn from the coronary sinus (CS) and from the peripheral vein (P) at baseline; three times during CABG surgery and on the first and fifth postoperative days. Plasma levels of L-arginine, SDMA, ADMA were determined with liquid chromatography-tandem mass spectrometry (LC-MS-MS).
Based on the intraoperative (CS) samples, the post-hoc ANOVA did not reveal a discernible increase of ADMA in the OPCAB group. In contrast ADMA levels rose in the CPB group (F=0.416, p<0.685 and F=14.751, p<0.001 for OPCAB and CPB groups respectively). A similar significant increase of ADMA was observed in the peripheral blood (F=30.738, p<0.001) during CPB, while ADMA levels remained largely unchanged in the peripheral blood during OPCAB. The time-course of L-arginine levels was significantly different in the blood samples from coronary sinus (F=3.255, p<0.05), when compared to samples from the peripheral blood (F=3.255, p<0.05). In the OPCAB group repeated measures ANOVA did not reveal a significant intrasubject time effect of L-arginine, either in blood samples from coronary sinus or in the samples from the peripheral vein. The values of the L-arginine/ADMA ratio were significantly higher in the OPCAB group at baseline and on the first postoperative day as compared to results of the CPB group (178.29±11.56 vs. 136.28±13.72 and 129.43±7.08 vs. 106.8±6.9 for OPCAB and CPB groups respectively).
Plasma levels of ADMA, SDMA, L-arginine and L-arginine/ADMA ratio are reliable and feasible markers of an early ischaemia-reperfusion injury. During CPB operation (but not during OPCAB), the plasma concentration of ADMA increased significantly and remained elevated until the first postoperative day due to extensive ischemia-reperfusion injury caused by CPB.
Hypertension provokes remodeling of large vessels and vasomotor dysfunction in resistance arteries via increased oxidative stress and consequent poly(ADP-ribose) polymerase (PARP) activation. Accordingly we assumed in our experiment the vasoprotective effect of L-2286 – a quinazoline-type PARP inhibitor – in spontaneously hypertensive rat (SHR) model.
One group of SHRs received no treatment (SHR-C, n=10) and the other (SHR-L, n=10) received 5 mg/kg/day L-2286 for 32 weeks. Normotensive age-matched groups were also included (CFY-C, n=10). In carotid arteries the following were measured: intima-media thickness (IMT), arterial stiffness index (ASI), acetylcholine-(ACh) and sodium nitroprusside (SNP) induced vasodilation. Blood pressure was assessed with the tail-cuff method. Ultrastructural morphology of aortas and with immunohistology the PARP activation were determined.
L-2286 treatment did not affect the blood pressure of SHR and CFY rats (SHR: 232±2 vs. CFY: 130±1 mmHg). At the beginning of the study IMT (CFY: 39±2.5 μm, SHR: 40±2.3) and ASI (CFY: 3.79±0.46, SHR: 3.86±0,41) were similar in SHR and CFY rats. However, at the end of the study IMT and ASI values were elevated in SHR-C compared to CFY-C (IMT: CFY: 41±2 μm, SHR: 78±5 μm; ASI: CFY: 4.1±0.1, SHR: 5.8±0.3, p<0.01), which were decreased significantly by L-2286 treatment (SHR-L: IMT: 63±1 μm, ASI: 4.3±0.4, p<0.05). Relaxation of carotid arteries to ACh was significantly increased in SHR-L group compared to SHR-C (22.3±8% vs. 41±5%, p<0.05), but responses to SNP did not differ. Electronmicroscopic examination showed increased fibroblast activation with collagen overproduction and lesions of the endothelial layer in aortic wall of SHR-C group. In contrast in SHR-L group activation of smooth muscle cells and decreased collagen growth were noticed. Furthermore in SHR-C increased PARP activation was detected which was reduced in SHR-L.
These data suggest that chronic inhibition of PARP decreases vascular remodeling and improves both endothelial and smooth muscle dependent vasomotor function.
Supported by: PTE ÁOK-KA 34039-21/2009, OTKA K71591, OTKA T67984 Grants
Glycogen synthase kinase-3beta (GSK-3beta) is a multifunctional Ser/Thr kinase playing crucial roles in cell death and survival. A major mechanism of cell necrosis after ischemia/reperfusion is opening of the mitochondrial permeability transition pore (mPTP) triggered by Ca2+ overload and production of reactive oxygen species upon reperfusion. Binding of a mitochondrial matrix protein, cyclophilin D (cypD) to adenine nucleotide translocase (ANT) in the mPTP complex increases sensitivity of the mPTP to Ca2+, facilitating cell necrosis. Inactivation of GSK-3beta by phosphorylation at Ser9 elevates the threshold for mPTP opening and increases myocyte tolerance to lethal reperfusion injury. Ischemic preconditioning, erythropoietin (EPO) receptor activation and activation of the mitochondrial KATP channel before ischemia similarly enhance GSK-3beta phosphorylation upon reperfusion, in which PI3K-Akt signaling triggered by activation of the adenosine A2b receptor appears to be involved. Mechanisms by which phospho-Ser9-GSK-3beta inhibits mPTP opening are unclear, but suppression of cypD-ANT interaction by binding of phospho-GSK-3beta to ANT is one of the possible mechanisms. Although GSK-3beta phosphorylation is crucial for a variety of interventions against ischemia/reperfusion injury to be protective, signaling to GSK-3beta is compromised by co-morbidities, including diabetes mellitus. In a rat model of obese type 2 diabetes, impairment of Jak2 phosphorylation by up-regulated calcineurin activity, interruption of ERK1/2-GSK-3beta signaling and up-regulation of non-phosphorylated GSK-3beta in mitochondria by endoplasmic reticulum stress make the myocardium refractory to protection afforded by Jak2 activating ligands such as EPO. Considering predominant roles of GSK-3beta in cardiomyocyte death, manipulation of this kinase and repair of defects in intracellular signaling leading to GSK-3beta phosphorylation are promising strategies for myocardial protection in coronary artery disease and heart failure.
The cardiac Sympathetic Nervous System (SNS) modulates coronary vasodilatation, heart frequency and contractility. A functional impairment of SNS occurs with aging and contributes to the pathophysiology of several cardiovascular diseases. However, it is not yet clear whether the interactions between sympathetic terminals (STs) and target cells change with aging and whether these alterations affect myocardial morphology and functionality.
We aimed to: i) provide a morphological description of the interaction between STs, vasculature and cardiomyocytes; ii) investigate how this relationship changes with aging and the functional consequences of such alteration.
Using immunofluorescence analysis we demonstrated that in normal adult mouse hearts STs are distributed at each anatomical level with a greater density in the atria and the right ventricle. STs were found in proximity to coronary vessels, cardiomyocytes and to capillaries. Similar analysis on hearts from aged mice demonstrated a significant reduction of sympathetic myocardial innervation with morphological alterations of STs. STs were shorter and fragmented in aged hearts. In addition, hearts from aged mice showed cardiomyocyte atrophy, increased capillary density and reduced capillary size. Sympathectomized hearts reproduced the same phenotype observed in aged hearts, with about a 20% decrease in cardiomyocyte size, as compared to controls, and altered capillary organization.
The structural and functional integrity of SNS is crucial in the maintenance of cardiomyocte trophism and capillary organization in the postnatal mouse heart. Future development of these studies will aim to determine the molecular pathways involved in the aging-related SNS impairment.
Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase. Increased ADMA plasma levels are associated with cardiovascular disease.
Two groups of patients undergoing percutaneous coronary intervention (PCI) with stenting were enrolled into the study. Group I consisted of 16 patients with, whereas group II included 24 patients without STEMI (controls). Before PCI and at 1 hour, 5 days and 30 days after reperfusion blood samples were taken for measurement of l-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and l-ornithine. L-arginine and methylarginines were measured by liquid chromatography-tandem mass spectrometry. Arginine methylation index (Arg-MI) was calculated according to the formula: Arg-MI = (ADMA+SDMA)/MMA.
In patients with elective stent implantation stenting induced a prompt and sustained depression of ADMA (p<0.000), and l-ornithine (p<0.000) with simultaneous increase of l-arginine (p<0.001) and l-arginine/ADMA ratio (p<0.000). By contrast, STEMI patients responded to stent placement with a variable increase in l-arginine (p<0.01) and l-ornithine (p<0.000), whereas there was an early fall of Arg-MI after stenting followed by a steady increase to approach the initial values. ADMA remained constant for STEMI patients after stent placement (p<0.069). The differences in the time-course for ADMA (p<0.000), Arg-MI (p<0.012) and l-ornithine (p<0.003) proved to be significant between the STEMI and control group.
Stent placement improves endothelial dysfunction in patients with obstructive coronary artery disease (OCAD) when it is not complicated by STEMI. Long-term follow-up of ADMA may be suitable to monitor the improvement of coronary function after revascularisation.
It has been observed that significant gender differences exist already in normal hearts. They involve among others cardiac growth, contractile function, calcium metabolism and function of mitochondria. Differences, characteristic of the normal myocardium, generate the logical presumption of the different reaction of the male and female heart to various pathogenic factors. Most of the experimental studies confirm the clinical observations: increased resistance of the female heart to ischemia/reperfusion injury was shown in dogs, rats, mice and rabbits. Furthermore, gender differences in the ischemic tolerance of the adult myocardium can be influenced by interventions (e.g. hypoxia) imposed during the early phases of ontogenetic development. The already high tolerance of the adult female heart can be increased by adaptation to chronic hypoxia and ischemic preconditioning. It seems that the protective effect depends on age: it was absent in young, highly tolerant heart but it appeared with the decrease of natural resistance during aging. Both experimental and clinical studies have indicated that female gender also favorably influences the remodeling and the adaptive response to myocardial infarction. It follows from the data available that male and female hearts differ significantly in many parameters under both physiological and pathological conditions. Detailed molecular and cellular mechanisms of these differences are still unknown; they involve genomic and non-genomic effects of sex steroid hormones, particularly the most frequently studied estrogens. The cardiovascular system is, however, influenced not only by estrogens but also by other sex hormones, e.g. androgens. Moreover, steroid hormone receptors do not act alone but interact with a broad array of co-regulatory proteins to alter transcription. The differences are so important that they deserve serious consideration in clinical practice in search for proper diagnostic and therapeutic procedures.
Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling after myocardial infarction (MI). However, the NOS isoform responsible for LV remodeling after MI has not been determined. We investigated whether inhibition of iNOS uncoupling prevents LV remodeling after MI. MI was created in wild-type (WT), inducible NOS-knockout (iNOS−/−), and endothelial NOS-knockout (eNOS−/−) mice by ligation of the left coronary artery. iNOS expression was markedly increased after MI in WT and eNOS−/− mouse hearts associated with an increase in nitrotyrosine (NT) formation, a marker for oxidative/nitrosative stress, without an increase in NOx, an index for bioavailability of NO. LV diastolic and systolic dimensions assessed by echocardiography 4 weeks after MI were not significantly different between all the mouse hearts. Oral administration of sepiapterin, a precursor of the NOS co-factor tetrahydrobiopterin, inhibited NT formation, increased NOx, and prevented LV dysfunction after MI in WT and eNOS−/− mice but not in iNOS−/− mice. These results suggest that inhibition of iNOS uncoupling by sepiapterin converts iNOS from detrimental to beneficial NOS and prevents LV remodeling after MI.
Embryonic stem (ES) cell-derived cardiomyocytes may play a special role in cardiac repair after myocardial infarction. Therefore, it is the most important to protect stem cells against ischemia/reperfusion (I/R) injury. The aim of this study was to test whether a nitric oxide (NO)-donor is protective against I/R injury of mouse ES cell-derived cardiomyocytes.
To reach our goal, we used mouse ES cell line (HM-1) derived embryonic bodies (EB). EBs were seeded onto 24-well plates (1 EB/well) and were kept in growth medium (containing ascorbic acid) under normoxic condition (at 37°C, 5% CO2) for 5 days. We used both hypoxic chamber and chemical conditions to stimulate ischemia. In the hypoxic group, the medium of the cultures was replaced with a “hypoxic” solution and plates were kept in a hypoxic chamber (gased with 95% N2 and 5% CO2 at 37°C) for 150 minutes in the presence or absence of the NO-donor S-nitrozo-penicillamine (SNAP, 10−6 M). The next step was the reoxygenation, when cells were covered with growth medium (without ascorbic acid) and kept in a normoxic incubator for 120 minutes. Subsequently, cell viability was measured by using Trypane blue staining and cells were counted in a hemocytometer. After simulated ischemia the cell death was 22.0±2.1%, which was attenuated by using the NO donor SNAP (13.3±1.9%, p<0.05).
As a control experiment, other series of EBs were maintained in a “normoxic” solution and plates were kept in the normoxic incubator for 150 minutes. All cells were subjected to reoxygenation and viability tests were performed similarly to simulated ischemia groups. In normoxic condition the cell death was 12.5±0.1%.
In conclusion, our results show that NO-donors may protect ESC-derived cardiomyocytes against ischemia/reoxygenation injury. Thus, NO-donors can be useful candidates for the enhancement of ES cell-derived cardiomyocyte survival after implantation into the ischemic myocardium.
NKFP_07_1-ES2HEART-HU, CLONET MRTN-CT-2006-035468, Partners PIAP-GA-2008-218205
Naturally occurring decline in cardiovascular reserve with age associates with a combination of the reduction in cardiomyocyte number and altered cardiomyocyte function. The total number of cardiomyocytes is estimated to be less by one third in the old heart than the number of cardiomyocytes at birth. Additionally, recent investigations suggested that about half of the cardiomyocytes is the same as at birth, while the other half of the cardiomyocytes is the result of cardiomyocyte renewal in the senescent heart. Thus, the reduction in the total number of cardiomyocytes of the aging heart cannot be fully compensated by cardiomyocyte renewal. Aging of long-lived differentiated myocardial cells, as well as of cardiac progenitor stem cells may contribute to an increased rate of apoptosis, and decreased capacity of cell duplication and/or cell differentiation into cardiomyocytes. In addition, differentiated cardiomycytes are prone to accumulating biological by-products of cellular metabolism and of incompletely processed oxidative insults. In this context, interactions between lysosomes and mitochondria appear to be crucial and may provide a mechanistic background for the age-dependent alterations in cardiac biomolecules. This reasoning postulates a direct relationship between the number of pro-oxidative, ill-functioning mitochondria and the amount of ballast-overloaded lysosomes in the long-lived cardiomyocytes. Accumulation of biological garbage (e.g. lipofuscin), and telomere shortening might be considered as hallmarks of cardiomyocyte aging with implications for depressed cardiac function and cardiomyocyte renewal. Moreover, an increasing oxidative stress may damage various subcellular targets and alter intracellular signaling. Changes in protein expression together with posttranslational modifications of myocardial proteins (e.g. membrane transporters and myofilaments) affect excitation-contraction coupling and explain the progressively declining mechanical function of the cardiomyocytes. Altogether, these changes are mainly responsible for the reduced cardiac reserve in aged individuals.
Prolactin-releasing peptide (PrRP), the endogenous ligand of the once “orphan” G-protein coupled receptor hGR3/GPR10 has been shown to have a wide range of action in the central nervous system: it modulates the hypothalamo-pituitary-adrenal axis, feeding behaviour and nociception, furthermore, central administration of the substance elevates the mean arterial pressure, heart rate and renal sympathetic nerve activity. Although, high levels of specific binding sites for PrRP have been found in the myocardium, the role of PrRP in the peripheral regulation of the cardiovascular system is not known so far. The aim of our study was to determine the effect of PrRP on the acute regulation of cardiac contractility.
Isolated hearts of 7-week-old male Sprague-Dawley rats were perfused by the Langendorff method in a constant flow system. Different drugs were added to the perfusate for 15 minutes after 45 minutes of equilibration. Contractile force was measured by connecting a force displacement transducer to the apex of the heart.
Administration of PrRP-31 (1 to 100 nM) resulted in a slowly developing, dose-dependent increase in cardiac contractility in the isolated rat heart preparation, with a maximal increase of 12.9±1.7%. This modest positive inotropic effect could not be further augmented by the non-specific phosphodiesterase inhibitor IBMX (10 μM). A marked enhancement in cardiac contractility was observed when PrRP was administered together with protein phosphatase (PP1 and PP2A) inhibitor calyculin A (1 nM). However, okadaic acid (10 nM), which only inhibits PP2A had no effect on PrRP-related positive inotropic effect. PKC-α inhibitor Ro-32-0432 (100 nM) significantly enhanced the inotropic response to PrRP. Parallelly, Ser-16 phosphorylation of phospholamban was increased by PrRP in the presence of Ro-32-0432, although the peptide alone had no effect on phospholamban phosphorylation.
Our data provide evidence on the hitherto unknown direct positive inotropic effect of PrRP. We could show that PrRP enhances contractility independently from the PKA-cAMP pathway. We also found that the PrRP-induced positive inotropy is limited by simultaneous activation of PKC-α and PP-1.
The direct atherogenic action of C. Pneumonia infection has now been identified. Circulating cholesterol can strongly modulate these atherogenic effects but the molecular mechanisms responsible for this are not clear. We have explored the mechanism whereby C pneumonia induces atherosclerosis and what factor within the high circulating cholesterol may be responsible for the effects. C. Pneumonia infection is associated with an increase in cell proliferation in the vasculature and this was strongly influenced by oxidized LDL. An induction of HSP60 expression was also involved in the mitogenic effects. The mitogenic effects of C. Pneumonia result in a thickening of the vascular wall under tissue culture conditions. This does not involve a host immune response but appears to be induced by a direct action on the vasculature. These effects are also accompanied by changes in vascular contractile function. Our data strongly support a role for infectious disease, and C Pneumonia specifically, in atherogenic cardiovascular disease.
Supported by CIHR
Circulatory short term assist devices were designed to support patients in acute hemodynamic collapse, when the patients are still considered salvageable and the pharmacologic support is ineffective. Now short term mechanical support is used in wide range of clinical conditions.
There are 3 common indications for the use of short-term mechanical support: a bridge to hemodynamic and myocardial recovery after acute MI or acute myocarditis; a bridge to await a transplant directly; and a bridge to upgrade to a long term assist device in 2 weeks, if the organ function has improved and the sepsis has resolved.
Inadequate preservation of the donor heart or RV failure after heart transplantation is also an indication for implantation of a right ventricular assist device (RVAD), left ventricular assist device (LVAD) or biventricular assist device (BIVAD). For the patients in acute cardiogenic shock after cardiac surgery this option and the plan must be disscussed before.
Several modalities exist for providing short-term mechanical circulatory support. These include counterpulsation, centrifugal, axial and pneumatic pumps. These devices are easy for implantation. Most of them are implanted not just surgically but also percutaneously. The second generation has constant flow instead of pulsatile flow. They are smaller and easier to implant, with less intraoperative dissection and blood loss, but these theoretical advantages are not validated yet.
The oldest device currently in use is intra-aortic balloon pump counterpulsation (IABP). The result is a reduction in myocardial work through afterload reduction and improvement in myocardial oxygen supply through augmentation coronary perfusion pressure.
ECMO is centrifugal pump with conventional cardiopulmonary bypass technology to support the circulation with continous nonpulsatile cardiac output and extracorporeal oxygenation. It can be provided in venous-arterial cannulation for both respiratory and circulatory support and veno-venous cannulation only for respiratory support. Most commonly it is used in neonatal intensive care unit, but now used also for adults mainly for pulmonary recovery. It can be inserted percutaneously in the ICU. Aggresive anticoagulation is needed.
Short-term ventricular assist devices (VAD) are mechanical pumps that serve to replace or augment the funtion of RV or LV or both ventricles. The primary indication is acute medically refractory heart failure: CI < 2.0 l/min/m2, WP > 20 mmHg, systolic BP < 80 mmHg despite pharmacological support. The following are currently in use:
This is a left atrial to femoral arterial bypass system. It can be inserted percutaneously and provides active flow via a centrifugal pump. It can provide up to 5 l/min of blood flow. Systemic anticoagulation is required. Contraindications include advanced right ventricular failure, ventricular septal defect, aortic insufficiency and severe peripheral vascular disease. In clinical trials it is more effective in improving hemodynamics, but not 30 days mortality comparing to IABP.
It is a percutaneously or surgically implanted VAD, with microaxial system for RVAD or LVAD. The inflow cannula with the pump is inserted across the aortic valve under fluoroscopic guidance. The device sits across the aortic valve and draws blood through distal port to unload the ventricle in the ascending aorta. Modest anticougulation is required.
This can be used for single RV or LV or for biventricular support. It is a pneumatically driven extracorporeal pump with pulsatile flow of up to 6 l/min. It was approved for postcardiotomy failure but the indications have expanded. Systemic anticoagulation is required.
This is a continuous flow centrifugal-type rotary blood pump. The only moving component within the pump is the impeller, which is magnetically levitated and rotated in a contact-free manner. It can provide flow rates of up to 9.9 l/min. It causes very little damage to the blood. It is the most often used in Europe. It is the pump of choice in acute post cardiotomy situations, if there is the likelihood of recovery. With TEE and haemodynamic monitoring it is important to determine whether LVAD, RVAD or BiVAD support is necessary. Anticoagulation is necessary with APTT 60–100 sec.
It doesn’t matter which device will be implanted, during the implantation and during the stay in the ICU monitoring of hemodynamic data, CVP, SVO2, VAD flows are mandatory. The patients are prone to bleeding. In signs of tamponade, TEE should be done. Regular hemoglobin and platelet measurements schould be made and the patients must be transfused if platelets are consumed by the device or hemoglobin is below 9 g/dl. There must be a special protocol for recovery assessment and a weaning protocol.
A novel concept of myocardial protection against ischemia/reperfusion injury is based on the exploitation of the heart’s own defensive mechanisms and relies on the evidence that the heart is able to protect itself by way of adaptation, either short-term or long-lasting, to transient episodes of stress (ischemia, hypoxia, free oxygen radicals, etc.) preceding sustained ischemia. Experimental data suggest that besides higher vulnerability to ischemia/reperfusion, diabetes mellitus may evoke development of adaptation leading to paradoxically enhanced ischemic tolerance that might share some molecular pathways with preconditioning in non-diseased myocardium, in particular, those related to oxidative state and activation of “pro-survival” cascades. We investigated ischemia-induced ROS formation (TBARS) in the Langendorff-perfused hearts of 1-wk diabetic (STZ, blood glucose>20 mM) and of non-diabetic rats preconditioned with ischemia and/or with KATP opening (diazoxide). Furthermore, we evaluated the involvement of PI3K/Akt in preconditioning-like effect in diabetic hearts and in normal hearts exposed to hyperglycemia (22 mM) and explored a potential link between cardiac response to I/R and gene expression of PPAR as main transcriptional regulators of lipid metabolism and energy production (RT-PCR).
In the hearts subjected to 30-min ischemia/2-h reperfusion for the determination of functional recovery (LVDP), infarct size (IS) and arrhythmias, lower susceptibility to I/R in the diabetics was documented by a 2-fold higher LVDP recovery, 60% decrease of IS, increased baseline levels of endogenous antioxidants and by reduced ROS production and arrhythmogenesis during ischemia, similar to that observed in the preconditioned non-diabetic hearts. Postischemic mRNA levels of PPARs were preserved in the diabetics in contrast to their marked downregulation in non-diabetics. In these hearts, efficient anti-infarct protection conferred by hyperglycemia or by diazoxide was abolished by PI3K inhibitor wortmannin similar to its effect in the diabetics indicating an important role of PI3K/Akt activation in protection against lethal myocardial injury. On the other hand, reduced incidence and severity of ventricular arrhythmias in the diabetic hearts as well in the non-diabetic preconditioned hearts were not reversed by application of wortmannin indicating that PI3K/Akt activity is not involved in the mechanisms of the anti-arrhythmic protection in this situation.
In conclusion, adaptive processes induced in the diabetic myocardium in the acute phase of the disease might be associated with the changes in PPAR gene expression and attenuation of oxidative load during I/R. Hyperglycemia and KATP opening might contribute to lower susceptibility to lethal injury via mechanisms involving PI3K/Akt activation.
Grants VEGA-2/0173/08, 1/0620/10, APVV-LPP-0393-09, APVV-0538-07, GSRT-5190/2005-759, GACR-305/07/0875
Heart failure is one of the most common causes of cardiovascular morbidity and mortality, and its prevalence is rapidly increasing as the mean age of the population advances. The molecular mechanisms responsible for heart failure are poorly defined, but disruption of coordinated tissue growth and angiogenesis may contribute to the progression from adaptive left ventricular hypertrophy to heart failure. Recently, it has been shown that a reduction in cardiac capillary density promotes contractile dysfunction in transgenic mice. It has also been concluded that as a response to hypertrophic stimuli both heart growth and function are angiogenesis dependent. Despite these observations implicating a beneficial role for angiogenesis in heart failure, little is known concerning the effects of key regulators of angiogenesis on diastolic dysfunction. We have tested the hypothesis whether local vascular endothelial growth factor-B (VEGF-B) gene therapy by intramyocardial delivery of adenoviral vector expressing VEGF-B167A, the predominant iso-form in humans, prevents left ventricular diastolic dysfunction. VEGF-B was overexpressed in healthy rat hearts and in hearts in rats subjected to pressure overload by angiotensin II infusion for 2 weeks using osmotic minipumps. Intramyocardial delivery of adenoviral vector expressing VEGF-B167A improved angiotensin II-induced diastolic dysfunction compared to LacZ control virus. Local VEGF-B gene transfer increased mean capillary area in the left ventricle in control and angiotensin II-infused animals, while the density of capillaries was not affected. Interestingly, significant increases were noted in Ki-67+ proliferating cells, expression of interleukin 1β, and c-kit+ cells in response to VEGF-B gene transfer. The increase in cardiac c-kit+ cells was not associated with an induction of stromal cell-derived factor 1α, suggesting no mobilization of cells from bone marrow. Also, the phosphatidylinositol-3 kinase/Akt-pathway was activated. These results demonstrate that VEGF-B gene transfer resulted in prevention of angiotensin II-induced diastolic dysfunction associated with induction of the Akt-pathway, increased proliferation and number of c-kit+ cells as well as an increase in capillary area in the left ventricle. VEGF-B may offer novel therapeutic possibilities for the prevention of the transition from compensated to decompensated cardiac hypertrophy and thereby for the treatment of heart failure.
Hypertension afflicts about 40% of the adult population in developed countries. Although successful therapy can usually be established, the number of idiopathic cases is high. Nevertheless, hypertension may occur via two major vascular mechanisms: (i) higher intracellular Ca2+ concentration in smooth muscle resulting in increased myogenic tone and (ii) increased Ca2+ sensitivity of the contractile system leading to increased vasoconstrictive response to agonists. The aim of this study was to characterize the Ca2+ sensitivity of force production in isolated arteries.
Arteria basilaris was isolated from the rat. Arterial rings (3–5 mm in length) were fixed on an isometric contractile measurement system. Contractile force (in mN) and intracellular Ca2+ concentrations ([Ca2+]i) of vascular rings expressed as Fura-2 (340/380 ratio) were measured simultaneously in the presence of agonists (KCl, serotonin, U-46619) and inhibitors of PKC (GF19209, 1 μM) and Rho kinase (fasudil, 2.5 μM).
KCl evoked a maximal contraction of 3.2±0.2 mN (n=97) with a 0.17±0.01 (340/380 ratio) increase in [Ca2+]i (n=97). The Ca2+ sensitivity of force production was 18.5 mN/340/380 ratio, which was independent of the dose. Serotonin evoked a higher contraction (6.7±0.5 mN) (n=19) with similar changes in [Ca2+]i (0.15±0.02) (n=19). The Ca2+ sensitivity of force production increased parallelly with the dose (EC50=32 nM) reaching its maximum at 38.7 mN/340/380 ratio. U-46619, a thromboxane A2 (TxA2) receptor agonist evoked a contraction of 4.2±0.5 mN with a significantly lower increase in [Ca2+]i (0.04±0.01 340/380 ratio, n=23) than that of KCl. As a result the increase in Ca2+ sensitivity of force production was more prominent (maximum: 97.9 mM/340/380 ratio, EC50=7.9 nM). The Ca2+ sensitivity of force production was not affected by PKC inhibition upon serotonin and U-46619 treatments (39.7 and 111.0 mM/340/380 ratio, respectively) but decreased upon Rho-kinase inhibition (to 24.2 and 42.4 mM/340/380 ratio for serotonin and U-46619, respectively).
We developed and validated a method for the determination of Ca2+ sensitivity of force production in isolated vascular preparations in vivo. It was revealed that Rho kinase, but not PKC mediated increase in Ca2+ sensitivity of force production contributes to vascular constriction to serotonin and TxA2 receptor stimulation.
Peroxynitrite-induced nitrosative stress has been shown to trigger ischemic stress adaptation of the rat myocardium. The aim of this study was to investigate the role of nitrosative stress in the mechanism of preconditioning in experimental hyperlipidemia.
Rats were fed with 2% cholesterol-enriched diet for 12 weeks. Hearts were than subjected to a preconditioning protocol: 3 intermittent periods of global ischemia/reperfusion or exogenous peroxynitrite (ONOO−) infusion (final 3 μM) of 5 min duration each) followed by a 30-min test ischemia and 120 min of reperfusion.
In the normocholesterolemic group preceding preconditioning induced either by global ischemia or ONOO− infusion decreased infarct size from 45±3.8% to 5.7±2.0% and 13.1±4.1%, respectively. ONOO− infusion did not induce preconditioning in the cholesterol-fed group (infarct size: 33.3±3.8%). To investigate the role of endogenously formed ONOO− we measured the ONOO− marker nitrotyrosine content during preconditioning and observed that the 1st period of ischemia/reperfusion increased nitrotyrosine formation, which was attenuated after the 3rd period of ischemia/reperfusion. This increased formation of nitrotyrosine was not observed in hyperlipidemic rats.
We conclude that nitrosative stress plays an important role in the triggering mechanism of preconditioning. This mechanism is deteriorated in cholesterol-diet induced hyperlipidemia.
Top athletes under sustained physical exercise may develop myocardial hypertrophy and dilation as an adaptation to exercise. Matrix metalloproteinases (MMPs) are key enzymes of extracellular matrix metabolism and may play a pivotal role in the development of these paraphysiological alterations. In this study we aimed to investigate the plasma levels of MMP-2 and MMP-9 gelatinases (two key enzymes of collagen turnover) and their correlation with left ventricular indices in male elite waterpolo players (n=20) and age-matched sedentary controls (n=16). Our results indicate significantly higher concentration of both MMP-2 (mean±SEM: 1230±29 vs. 1092±22 ng/mL, p<0.03) and MMP-9 (63±7 vs. 35±8 ng/ml, p<0.01) in top athletes. Increased body surface area adjusted left ventricular mass (LVMi: 76±5 vs. 60±3 g/m2, p<0.01), end-diastolic (LVEDVi: 127±4 vs. 95±2 ml/m2, p<0.01) and end-systolic (LVESVi: 55±2 vs. 38±2 ml/m2, p<0.01) volume indices were observed on cardiac MRI images of athlete hearts as compared to the controls. No late gadolinium enhancement could be observed. Significant positive correlation could be found between LVMi and MMP-9 level (r=0.45, p=0.01), and both MMP-2 and MMP-9 levels and LVESVi (MMP-2: r=0.38, p<0.05; MMP-9: r=0.49, p=0.01) and LVEDVi (MMP-2: r=0.47, p<0.03; MMP-9: r=0.47, p=0.03). Our results indicate elevated MMP-2 and MMP-9 enzyme levels that correlate with significant myocardial hypertrophy and ventricular dilation in top elite athletes which refers to the remodeling of the athlete’s heart. Similar elevated enzyme levels and correlations were described previously in patients with hypertrophic cardiomyopathy and heart failure. In our case, no fibrosis indicating late gadolinium enhancement was observed in the study population which may refer to the role of MMPs not only in pathological but also in (para)physiological hypertrophy.
This study was granted by TAMOP 4.2.2. – 08/1/KMR-2008-0004
Endothelium is an essential structural and functional element of the cardiovascular system. Cardiac endothelial activation and subsequent dysfunction are probably an early event in the progression toward heart failure. The aim of our recent work is to establish reproducible in vitro culture systems of both cardiac microvascular endothelial (CMVE) and endocardial endothelial (EE) cells to investigate the endothelial function in human hearts.
Endothelial cells were isolated from human heart samples obtained during heart surgery. In case of CMVE isolation the tissue specimens were digested with collagenase and trypsin. During the isolation of EE cells tissue sample was digested by collagenase only. After filtering and centrifugation the pellets were seeded to culture flasks and grown in a medium containing endothelial cell growth supplement. Culture conditions were optimized on different artificial extracellular surface components (collagen, laminin, gelatin, fibronectin). The primary cultures of CMVE were separated from nonendothelial cells with anti-CD31 immunomagnetic beads or puromycin treatment. The CMVE and EE cells were characterized by formation of tubes on Matrigel (solubilized extract of basement membrane), uptake of acetylated LDL (DiI-Ac-LDL) and immunostaining of von Willebrand factor and CD31.
Primary isolated CMVE cells exhibited different cell morphologies, they were multilayered and only the 50% of the cells labelled with DiI-Ac-LDL. The cell separation with CD31 antibody coated magnetic beads was very efficient both in primary culture and after the passage of the CMVE cells. The 2 ug/mL 24-hour puromycin treatment was effective only in the primary culture. Numerous cell colonies were observed within 2–3 days on gelatin coated surface and confluence was achieved within 10–14 days. Primary isolated EE cells formed a confluent, homogeneous monolayer within 4 days without any separation process. Both cell types displayed the typical markers and formed capillary tubes on Matrigel.
With use of these efficient separation techniques and culture conditions in 95% pure cultures were obtained which are applicable for further functional investigation.
Physiological investigations of smooth muscle vascular relaxation (or contractility) produce curves with a sigmoidal pattern which can be described with a four-parameter logistic equation or with a bell-shaped dose-response curve, which combine two sigmoidal equations (for more complicated non-monotonic sigmoidal relationship). Comparisons of these curves determine relative potencies of the relaxant (contractile) agent. In our presentation, we will 1st focus on the limitations and pitfalls of the methods commonly used for comparing dose-response curves such as ANOVA with multiple vertical contrasts, or T-test for comparisons of a model parameters, 2nd propose more correct ways of curve-fitting and evaluating, and 3rd demonstrate usefulness of the proposed statistical procedures for analyses of the acetylcholine-induced relaxation dose-response curves obtained from our measurements.
The femoral artery reactivity from the left hind limb was assessed by a wire myograph in a total of ten Wistar-Kyoto rats equally divided into two groups, the control and the treated. The data were analyzed with the most frequently used statistical methods in order to assess between-group differences in the maximal acetylcholine-induced relaxation. Simultaneously the data were analyzed with less known methods such as the nonlinear regression followed with F-test and Akaike’s information criterion (AIC).
The results of analysis of variance (ANOVA) with the multiple contrasts using Bonferroni adjustments do not confirm any significant differences between the groups at α = 0.05. The nonlinear regression followed by F-test and AIC showed between-group differences in the selected best-fit model parameters, e.g. in the values of maximal response, and also in the concentration that produced half-maximal response. Performing a response-curve analysis, we should think about the limitation of ANOVA when one (or more) of the factors is a continuous variable. In such a case the interpretation of the ANOVA results can essentially confuse the findings. Instead, if it is possible, it is recommended to perform a global model-fitting procedure and the AIC calculation. These methods can easily be learned and their outcomes can be directly translated into concrete decisive statements.
Recent studies suggest that H2S is a potent antioxidant that can improve the cardiovascular functions in several diseases, such as myocardial ischemia/reperfusion, infarction, and coronary circulation. Thus we aimed to compare the antioxidant effect of H2S to the well established effect of SOD on superoxide-induced vasomotor activity elicited by pyrogallol.
Small veins (n=12) were isolated from the gracilis muscle of male Wistar rats (3 months). The veins were mounted on wires in the chambers of the myograph filled with Krebs buffer (37°C) and gassed with 95% O2 and 5% CO2 and their isometric force was measured (Experimetria-WPI wire-myograph chamber). The vessels were exposed to ~1,5 mN to establish a basal tone, then 60 mM KCl was used to induce pre-contraction. Then the effect of pyrogallol (10−5 mM) was measured. Next, the chamber was washed out with Krebs solution and the vessels were pre-contracted again with KCl and incubated with SOD (15 minutes, 120 U/ml). Then vasocontraction to pyrogallol were obtained again. Subsequently, on KCl-induced tone, the effect of the H2S donor NaHS (10−5 mM) on pyrogallol-induced contraction was measured.
In isolated small veins KCl elicited substantial vasomotor tone (0.63±0.1 mN). Additional administration of the superoxide generator pyrogallol further increased the vasomotor tone significantly (1.3±0.2 mN, 219.5+21.6 %, n=12, p <0.05). Next, after KCl pre-contraction, in presence of SOD pyrogallol elicited significantly less contraction (0.9±0.2 mN p <0.05). Interestingly, after KCl pre-contraction in the presence of H2S, pyrogallol elicited a contraction, in a magnitude similar to the control (1.3±0.2 mN). Also, SOD significantly decreased the KCl induced vasomotor tone (0.5±0.1 mN, 68.2±16.5 %, p <0.05), whereas H2S did not (115.9±24.1 %, 0.6±0.1 mN).
In the present study, in small veins isolated from rat gracilis muscle, H2S – in the concentration used – did not change the vasomotor effect of pyrogallol, whereas SOD significantly decreased the pyrogallol induced contractions. These results suggest that the previously described antioxidant effects of H2S are unlikely to be mediated by its direct effect on superoxide.
Supported by OTKA K71591 and K67984
Extracellular signal-regulated kinases 1/2 (ERK1/2) have been shown to regulate various cellular processes including cell growth, proliferation and apoptosis in the heart. However, the precise function of ERK1/2 signaling in the control of cardiac contractile force is not well defined. Here, we examined whether and how ERK1/2 signaling contributes to the inotropic effect of adrenomedullin (AM), one of the most potent endogenous stimulators of cardiac contractility.
In the isolated perfused, paced rat heart preparation, intracoronary infusion of AM (1 nmol/L) for 30 min increased developed tension by 24±1% (P<0.001). Western blot analysis revealed that AM significantly increased the levels of phosphorylated ERK1/2. The MEK1/2 inhibitor U0126 (1.5 μmol/L) significantly reduced the levels of phospho-ERK1/2 and attenuated the inotropic response to AM by 40% (P<0.01). AG1478 (1 μmol/L), an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, abolished the AM-stimulated increase in the levels of phospho-ERK1/2 and attenuated the inotropic response by 45% (P<0.001). Moreover, the Na+/H+ exchanger (NHE) inhibitor zoniporide (1 μmol/L) decreased the AM-induced increase in contractility by 46% (P<0.001).
Our data are the first to demonstrate that AM increases cardiac contractility via activation of the MEK1/2–ERK1/2 pathway. Moreover, EGFR acts as the upstream regulator and NHE1 as the downstream effector of ERK1/2 signaling.
There are controversial findings in the literature regarding whether postconditioning (PostC), intermittent interruption of coronary flow in the very early phase of reperfusion, is capable of reducing reperfusion injury. Therefore, our aim was to study whether PostC is able to limit infarct size via activation of matrix metalloproteinase-2 (MMP-2) following global or regional ischemia.
Hearts isolated from male Wistar rats were randomized into four groups. Hearts were subjected to either 30-min global or coronary occlusion-induced regional ischemia followed by 120-min reperfusion with or without postconditioning protocol induced by six intermittent periods of ischemia/reperfusion of 10-s duration each. At the end of reperfusion infarct size was determined by standard TTC staining. In separate experiments cardiac MMP-2 activities were measured at the tenth minute of reperfusion.
We found that postconditioning applied after coronary occlusion significantly decreased infarct size (12.6±3.2% vs. 43.0±3.1%, p<0.05) and MMP-2 activity while postconditioning protocol after global ischemia failed to affect infarct size (41.3±3.6% vs. 42.9±2.5%) and MMP-2 activity.
We conclude that PostC offers protection after regional ischemia by a mechanism that likely involves decreased activation of MMP-2. However, PostC remains ineffective when applied after global ischemia in rats.
Cytosolic calcium signals are central in cardiac muscle cells to activate contraction and recruit transcription factors, many of which are endpoints of calcium-dependent signalling cascades. One central cascade is regulated by calcium-calmodulin-dependent protein kinase II (CaMKII) capable of inducing hypertrophic and fetal cardiac gene expression. We have identified a novel CaMKII-dependent pathway for controlling cardiomyocyte gene expression. We show that CaMKII potentiates calcium-induced nuclear translocation of the transcriptional repressor DREAM, which binds to a downstream regulatory element (DRE) in the promoter of the L-type calcium channel (LTCC) pore-forming α1C-subunit gene (Cacna1c). We demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCC to the amount of intracellular calcium detected by CaMKII. This mechanism is likely to participate in cardiomyocyte remodelling during development of cardiac hypertrophy and failure as well as normal development of cardiac myocytes.
Elevation of cytosolic calcium plays a major role in the stimulus – energetic coupling of tissues. In the working heart elevation of cytosolic [Ca2+] to high nanomolar range stimulates respiration and ATP synthesis. The factors playing a role in the stimulating effects of calcium are the activation of dehydrogenases, the activation of substrate transporters and the activation of ATP synthesis. The activation of mitochondrial ATP production represents an adaptation to the growing energy demand. With the increase of cytosolic [Ca2+] the beneficial effects will gradually disappear. Due to the low affinity of Ca2+-uniporter and the low capacity of Ca2+-release mechanisms the net uptake of Ca2+ is much higher at micromolar concentrations. The stimulation of ATP synthesis will be stopped and mitochondria will be gradually depolarized. At high micromolar [Ca2+]s calcium becomes toxic to mitochondria. Calcium toxicity is associated with increased formation of calcium-phosphate precipitates in the matrix with membrane depolarization and with a decay in the ATP synthesis and finally with a general failure of calcium handling. The terminal phase of calcium toxicity is associated with permeability transition pore opening, and with an irreversible collapse of mitochondrial membrane potential. Mitochondria will become net ATP consumers instead of being ATP producers. The mitochondrial impairment can be a major factor in cell death.
Angiotensin II (Ang II) and norepinephrine (NE) are potent vasoconstrictors and play crucial roles in the regulation of blood pressure. In addition, Ang II has been shown to exhibit tachyphylaxis. The potential aging-induced changes in Ang II and NE-induced vasomotor responses have not yet been explored. We hypothesized that aging induces changes in the magnitude of vasomotor responses to Ang II and NE. Thus carotid arteries from newborn (8days: 8d), young (2month: 2m), adult (6m) and aged (12m and 24m) rats were isolated and placed in a wire myograph to measure changes in their isometric tension. Two dose response curves to Ang II and NE were obtained in a sequential manner (1.-, and 2.-administration (Adm)). Contractions of vessels to 1.-Adm of Ang II in 8d vessels were significantly less compared to those of 2m, 6m, 12m and 24m, which were similar in magnitude (1.4±0.2 and 5.2±0.6, 5.6±0.4, 5.2±0.5, 5.7±0.4 mN, respectively). Contractions of vessels to 2.-Adm of Ang II were significantly reduced in 2m, 6m, 12m and 24m vessels (4.4±0.5; 3.6±0.4; 1.9±0.4; 0.5±0.2 mN), whereas it didn’t change in vessels of 8d rats (1.28±0,2 mN). Contractions to 1.-Adm of NE increased to the age of 2m (8d: 0.7±0.3 and 2m: 6,9±0,7 mN), then it did not change (6m: 5.9±0.5, 12m: 5.7±0.9, 24m: 6.0±0.8mN). Compared to the 1.-Adm, contractions to 2.-Adm of NE did not change significantly (8d: 0.3±0.2mN, 2m: 6.4±0.8; 6m: 5.4±1.2, 12m: 5.0±1.7, 24m: 6.3±0.4mN). In summary, we have found that: 1) Ang II-induced contraction increases with age, and 2) repeated administration of Ang II elicits tachyphylaxis, which increases with age. The magnitude of NE-induced contractions did not change with age and did not exhibit tachyphylaxis. These findings suggest that aging may induce specific changes in the functional availability of AT1-receptor, which could be due to altered internalization of its receptors.
We have shown previously that nitric oxide (NO) plays an important role in both the early and the delayed protective effects of preconditioning (PC) against the acute ischaemia and reperfusion (I/R)-induced ventricular arrhythmias. The evidence for this derived from studies in anaesthetised dogs in which we demonstrated that (i) the protection was abolished if the generation of NO had been inhibited, and that (ii) drugs which are able to donate NO provided an antiarrhythmic effect similar to PC. More recent evidence suggests that peroxynitrite (PN), given locally into the coronary artery in low micromolar concentrations, as well as sodium nitrite (NaNO2) administered in intravenous infusion may also provide protection against arrhythmias. Thus, both the short (5 min) periods of the intracoronary infusion of PN (100 nM) prior to a 25 min occlusion of the left anterior descending coronary artery (LAD) and the intravenous infusion of NaNO2 (0.2 μM kg−1 min−1) given either prior to and during the occlusion or just prior to reperfusion markedly suppressed the occurrence of ventricular arrhythmias and increased survival, compared to the controls (e.g. 50, 60. 85 % cp. 0%). Furthermore, all these interventions maintained or even increased plasma nitrite/nitrate (NOx) levels during the occlusion in contrast to the controls in which plasma NOx levels were markedly reduced by the end of the occlusion period. However, both PN and NaNO2 as well as PC markedly suppressed the reperfusion-induced superoxide and nitrotyrosine (NT) productions. These results suggest that under in vivo conditions, preservation of NO availability during I/R is important for the antiarrhythmic protection, and that this effect may result from an NO-mediated reduction in superoxide production. There are, of course, a number of possible ways by which NO may result in antiarrhythmic protection. One of these, which was also examined, the regulation of gap junction channels by NO. The role of gap junctions in the antiarrhythmic protection will also be discussed.
The histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is the leading cardioplegic solution in the field of cardiac surgery. However, Custodiol is unable to effectively reduce reperfusion injury after a cold ischemic period. On the basis of this solution, novel HTK cardioplegic solutions were developed. In this study, we investigated whether their reduced cytotoxicity and their ability to reduce reactive oxygen generation after ischemia/reperfusion injury have beneficial effects of canine model of CPB.
24 dogs underwent hypothermic cardiopulmonary bypass with 60 minutes of hypothermic cardiac arrest. Dogs were divided into 3 groups: traditional HTK (n=8) and Custodiol-B (addition of L-arginine and N-α-acetyl-L-histidine), Custodiol-N (Custodiol-B supplemented with iron chelators deferoxamine and LK-614). Biventricular hemodynamic variables were measured by a combined pressure-volume conductance catheter at baseline and after 60 minutes of reperfusion. Coronary blood flow, ATP content, nitrite and myeloperoxidase levels were also determined.
Traditional HTK solution failed to prevent cardiac and endothelial dysfunction, however both Custodiol-B and N improved coronary blood flow, but only Custodiol-N2 was able to effectively prevent cardiac dysfunction after cardiac arrest. In addition, the ATP content, nitrite was significantly higher after application of the new solutions. Furthermore, myeloperoxidase level significantly decreased in the new HTK-groups.
The new HTK cardioplegic solutions improved myocardial and endothelial function after cardiopulmonary bypass with hypothermic cardiac arrest. The observed protective effects imply that the Custodiol-N could be the next generation cardioplegic solution in the protection against ischemia-reperfusion injury in cardiac surgery.
Statins reduce cholesterol levels by inhibiting enzyme HMG-CoA reductase. This results in diminished synthesis of other products downstream of the mevalonate pathway, namely coenzyme Q (CoQ), which may be involved in some adverse effects of statins. Since CoQ is an essential part of the mitochondrial respiratory chain responsible for ATP production, we were interested in 1) whether and to what extent statins reduce CoQ content and 2) if so, whether or not it is associated with changes in mitochondrial function. We have examined the effect of two different doses of atorvastatin on the function of myocardial mitochondria and CoQ content in control (C) and hypercholesterolemic (CH) rats.
Wistar rats were feeding with high cholesterol (4%) and saturated fat (10%) diet for 8 weeks. Atorvastatin was administered orally by a gastric tube either at a low dose (10 mg/kg/day) or at a high dose (80 mg/kg/day) for 4 weeks. Biochemical parameters (analyser Hitachi 911), lipid peroxidation (spectrophotometrically), functional parameters of mitochondria (Clark oxygen electrode), CoQ9, CoQ10 (HPLC) were also determined. The results were analysed with StatsDirect 2.7.7. software.
Atorvastatin at a high dose decreased plasma concentration of CoQ9, and CoQ9/tChol ratio in both, C and CH groups. Decreased TBARS formation in plasma was found only in CH rats given the high dose of atorvastatin. Both doses decreased mitochondrial respiration, rate of ATP production, and concentrations of CoQ9 and CoQ10 in CH rats. High dose of atorvastatin decreased mitochondrial function and CoQ content in both, C and CH groups of rats.
Our results imply that atorvastatin impaired oxidative phosphorylation in myocardial mitochondria in both, healthy and hypercholesterolemic rats likely by inhibition of coenzyme Q biosynthesis. Atorvastatin at both doses decreased the capacity of the mitochondrial respiratory chain and rate of ATP formation in association with a decreased mitochondrial CoQ content. Concentration of CoQ9 as well as CoQ9/tChol ratio in plasma was diminished significantly in hypercholesterolemic rats given the high dose of atorvastatin. We conclude that although high dose of atorvastatin undoubtedly exerted a beneficial effect on lipid peroxidation, coenzyme Q content was reduced and mitochondrial function was impaired. Physicians should be aware when prescribing statins mainly in higher doses to patients with coexisting proved CoQ10 deficiency resulting from age-related decline, and metabolic or mitochondrial diseases.
Financially supported by Scientific Grant Agency of the Ministry of Education of the Slovak Republic, VEGA 1/0328/10. Technical assistance: A. Štetková, L’. Butašová
In spite of numerous studies on hypertension (HYP)-induced functional abnormalities on the subcellular level, there are still only scarce data available about HYP-induced alterations in function and properties of the mitochondria (MIT). The present study deals with HYP-induced alterations in heart, kidney and liver MIT of 16 week old male spontaneously hypertensive (SH) rats treated for the next 4 weeks, daily per os, with the ACE inhibitor captopril (CAP, 80 mg·kg−1) or CAP + the Ca antagonist nifedipine (10 mg·kg−1 (CAP+NIF)).
Systolic blood pressure (SBP) and heart rate (HR) were monitored during the experiment and the heart to body weight ratio (HW/BW) at termination of the experiment. Heart, kidney and liver MIT were isolated by differential centrifugation. MIT membrane fluidity (MF) was estimated by means of the fluorescence probe DPH. Using succinate (SUC) as well as glutamate/malate (GLUT/MAL) as substrates the following parameters of oxidative ATP production (OATPP) were assessed: O2 consumption (QO2 (oxygen electrode)) in the presence and absence of exogenous ADP (states S3 and S4), the respiratory control index (RCI), the rate of oxidative phosphorylation (OPR) and the ADP:O ratio.
CAP and CAP+NIF treatments prevented almost similarly the increase in SBP, HR and partially also in HW/BW (p<0.05 or more) in SH rats. Mitochondria from SH hearts, and to a moderate extent surprisingly also from the liver, exhibited with both substrates a compensatory increase in most parameters of the OATPP (p<0.05), but a depression in ADP:O, in response to a HYP-induced increase in energy demands. Prevention of increase in SBP and HR by CAP and CAP+NIF treatments also eliminated the reasons for elevation of OATPP and decrease in the ADP:O (p<0.05 – 0.01). In contrast to heart and liver the kidney MIT from SH rats exhibited considerable perturbations in QO2 values and depression in RCI, OPR and ADP:O values (p<0.05– 0.01) with all substrates. CAP and also CAP+NIF treatments both failed in preventing the HYP-induced alterations of OATPP in kidney MIT. Oppositely, they aggravated their deterioration, particularly with SUC as substrate (p<0.05). Combined treatment with CAP+NIF was in this respect more detrimental than the monotherapy with CAP. In SH rats the heart, liver and kidney MIT all exhibited a moderate increase in MF that was more potentiated by CAP than by the CAP+NIF treatment.
Support: Grants VEGA 2/7126/27; 1/0755/09; 1/0620/10; 2/0173/08 and 1/0142/09
Phosphodiesterase-5 (PDE5) catalyzes cyclic GMP (cGMP) degradation, as well as its intracellular content. Specific inhibition of that isoenzyme induces increase of cGMP concentration, relaxation of smooth muscle cells and consequetly vasodilation. Regarding information above, the aim of our study was to compare effects of different PDE5 inhibitors on coronary flow and L-arginine/NO system in isolated rat heart. Hearts were isolated from male Wistar albino rats (n=12 rats) and perfused with buffer at constant pressure. The coronary autoregulation (CA) was investigated with follow-up of coronary perfusion pressure (CPP) changes from 40 to 120 cm H2O. After the first sequence of CPP changes (basic protocol), the hearts were perfused with Sildenafil, Tadalafil and Vardenafil, all in different doses (10, 20, 50, 200 nM), alone or in combination with nitric oxide synthase inhibitor (L-NAME, 30 μM). During control condition the hearts exhibit CA between 50 and 90 cm H2O, with basal coronary flow (at 60 cm H2O) of 6.63±0.30 ml/min. 20 nM Sildenafil induced significant vasodilation (from 76% at 40 to 23% at 80 cm H2O), also in 50 nM dose (51% at 40 cm H2O). 20 nM Tadalafil also induced significant vasodilation (from 32 % at 60 to 19 % at 120 cm H2O), as did 50 nM (from 23% at 60 to 40 % at 120 cm H2O). Furthermore Vardenafil induced significant vasodilation in doses of 200 nM (about 20% at all CPP-values). Additional application of L-NAME induced a decrease of coronary flow (CF) in all treated groups. All hemodynamic changes were accompanied with parallel NO-dynamic.
Our findings clearly show that all estimated PDE5 inhibitors affect coronary autoregulation, but in different doses, mediated by L-arginine/NO system.