Increasing evidence indicates that high levels of serum erythropoietin (Epo) can lessen ischemia-reperfusion injury in the heart and multiple cardiac cell types have been suggested to play a role in this Epo effect. To clarify the mechanisms underlying this cardioprotection, we explored Epo treatment of coronary artery endothelial cells and Epo cardioprotection in a Mus musculus model with Epo receptor expression restricted to hematopoietic and endothelial cells (ΔEpoR). Epo stimulation of coronary artery endothelial cells upregulated endothelial nitric oxide synthase (eNOS) activity in vitro and in vivo, and enhanced nitric oxide (NO) production that was determined directly by real time measurements of gaseous NO release. Epo stimulated phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathways, and inhibition of PI3K, but not MEK activity, blocked Epo-induced NO production. To verify the potential of this Epo effect in cardioprotection in vivo, ΔEpoR-mice with Epo response in heart restricted to endothelium were treated with Epo. These mice exhibited a similar increase in eNOS phosphorylation in coronary artery endothelium as that found in wild type (WT) mice. In addition, in both WT- and ΔEpoR-mice, exogenous Epo treatment prior to myocardial ischemia provided comparable protection. These data provide the first evidence that endothelial cell response to Epo is sufficient to achieve an acute cardioprotective effect. The immediate response of coronary artery endothelial cells to Epo stimulation by NO production may be a critical mechanism underlying this Epo cardioprotection.

The inflammatory cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α play an important role in left ventricular (LV) remodeling after myocardial infarction (MI). Phosphatase and tensin homologue deleted on chromosome ten (PTEN) inactivates protein kinase Akt and promotes cell death in the heart. However, it is not known whether PTEN promotes post-MI remodeling by regulating IL-10 and TNF-α. MI was induced in wildtype (WT) mice and Pten heterozygous mutant (HET) mice. Pten adenoviruses (adPten) or empty vectors (adNull) were injected into the peri-infarct area of WT mice. LV dilation was attenuated and fractional shortening was increased in HET mice compared with WT mice. Survival rate and fractional shortening were decreased in adPten mice compared with adNull mice. Leukocyte infiltration into the peri-infarct area was attenuated in HET mice and worsened in adPten mice. PTEN expression was upregulated in the infarcted heart of WT mice. Partial inactivation of PTEN increased production of IL-10 and decreased expression of TNF-α and matrix metalloproteinase (MMP)-2 and -9 after MI in HET mice. PTEN overexpression caused opposite effects in the infarcted heart. Moreover, in the infarcted heart of HET mice, Akt inhibition decreased Stat3 phosphorylation and IL-10 expression, and blockade of the IL-10 receptor increased TNF-α and MMP-2 expression. Both Akt inhibition and IL-10 receptor blockade abolished the attenuation of post-MI remodeling in HET mice. In conclusion, PTEN is critically involved in post-MI remodeling through the Akt/IL-10 signaling pathway. Therefore, targeting PTEN may be an effective approach to post-MI remodeling.

The cardiac Na+/Ca2+ exchanger (NCX) generates an inward electrical current during SR-Ca2+ release, thus possibly promoting afterdepolarizations of the action potential (AP). We used transgenic mice 12.5 weeks or younger with cardiomyocyte-directed overexpression of NCX (NCX-Tg) to study the proarrhythmic potential and mechanisms of enhanced NCX activity. NCX-Tg exhibited normal echocardiographic left ventricular function and heart/body weight ratio, while the QT interval was prolonged in surface ECG recordings. Langendorff-perfused NCX-Tg, but not wild-type (WT) hearts, developed ventricular tachycardia. APs and ionic currents were measured in isolated cardiomyocytes. Cell capacitance was unaltered between groups. APs were prolonged in NCX-Tg versus WT myocytes along with voltage-activated K+ currents (Kv) not being reduced but even increased in amplitude. During abrupt changes in pacing cycle length, early afterdepolarizations (EADs) were frequently recorded in NCX-Tg but not in WT myocytes. Next to EADs, delayed afterdepolarizations (DAD) triggering spontaneous APs (sAPs) occurred in NCX-Tg but not in WT myocytes. To test whether sAPs were associated with spontaneous Ca2+ release (sCR), Ca2+ transients were recorded. Despite the absence of sAPs in WT, sCR was observed in myocytes of both genotypes suggesting a facilitated translation of sCR into DADs in NCX-Tg. Moreover, sCR was more frequent in NCX-Tg as compared to WT. Myocardial protein levels of Ca2+-handling proteins were not different between groups except the ryanodine receptor (RyR), which was increased in NCX-Tg versus WT. We conclude that NCX overexpression is proarrhythmic in a non-failing environment even in the absence of reduced KV. The underlying mechanisms are: (1) occurrence of EADs due to delayed repolarization; (2) facilitated translation from sCR into DADs; (3) proneness to sCR possibly caused by altered Ca2+ handling and/or increased RyR expression.

The heart’s rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2−/− hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2−/− hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.

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Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3−/− and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160 % of baseline at 10 h after sepsis induction followed by a decline to 63 % of baseline after 18 h in wildtype mice. CO was unaltered in septic NOS3−/− mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6 h post CLP. At 12 h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3−/− mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3−/− mice diminished this survival benefit. Plasma NOx- and local myocardial NOx- and NO levels (via NO spin trapping) demonstrated enhanced NOx- and bioactive NO levels in septic wildtype as compared to NOS3−/− mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility.

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Pulmonary arterial hypertension (PAH) is a fatal disease for which no cure is yet available. The leading cause of death in PAH is right ventricular (RV) failure. Previously, the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) has been associated with different fibrotic diseases. However, so far there is no study demonstrating a causal role for endogenous Fn14 signaling in RV or LV heart disease. The purpose of this study was to determine whether global ablation of Fn14 prevents RV fibrosis and remodeling improving heart function. Here, we provide evidence for a causative role of Fn14 in pulmonary artery banding (PAB)-induced RV fibrosis and dysfunction in mice. Fn14 expression was increased in the RV after PAB. Mice lacking Fn14 (Fn14−/−) displayed substantially reduced RV fibrosis and dysfunction following PAB compared to wild-type littermates. Cell culture experiments demonstrated that activation of Fn14 induces collagen expression via RhoA-dependent nuclear translocation of myocardin-related transcription factor-A (MRTF-A)/MAL. Furthermore, activation of Fn14 in vitro caused fibroblast proliferation and myofibroblast differentiation, which corresponds to suppression of PAB-induced RV fibrosis in Fn14−/− mice. Moreover, our findings suggest that Fn14 expression is regulated by endothelin-1 (ET-1) in cardiac fibroblasts. We conclude that Fn14 is an endogenous key regulator in cardiac fibrosis and suggest this receptor as potential new target for therapeutic interventions in heart failure.

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Aim

Protection achieved by ischemic preconditioning is dependent on A2B adenosine receptors (A2BAR) in rabbit and mouse hearts and, predictably, an A2BAR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A2BAR. The present study tested whether A2BAR could be demonstrated on rat cardiomyocytes.

Methods and Results

Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A2BAR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4±2.0 % of the risk zone in control hearts to 19.9±2.8 % indicating that A2BAR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A2BAR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A2BAR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A2BAR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A2BAR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A2BAR band.

Conclusions

Thus the present study demonstrates that activation of A2BAR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A2BAR are expressed in individual cardiomyocytes. However, surprisingly, A2BAR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A2BAR signalling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

Little is known about the vascular function and expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in Duchenne muscular dystrophy (DMD). Bradykinin is involved in the regulation of eNOS expression induced by angiotensin-converting enzyme inhibitors. We characterized the vascular function and eNOS and nNOS expression in a canine model of DMD and evaluated the effects of chronic bradykinin treatment. Vascular function was examined in conscious golden retriever muscular dystrophy (GRMD) dogs with left ventricular dysfunction (measured by echocardiography) and in isolated coronary arteries. eNOS and nNOS proteins in carotid arteries were measured by western blot and cyclic guanosine monophosphate (cGMP) content was analyzed by radioimmunoassay. Compared with controls, GRMD dogs had an impaired vasodilator response to acetylcholine. In isolated coronary artery, acetylcholine-elicited relaxation was nearly absent in placebo-treated GRMD dogs. This was explained by reduced nNOS and eNOS proteins and cGMP content in arterial tissues. Chronic bradykinin infusion (1 μg/min, 4 weeks) restored in vivo and in vitro vascular response to acetylcholine to the level of control dogs. This effect was NO-mediated through upregulation of eNOS and nNOS expression. In conclusion, this study is the first to demonstrate that DMD is associated with NO-mediated vascular endothelial dysfunction linked to an altered expression of eNOS and nNOS, which can be overcome by bradykinin.

Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients; however, the precise mechanisms of its effects on hypertension are largely unknown. Therefore, this study aimed to investigate the mechanisms within the brain that can influence exercise-induced effects in an animal model of human essential hypertension. Young normotensive WKY and SHR rats were given moderate-intensity exercise for 16 weeks. Blood pressure was measured bi-weekly by tail-cuff method. Animals were then euthanized; paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), important cardiovascular regulatory centers in the brain, were collected and analyzed by Real-time RT-PCR, western blot, EIA, and fluorescent microscopy. Exercise of 16 weeks duration attenuated systolic, diastolic, and mean arterial pressure in SHR. Sedentary SHR exhibited increased proinflammatory cytokines (PICs) and decreased anti-inflammatory IL-10 levels in the PVN and RVLM. Furthermore, SHRsed rats exhibited elevated levels of ACE, AT1R, and decreased levels of ACE2 and receptor Mas in the PVN and RVLM. Chronic exercise not only prevented the increase in PICs (TNF-α, IL-1β), ACE, and AT1R protein expression in the brain of SHR, but also dramatically upregulated IL-10, ACE2, and Mas receptor expression in SHR. In addition, these changes were associated with reduced plasma AngII levels, reduced neuronal activity, reduced NADPH-oxidase subunit gp91phox and inducible NO synthase (iNOS) in trained SHRs indicating reduced oxidative stress. These results suggest that chronic exercise not only attenuates PICs and the vasoconstrictor axis of the RAS but also improves the anti-inflammatory defense mechanisms and vasoprotective axis of the RAS in the brain, which, at least in part, explains the blood pressure-lowering effects of exercise in hypertension.

Heart failure may lead to hypoperfusion and hypooxygenation of tissues and this is often exacerbated by central and obstructive sleep apnoeas associated with recurrent episodes of systemic hypoxia which triggers release of ATP within the CNS circuits controlling sympathetic outflow. Using in vitro and in vivo models we tested two hypotheses: (1) activated brainstem astroglia release ATP and via release of ATP activate sympathoexcitatory neurones of the rostral ventrolateral medulla (RVLM); and (2) ATP actions in the RVLM contribute to sympathoexcitation, progression of left ventricular (LV) remodelling and development heart failure secondary to myocardial infarction. In vitro, optogenetic activation of RVLM astrocytes transduced to express light-sensitive channelrhodopsin-2 activated sympathoexcitatory RVLM neurones in ATP-dependent manner. In anaesthetised rats in vivo, similar optogenetic activation of RVLM astrocytes increased sympathetic renal nerve activity, arterial blood pressure and heart rate. To interfere with ATP-mediated signalling by promoting its extracellular breakdown, we developed a lentiviral vector to express an ectonucleotidase—transmembrane prostatic acid phosphatase (TMPAP) on the cellular membranes. In rats with myocardial infarction-induced heart failure, expression of TMPAP bilaterally in the RVLM led to lower plasma noradrenaline concentration, maintained left ventricular end diastolic pressure, attenuated decline in dP/dTmax and shifted the LV pressure–volume relationship curve to the left. These results show that activated RVLM astrocytes are capable of increasing sympathetic activity via release of ATP while facilitated breakdown of ATP in the RVLM attenuates the progression of LV remodelling and heart failure secondary to myocardial infarction.

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Aims

Findings from our laboratory indicate that proinflammatory cytokines and their transcription factor, nuclear factor-kappaB (NF-κB), are increased in the hypothalamic paraventricular nucleus (PVN) and contribute towards the progression of heart failure. In this study, we determined whether NF-κB activation within the PVN contributes to sympathoexcitation via interaction with neurotransmitters in the PVN during the pathogenesis of heart failure.

Methods and results

Heart failure was induced in rats by left anterior descending coronary artery ligation. Sham-operated control (SHAM) or heart failure rats were treated for 4 weeks through bilateral PVN infusion with SN50, SN50M or vehicle via osmotic minipump. Rats with heart failure treated with PVN vehicle or SN50M (inactive peptide for SN50) had increased levels of glutamate, norepinephrine, tyrosine hydroxylase (TH), superoxide, gp91phox (a subunit of NAD(P)H oxidase), phosphorylated IKKβ and NF-κB p65 activity, and lower levels of gamma-aminobutyric acid (GABA) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN compared with those of SHAM rats. Plasma levels of cytokines, norepinephrine, epinephrine and angiotensin II, and renal sympathetic nerve activity (RSNA) were increased in heart failure rats. Bilateral PVN infusion of SN50 prevented, the decreases in PVN GABA and GAD67, and the increases in RSNA and PVN glutamate, norepinephrine, TH, superoxide, gp91phox, phosphorylated IKKβ and NF-κB p65 activity observed in vehicle or SN50M treated heart failure rats. A same dose of SN50 given intraperitoneally did not affect neurotransmitters concentration in the PVN and was similar to vehicle treated heart failure rats.

Conclusion

These findings suggest that NF-κB activation in the PVN modulates neurotransmitters and contributes to sympathoexcitation in rats with ischemia-induced heart failure.

Background

Little is known about the impact of type 2 diabetes mellitus (DM) on coronary arteriole remodeling. The aim of this study was to determine the mechanisms that underlie coronary arteriole structural remodeling in type 2 diabetic (db/db) mice.

Methods and Results

Passive structural properties of septal coronary arterioles isolated from 12- and 16-wk-old diabetic db/db and control mice were assessed by pressure myography. Coronary arterioles from 12-wk-old db/db mice were structurally similar to age-matched controls. By 16-wks of age, coronary wall thickness was increased in db/db arterioles (p < 0.01), while luminal diameter was reduced (Control: 118±5μm; db/db: 102±4μm, p < 0.05), augmenting the wall-to-lumen ratio by 58% (Control: 5.9±0.6; db/db: 9.5±0.4, p < 0.001). Inward hypertrophic remodeling was accompanied by a 56% decrease in elastic modulus (p < 0.05, indicating decreased vessel coronary wall stiffness) and a ~30% reduction in coronary flow reserve in diabetic mice. Interestingly, aortic pulse wave velocity and femoral artery incremental modulus were increased (p < 0.05) in db/db mice, indicating macrovascular stiffness. Molecular tissue analysis revealed increased elastin-to-collagen ratio in diabetic coronaries when compared to control and a decrease in the same ratio in the diabetic aortas.

Conclusions

These data show that coronary arterioles isolated from type 2 diabetic mice undergo inward hypertrophic remodeling associated with decreased stiffness and increased elastin-to-collagen ratio which results in a decreased coronary flow reserve. This study suggests that coronary microvessels undergo a different pattern of remodeling from macrovessels in type 2 DM.

Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO−). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.

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Increased afterload results in ‘pathological’ cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner.

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The strategy to reprogram somatic stem cells to pluripotency status has provided an alternative source of surrogate ES cells (ESC). We report efficient reprogramming of multipotent bone marrow (BM) mesenchymal stem cells (MSC) to pluripotent status and the resultant MSC derived iPS cells (MiPS) and their derived progenitors effectively repaired the infarcted heart. MSC from young, male, Oct4-GFP transgenic mice were reprogrammed by retroviral transduction with Oct4, Sox2, Klf4, and c-Myc stemness factors. MiPS thus generated displayed characteristics of mouse ESC including morphology, surface antigens, gene and miR expression profiles. MiPS also formed spontaneously beating cardiac progenitors which expressed cardiac specific transcription factors and protein markers including Gata4, Mef2c, Nkx2.5, myosin heavy chain, troponin-I, and troponin-T, and showed ultra structural characteristics typical of cardiomyocytes. Intramyocardial delivery of MiPS (group-2) and their derivative cardiac-like cells (MiPS-CP; group-3) in a mouse model of acute myocardial infarction showed extensive survival and engraftment at 4 weeks with resultant attenuation of infarct size (p < 0.001 vs. DMEM injected control; n = 4). Engraftment of MiPS-CP was without cardiac tumorigenesis as compared to 21 % in MiPS transplanted animals. Furthermore, angiogenesis was improved in groups-2 and 3 (p < 0.001 vs. control). Transthoracic echocardiography revealed significantly preserved indices of cardiac contractility (ejection fraction p < 0.001 and fractional shortening p < 0.001 vs. control; n = 7). MSC were successfully reprogrammed into MiPS that displayed ESC-like characteristics and differentiated into spontaneously beating cardiomyocytes. Cardiac progenitors derived from MiPS repopulated the infarcted heart without tumorigenesis and improved global cardiac function.

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Impairment of vascular growth is a hallmark of diabetic complications, but the progression and mechanisms are poorly understood. To determine whether obesity and early diabetes impair endothelium-dependent vasodilatation and vascular response to ischemia, microvascular function as well as angiogenic responses to ischemia were assessed in young (C57) and 6-month-old lean mice (old C57), in obese (db-C57) mice, and in mice suffering an early (db-KsJ) and sustained type 2 diabetes (old db-KsJ). Glycemia gradually increased from the db-C57 to the old db-KsJ. Early and established type II diabetes significantly reduced the level of insulin that was significantly increased in obese mice. Endothelial function was assessed in isolated resistance arteries while the angiogenic response induced by unilateral hindlimb ischemia was analyzed, after 28 days, with a laser Doppler flowmeter and angiography. Aging (−21%), obesity (−45%), as well as early (−58%) and sustained type II diabetes (−69%) induced a progressive impairment of the endothelium-dependent relaxation of the gracilis artery. Laser Doppler measurements demonstrated that only early and sustained type II diabetes impaired skin blood flow recovery. Vascular collateralization was reduced with aging and severely impaired in older db-KsJ mice, the two strains of mice in which ischemia reduced eNOS expression. These results demonstrate that endothelial dysfunction induced by obesity is insufficient to alter the angiogenic response to ischemia. Furthermore, the development of frank type II diabetes or increasing age is required to impair the vascular response to hindlimb ischemia. We conclude that additional risk factors or severe endothelial dysfunction may be requisite to impede the angiogenic response to ischemia.

Summary

We have examined the sodium-calcium exchange stoichiometry in Langendorff-perfused rabbit hearts using gamma-emitting tracers under conditions of sodium pump inhibition. Following a 60-min perfusion with 10−5 acetylstrophanthidin, and extracellular concentrations [Na]o = 70 mM and [Ca]o = 300 µM, intracellular sodium rose to 59.2 mM. At this point an increase in extracellular calcium [Ca]o = 1.52 mM) caused a net efflux of sodium, but an increase in sodium [Na]o = 105 mM) caused no measurable change. When sodium and calcium were simultaneously increased according to the ratio [Na]o)n/[Ca]o = [Na]′o)n/[Ca]′o, a sodium efflux is observed when n = 4, but not when n = 3. These results are consistent with an exchange stoichiometry of 3 Na+ for each Ca2+ ion, but not values of 4 or more.

Objective

Pressure-overload hypertrophy is associated with decreased capillary density in myocardium resulting in impaired substrate delivery. Treatment of hypertrophied hearts with vascular endothelial growth factor (VEGF) induces angiogenesis. Since angiogenesis is associated with extracellular matrix degradation, we sought to determine whether VEGF induced angiogenesis in hypertrophy required matrix metalloproteinases (MMP) activation.

Methods

Newborn rabbits underwent aortic banding. Progression of hypertrophy (mass-to-volume (M/V) ratio) and mid-wall contractility index was monitored by echocardiography. At 4 and 6 weeks, VEGF (2 μg/kg), vehicle or VEGF combined with GM6001 (5 mg/kg), a MMP inhibitor, was administered intrapericardially. CD-31 (indicator of angiogenesis), MMP-2, MT1-MMP and TIMPs (endogenous MMP inhibitors) expression were measured by immunoblotting. MMP-2 activity was determined by gelatin zymography.

Results

Untreated hypertrophied hearts progressed to ventricular dilatation at 7 wks (M/V ratio: 0.75 ± 0.07), but compensatory hypertrophy was maintained with VEGF (0.91±0.07; p<0.05). LV contractility declined in untreated hearts from −0.41 ± 0.9 (5 wks) to −0.73 ± 0.5 (7 wks; p < 0.05) but remained normal with VEGF (+1.61 ± 0.6 vs. +0.47 ± 0.2). MMP-2 expression and activity were significantly elevated in VEGF treated hypertrophied hearts (p < 0.05) and were blocked by concomitant administration of GM6001. VEGF induced neovascularization was inhibited by addition of GM6001. MT1-MMP showed a trend to higher levels in VEGF treated hearts. TIMPs were unchanged in all three groups.

Conclusions

Exogenous VEGF and resultant MMP-2 activation leads to increased capillary formation in severe hypertrophy, preventing progression to ventricular dilation and dysfunction. VEGF and the associated MMP-2 activation play an important and potentially therapeutic role in vascular remodeling of hypertrophied hearts.

Three bioactive sphingolipids, namely sphingosine-1-phosphate (S1P), ceramide (CER) and sphingosine (SPH) were shown to be involved in ischemia/reperfusion injury of the heart. S1P is a powerful cardioprotectant, CER activates apoptosis and SPH in a low dose is cardioprotective whereas in a high dose is cardiotoxic. The aim of the present study was to examine effects of experimental myocardial infarction on the level of selected sphingolipids in plasma, erythrocytes and platelets in the rat. Myocardial infarction was produced in male Wistar rats by ligation of the left coronary artery. Blood was taken from the abdominal aorta at 1, 6 and 24 h after the ligation. Plasma, erythrocytes and platelets were isolated and S1P, dihydrosphingosine-1-phosphate (DHS1P), SPH, dihydrosphingosine (DHS) and CER were quantified by means of an Agilent 6460 triple quadrupole mass spectrometer using positive ion electrospray ionization source with multiple reaction monitoring. The infarction reduced the plasma level of S1P, DHS1P, SPH and DHS but increased the level of total CER. In erythrocytes, there was a sharp elevation in the level of SPH and DHS early after the infarction and a reduction after 24 h whereas the level of S1P, DHS1P and total CER gradually increased. In platelets, the level of each of the examined compounds profoundly decreased 1 and 6 h after the infarction and partially normalized in 24 h. The results obtained clearly show that experimental heart infarction in rats produces deep changes in metabolism of sphingolipids in the plasma, platelets and erythrocytes.

Although epicardial blood flow can be restored by an early intervention in most cases, a lack of adequate reperfusion at the microvascular level is often a limiting prognostic factor of acute myocardial infarction (AMI). Our group has recently found that paracrine factors secreted from apoptotic peripheral blood mononuclear cells (APOSEC) attenuate the extent of myocardial injury. The aim of this study was to determine the influence of APOSEC on microvascular obstruction (MVO) in a porcine AMI model. A single dose of APOSEC was intravenously injected in a closed chest reperfused infarction model. MVO was determined by magnetic resonance imaging and cardiac catheterization. Role of platelet function and vasodilation were monitored by means of ELISA, flow cytometry, aggregometry, western blot and myographic experiments in vitro and in vivo. Treatment of AMI with APOSEC resulted in a significant reduction of MVO. Platelet activation markers were reduced in plasma samples obtained during AMI, suggesting an anti-aggregatory capacity of APOSEC. This finding was confirmed by in vitro tests showing that activation and aggregation of both porcine and human platelets were significantly impaired by co-incubation with APOSEC, paralleled by vasodilator-stimulated phosphoprotein (VASP)-mediated inhibition of platelets. In addition, APOSEC evidenced a significant vasodilatory capacity on coronary arteries via p-eNOS and iNOS activation. Our data give first evidence that APOSEC reduces the extent of MVO during AMI, and suggest that modulation of platelet activation and vasodilation in the initial phase after myocardial infarction contributes to the improved long-term outcome in APOSEC treated animals.

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Our novel proposal is that TNFα exerts a direct effect on mitochondrial respiratory function in the heart, independently of its cell surface receptors. TNFα-induced cardioprotection is known to involve reactive oxygen species (ROS) and sphingolipids. We therefore further propose that this direct mitochondrial effect is mediated via ROS and sphingolipids. The protective concentration of TNFα (0.5 ng/ml) was added to isolated heart mitochondria from black 6 × 129 mice (WT) and double TNF receptor knockout mice (TNFR1&2−/−). Respiratory parameters and inner mitochondrial membrane potential were analyzed in the presence/absence of two antioxidants, N-acetyl-l-cysteine or N-tert-butyl-α-(2-sulfophenyl)nitrone or two antagonists of the sphingolipid pathway, N-oleoylethanolamine (NOE) or imipramine. In WT, TNFα reduced State 3 respiration from 279.3 ± 3 to 119.3 ± 2 (nmol O2/mg protein/min), increased proton leak from 15.7 ± 0.6% (control) to 36.6 ± 4.4%, and decreased membrane potential by 20.5 ± 3.1% compared to control groups. In TNFR1&2−/− mice, TNFα reduced State 3 respiration from 205.2 ± 4 to 75.7 ± 1 (p < 0.05 vs. respective control). In WT mice, both antioxidants added with TNFα restored State 3 respiration to 269.2 ± 2 and 257.6 ± 2, respectively. Imipramine and NOE also restored State 3 respiration to 248.4 ± 2 and 249.0 ± 2, respectively (p < 0.01 vs. TNFα alone). Similarly, both antioxidant and inhibitors of the sphingolipid pathway restored the proton leak to pre-TNF values. TNFα-treated mitochondria or isolated cardiac muscle fibers showed an increase in respiration after anoxia–reoxygenation, but this effect was lost in the presence of an antioxidant or NOE. Similar data were obtained in TNFR1&2−/− mice. TNFα exerts a protective effect on respiratory function in isolated mitochondria subjected to an anoxia–reoxygenation insult. This effect appears to be independent of its cell surface receptors, but is likely to be mediated by ROS and sphingolipids.

Ossabaw miniswine have been naturally selected to efficiently store large amounts of lipids offering them a survival advantage. Our goal was to evaluate the myocardial response to chronic ischemia of the Ossabaw consuming a hypercaloric, high-fat/cholesterol diet with and without metformin supplementation. At 6 weeks of age animals were fed either a regular diet (OC, n = 9), a hypercaloric high-fat/cholesterol diet (OHC, n = 9), or a hypercaloric high-fat/cholesterol diet supplemented with metformin (OHCM, n = 8). At 9 weeks, all animals underwent ameroid constrictor placement to the left circumflex coronary artery to simulate chronic ischemia. Seven weeks after ameroid placement, all animals underwent hemodynamic and functional measurements followed by cardiac harvest. Both OHC and OHCM animals developed significantly greater weight gain, total cholesterol, and LDL:HDL ratio compared to OC controls. Metformin administration reversed diet-induced hypertension and glucose intolerance. There were no differences in global and regional contractility, myocardial perfusion, capillary and arteriolar density, or total protein oxidation between groups. Myocardial protein expression of VEGF, PPAR-α, γ, and δ was significantly increased in the OHC and OHCM groups. Microvessel reactivity was improved in the OHC and OHCM groups compared to controls, and correlated with increased p-eNOS expression. Overfed Ossabaw miniswine develop several components of metabolic syndrome. However, impairments of myocardial function, neovascularization and perfusion were not present, and microvessel reactivity was paradoxically improved in hypercholesterolemic animals. The observed cardioprotection despite metabolic derangements may be due to lipid-dependant upregulation of the PPAR pathway which is anti-inflammatory and governs myocardial fatty acid metabolism.

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K+- and Mg2+-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca2+ and Mg2+, and the current increase could be inhibited by Ni2+ or Ba2+. The TRPM7-like current was potentiated by acidic pH and inhibited by La3+ and 2-aminoethoxydiphenyl borate. In addition, Ca2+-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca2+ ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.