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1.  The Effects of Propofol Cardioplegia on Blood and Myocardial Biomarkers of Stress and Injury in Patients With Isolated Coronary Artery Bypass Grafting or Aortic Valve Replacement Using Cardiopulmonary Bypass: Protocol for a Single-Center Randomized Controlled Trial 
JMIR Research Protocols  2014;3(3):e35.
Background
Despite improved myocardial protection strategies, cardioplegic arrest and ischemia still result in reperfusion injury. We have previously published a study describing the effects of propofol (an anesthetic agent commonly used in cardiac surgery) on metabolic stress, cardiac function, and injury in a clinically relevant animal model. We concluded that cardioplegia supplementation with propofol at a concentration relevant to the human clinical setting resulted in improved hemodynamic function, reduced oxidative stress, and reduced reperfusion injury when compared to standard cardioplegia.
Objective
The Propofol cardioplegia for Myocardial Protection Trial (ProMPT) aims to translate the successful animal intervention to the human clinical setting. We aim to test the hypothesis that supplementation of the cardioplegic solution with propofol will be cardioprotective for patients undergoing isolated coronary artery bypass graft or aortic valve replacement surgery with cardiopulmonary bypass.
Methods
The trial is a single-center, placebo-controlled, randomized trial with blinding of participants, health care staff, and the research team. Patients aged between 18 and 80 years undergoing nonemergency isolated coronary artery bypass graft or aortic valve replacement surgery with cardiopulmonary bypass at the Bristol Heart Institute are being invited to participate. Participants are randomly assigned in a 1:1 ratio to either cardioplegia supplementation with propofol (intervention) or cardioplegia supplementation with intralipid (placebo) using a secure, concealed, Internet-based randomization system. Randomization is stratified by operation type and minimized by diabetes mellitus status. Biomarkers of cardiac injury and metabolism are being assessed to investigate any cardioprotection conferred. The primary outcome is myocardial injury, studied by measuring myocardial troponin T. The trial is designed to test hypotheses about the superiority of the intervention within each surgical stratum. The sample size of 96 participants has been chosen to achieve 80% power to detect standardized differences of 0.5 at a significance level of 5% (2-tailed) assuming equal numbers in each surgical stratum.
Results
A total of 96 patients have been successfully recruited over a 2-year period. Results are to be published in late 2014.
Conclusions
Designing a practicable method for delivering a potentially protective dose of propofol to the heart during cardiac surgery was challenging. If our approach confirms the potential of propofol to reduce damage during cardiac surgery, we plan to design a larger multicenter trial to detect differences in clinical outcomes.
Trial Registration
International Standard Randomized Controlled Trial Number (ISRCTN): 84968882; http://www.controlled-trials.com/ISRCTN84968882/ProMPT (Archived by WebCite at http://www.webcitation.org/6Qi8A51BS).
doi:10.2196/resprot.3353
PMCID: PMC4115261  PMID: 25004932
cardiac surgery; anesthetics; cardiopulmonary bypass; ischemia; reperfusion; cardioplegia; aortic valve; coronary artery; troponin; clinical trials, randomized
2.  Cardiac Phosphoproteomics during Remote Ischemic Preconditioning: A Role for the Sarcomeric Z-Disk Proteins 
BioMed Research International  2014;2014:767812.
Remote ischemic preconditioning (RIPC) induced by brief ischemia/reperfusion cycles of remote organ (e.g., limb) is cardioprotective. The myocardial cellular changes during RIPC responsible for this phenomenon are not currently known. The aim of this work was to identify the activation by phosphorylation of cardiac proteins following RIPC. To achieve our aim we used isobaric tandem mass tagging (TMT) and reverse phase nanoliquid chromatography tandem spectrometry using a Linear Trap Quadropole (LTQ) Orbitrap Velos mass spectrometer. Male C57/Bl6 mice were anesthetized by an intraperitoneal injection of Tribromoethanol. A cuff was placed around the hind limb and inflated at 200 mmHg to prevent blood flow as confirmed by Laser Doppler Flowmetry. RIPC was induced by 4 cycles of 5 min of limb ischemia followed by 5 min of reperfusion. Hearts were extracted for phosphoproteomics. We identified approximately 30 phosphoproteins that were differentially expressed in response to RIPC protocol. The levels of several phosphoproteins in the Z-disk of the sarcomere including phospho-myozenin-2 were significantly higher than control. This study describes and validates a novel approach to monitor the changes in the cardiac phosphoproteome following the cardioprotective intervention of RIPC and prior to index ischemia. The increased level of phosphorylated sarcomeric proteins suggests they may have a role in cardiac signaling during RIPC.
doi:10.1155/2014/767812
PMCID: PMC3985148  PMID: 24795895
3.  Remote ischemic conditioning: from experimental observation to clinical application: report from the 8th Biennial Hatter Cardiovascular Institute Workshop 
Basic Research in Cardiology  2014;110(1):453.
In 1993, Przyklenk and colleagues made the intriguing experimental observation that ‘brief ischemia in one vascular bed also protects remote, virgin myocardium from subsequent sustained coronary artery occlusion’ and that this effect ‘…. may be mediated by factor(s) activated, produced, or transported throughout the heart during brief ischemia/reperfusion’. This seminal study laid the foundation for the discovery of ‘remote ischemic conditioning’ (RIC), a phenomenon in which the heart is protected from the detrimental effects of acute ischemia/reperfusion injury (IRI), by applying cycles of brief ischemia and reperfusion to an organ or tissue remote from the heart. The concept of RIC quickly evolved to extend beyond the heart, encompassing inter-organ protection against acute IRI. The crucial discovery that the protective RIC stimulus could be applied non-invasively, by simply inflating and deflating a blood pressure cuff placed on the upper arm to induce cycles of brief ischemia and reperfusion, has facilitated the translation of RIC into the clinical setting. Despite intensive investigation over the last 20 years, the underlying mechanisms continue to elude researchers. In the 8th Biennial Hatter Cardiovascular Institute Workshop, recent developments in the field of RIC were discussed with a focus on new insights into the underlying mechanisms, the diversity of non-cardiac protection, new clinical applications, and large outcome studies. The scientific advances made in this field of research highlight the journey that RIC has made from being an intriguing experimental observation to a clinical application with patient benefit.
doi:10.1007/s00395-014-0453-6
PMCID: PMC4250562  PMID: 25449895
Ischemia; Organ protection; Remote ischemic conditioning; Reperfusion
4.  Cardiac taurine and principal amino acids in right and left ventricles of patients with either aortic valve stenosis or coronary artery disease: the importance of diabetes and gender 
SpringerPlus  2014;3:523.
Free intracellular taurine and principal α-amino acids (glutamate, glutamine, aspartate, asparagine and alanine) are abundant in human heart. They are cellular regulators and their concentration can change in response to disease and cardiac insults and have been shown to differ between hypertrophic left ventricle (LV) and the relatively “normal” right ventricle (RV) in patients with aortic valve stenosis (AVS). This difference has not been shown for coronary artery disease (CAD) and there are no studies that have simultaneously compared amino acid content in LV and RV from different pathologies. In this study we investigated the effect of disease on taurine and principal amino acids in both LV and RV, measured in myocardial biopsies collected from patients with either AVS (n = 22) or CAD (n = 36). Amino acids were extracted and measured using HPLC. Intra- and inter-group analysis was performed as well as subgroup analysis focusing on gender in AVS and type 2 diabetes in CAD. LV of both groups has significantly higher levels of taurine compared to RV. This difference disappears in both diabetic CAD patients and in male AVS patients. Alanine was the only α-amino acid to be altered by diabetes. LV of female AVS patients had significantly more glutamate, aspartate and asparagine than corresponding RV, whilst no difference was seen between LV and RV in males. LV of females has higher glutamate and glutamine and less metabolic stress than LV of males. This work shows that in contrast to LV, RV responds differently to disease which can be modulated by gender and diabetes.
doi:10.1186/2193-1801-3-523
PMCID: PMC4176845  PMID: 25279314
Left and right ventricle; Aortic valve stenosis; Coronary artery disease; Hypertrophy; Taurine; Glutamine; Glutamate; Alanine
5.  The effect of disease on human cardiac protein expression profiles in paired samples from right and left ventricles 
Clinical Proteomics  2014;11(1):34.
Background
Cardiac diseases (e.g. coronary and valve) are associated with ventricular cellular remodeling. However, ventricular biopsies from left and right ventricles from patients with different pathologies are rare and thus little is known about disease-induced cellular remodeling in both sides of the heart and between different diseases. We hypothesized that the protein expression profiles between right and left ventricles of patients with aortic valve stenosis (AVS) and patients with coronary artery disease (CAD) are different and that the protein profile is different between the two diseases. Left and right ventricular biopsies were collected from patients with either CAD or AVS. The biopsies were processed for proteomic analysis using isobaric tandem mass tagging and analyzed by reverse phase nano-LC-MS/MS. Western blot for selected proteins showed strong correlation with proteomic analysis.
Results
Proteomic analysis between ventricles of the same disease (intra-disease) and between ventricles of different diseases (inter-disease) identified more than 500 proteins detected in all relevant ventricular biopsies. Comparison between ventricles and disease state was focused on proteins with relatively high fold (±1.2 fold difference) and significant (P < 0.05) differences. Intra-disease protein expression differences between left and right ventricles were largely structural for AVS patients and largely signaling/metabolism for CAD. Proteins commonly associated with hypertrophy were also different in the AVS group but with lower fold difference. Inter-disease differences between left ventricles of AVS and CAD were detected in 9 proteins. However, inter-disease differences between the right ventricles of CAD and AVS patients were associated with differences in 73 proteins. The majority of proteins which had a significant difference in one ventricle compared to the other pathology also had a similar trend in the adjacent ventricle.
Conclusions
This work demonstrates for the first time that left and right ventricles have a different proteome and that the difference is dependent on the type of disease. Inter-disease differential expression was more prominent for right ventricles. The finding that a protein change in one ventricle was often associated with a similar trend in the adjacent ventricle for a large number of proteins suggests cross-talk proteome remodeling between adjacent ventricles.
doi:10.1186/1559-0275-11-34
PMCID: PMC4158351  PMID: 25249829
Proteomics; Human; Cardiac; Coronary artery disease; Aortic valve stenosis; Ventricular biopsies; TMT tag; Mass spectrometry
6.  Hearts from Mice Fed a Non-Obesogenic High-Fat Diet Exhibit Changes in Their Oxidative State, Calcium and Mitochondria in Parallel with Increased Susceptibility to Reperfusion Injury 
PLoS ONE  2014;9(6):e100579.
Rationale
High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown.
Objectives
To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury.
Methods and Results
Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet.
Conclusions
This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults.
doi:10.1371/journal.pone.0100579
PMCID: PMC4065057  PMID: 24950187
7.  Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion 
Background
Consecutive treatment of normal heart with a high dose of isoproterenol and adenosine (Iso/Ade treatment), confers strong protection against ischaemia/reperfusion injury. In preparation for translation of this cardioprotective strategy into clinical practice during heart surgery, we further optimised conditions for this intervention using a clinically-relevant dose of Iso and determined its cardioprotective efficacy in hearts isolated from a model of surgically-induced heart failure.
Methods
Isolated Langendorff-perfused rat hearts were treated sequentially with 5 nM Iso and 30 μM Ade followed by different durations of washout prior to 30 min global ischaemia and 2 hrs reperfusion. Reperfusion injury was assessed by measuring haemodynamic function, lactate dehydrogenase (LDH) release and infarct size. Protein kinase C (PKC) activity and glycogen content were measured in hearts after the treatment. In a separate group of hearts, Cyclosporine A (CsA), a mitochondria permeability transition pore (MPTP) inhibitor, was added with Iso/Ade. Failing hearts extracted after 16 weeks of ligation of left coronary artery in 2 months old rats were also subjected to Iso/Ade treatment followed by ischaemia/reperfusion.
Results
Recovery of the rate pressure product (RPP) in Iso/Ade-treated hearts was significantly higher than in controls. Thus in Iso/Ade treated hearts with 5 nM Iso and no washout period, RPP recovery was 76.3 ± 6.9% of initial value vs. 28.5 ± 5.2% in controls. This was associated with a 3 fold reduction in LDH release irrespective to the duration of the washout period. Hearts with no washout of the drugs (Ade) had least infarct size, highest PKC activity and also showed reduced glycogen content. Cardioprotection with CsA was not additive to the effect of Iso/Ade treatment. Iso/Ade treatment conferred significant protection to failing hearts. Thus, RPP recovery in failing hearts subjected to the treatment was 69.0 ± 16.3% while in Control hearts 19.7 ± 4.0%. LDH release in these hearts was also 3 fold lower compared to Control.
Conclusions
Consecutive Iso/Ade treatment of normal heart can be effective at clinically-relevant doses and this effect appears to be mediated by glycogen depletion and inhibition of MPTP. This intervention protects clinically relevant failing heart model making it a promising candidate for clinical use.
doi:10.1186/1479-5876-12-139
PMCID: PMC4045901  PMID: 24885907
Ischaemia/reperfusion; Cardioprotection; Isoproterenol; Adenosine; Heart failure; Mitochondria permeability transition pore
8.  Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes☆ 
L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.
Graphical abstract
Highlights
•Basal L type calcium current was reduced by interfering with caveolin-3 binding.•L type calcium current is tonically regulated by PKA phosphorylation.•Interfering with caveolin-3 binding reduced beta2 adrenergic stimulation of ICa.
doi:10.1016/j.yjmcc.2013.12.026
PMCID: PMC3980375  PMID: 24412535
t-tubules; Ca; Phosphorylation; β2-Adrenoceptors
9.  Local Inhibition of MicroRNA-24 Improves Reparative Angiogenesis and Left Ventricle Remodeling and Function in Mice With Myocardial Infarction 
Molecular Therapy  2013;21(7):1390-1402.
Myocardial infarction (MI) is the leading cause of death worldwide. MicroRNAs regulate the expression of their target genes, thus mediating a plethora of pathophysiological functions. Recently, miRNA-24 emerged as an important but controversial miRNA involved in post-MI responses. Here, we aimed at clarifying the effect of adenovirus-mediate intra-myocardial delivery of a decoy for miRNA-24 in a mouse MI model and to investigate the impact of miRNA-24 inhibition on angiogenesis and cardiovascular apoptosis. After MI induction, miRNA-24 expression was lower in the peri-infarct tissue and its resident cardiomyocytes and fibroblasts; while it increased in endothelial cells (ECs). Local adenovirus-mediated miRNA-24 decoy delivery increased angiogenesis and blood perfusion in the peri-infarct myocardium, reduced infarct size, induced fibroblast apopotosis and overall improved cardiac function. Notwithstanding these beneficial effects, miRNA-24 decoy increased cardiomyocytes apoptosis. In vitro, miRNA-24 inhibition enhanced ECs survival, proliferation and networking in capillary-like tubes and induced cardiomyocyte and fibroblast apoptosis. Finally, we identified eNOS as a novel direct target of miR-24 in human cultured ECs and in vivo. Our findings suggest that miRNA-24 inhibition exerts distinct biological effects on ECs, cardiomyocytes and fibroblasts. The overall result of post-infarction local miRNA-24 inhibition appears to be therapeutic.
doi:10.1038/mt.2013.89
PMCID: PMC3702112  PMID: 23774796
10.  Immediate hypothermia reduces cardiac troponin I following hypoxic-ischemic encephalopathy in newborn pigs 
Pediatric research  2011;70(4):352-356.
Neonatal hypoxic-ischemic encephalopathy (HIE) is a clinically defined neurological condition following lack of oxygen and often associated with cardiac dysfunction in term infants. Therapeutic hypothermia after birth is neuroprotective in infants with HIE. However, it is not known whether hypothermia (HT) is also cardioprotective. Four newborn pigs were used in the pilot study and a further 18 newborn pigs (randomly assigned to 72h-normothermia (NT) or 24h-HT followed by 48h-NT) were subjected to global HIE insults. Serum cTnI was measured prior to and post the HIE insult. Blood pressure, inotropic support, blood gases and heart rate (HR) were recorded throughout. Cardiac pathology was assessed from histological sections. Cooling reduced serum cTnI levels significantly in HT pigs by 6h (NT, 1.36±0.67; HT 0.34±0.23 ng/ml, p=0.0009). After rewarming, from 24 to 30h post insult, HR and cTnI increased in the HT group; from HR[24h]=117±22 to HR[30h]=218±32 beats/minute (p=0.0002) and from cTnI[24h]=0.23±0.12 to cTnI[30h]=0.65±0.53ng/ml, (p=0.05). There were fewer ischemic lesions on cardiac examination (37%) in the HT group compared to the NT group (70%). Hypothermia (24h) pigs did not have the post-insult cTnI increase seen in NT treated pigs. There was a trend that HT improved cardiac pathology in this 3-day survival model.
doi:10.1203/PDR.0b013e31822941ee
PMCID: PMC3173864  PMID: 21691250
11.  Warm-blood cardioplegia with low or high magnesium for coronary bypass surgery: a randomised controlled trial☆ 
Objective: Magnesium (Mg2+) is cardioprotective and has been routinely used to supplement cardioplegic solutions during coronary artery bypass graft (CABG) surgery. However, there is no consensus about the Mg2+ concentration that should be used. The aim of this study was to compare the effects of intermittent antegrade warm-blood cardioplegia supplemented with either low- or high-concentration Mg2+. Methods: This study was a randomised controlled trial carried out in two cardiac surgery centres, Bristol, UK and Cuneo, Italy. Patients undergoing isolated CABG with cardiopulmonary bypass were eligible. Patients were randomised to receive warm-blood cardioplegia supplemented with 5 or 16 mmol l−1 Mg2+. The primary outcome was postoperative atrial fibrillation. Secondary outcomes were serum biochemical markers (troponin I, Mg2+, potassium, lactate and creatinine) and time-to-plegia arrest. Intra-operative and postoperative clinical outcomes were also recorded. Results: Data from two centres for 691 patients (342 low and 349 high Mg2+) were analysed. Baseline characteristics were similar for both groups. There was no significant difference in the frequency of postoperative atrial fibrillation in the high (32.8%) and low (32.0%) groups (risk ratio 1.03, 95% confidence interval, CI, 0.82–1.28). However, compared with the low group, troponin I release was 28% less (95% CI 55–94%, p = 0.02) in the high-Mg2+ group. The 30-day mortality was 0.72% (n = 5); all deaths occurred in the high-Mg2+ group but there was no significant difference between the groups (p = 0.06). Frequencies of other major complications were similar in the two groups. Conclusions: Warm-blood cardioplegia supplemented with 16 mmol l−1 Mg2+, compared with 5 mmol l−1 Mg2+, does not reduce the frequency of postoperative atrial fibrillation in patients undergoing CABG but may reduce cardiac injury. (This trial was registered as ISRCTN95530505.)
doi:10.1016/j.ejcts.2010.09.049
PMCID: PMC3162136  PMID: 21353585
Myocardial protection; Cardioplegia; CABG; Surgery; Atrial fibrillation; Flutter
12.  Oxidative stress increases SNAT1 expression and stimulates cysteine uptake in freshly isolated rat cardiomyocytes 
Amino acids  2010;40(2):517-526.
Intracellular cysteine availability is an important rate-limiting factor governing glutathione synthesis in the heart. This is also dependent on the magnitude and rate of cysteine uptake into cardiomyocytes, which has been little studied. This study investigated the hypothesis that changes to cysteine transporter expression and activity during oxidative stress influence cardiomyocyte glutathione levels. The uptake of 0–3 mM l-[35S]cysteine into ventricular cardiomyocytes isolated from adult male Wistar rats was measured using oil filtration. Cysteine transporter expression was investigated by conventional and real-time quantitative reverse-transcription polymerase chain reaction and Western blotting. Glutathione levels were measured enzymatically. Oxidative stress was induced via 0–6 h incubation with 0.05 mM H2O2. Cysteine uptake was greatest in sodium-containing media and was inhibited by glutamine, 2-(methylamino)-isobutyric acid (αMeAIB), serine or alanine. The Km and Vmax of the αMeAIB insensitive and sensitive portions were 0.133 ± 0.01 mM and 468.11 ± 9.04 pmol/μl cell vol/min, and 0.557 ± 0.096 mM and 279.87 ± 16.06 pmol/μl cell vol/min, respectively. Cardiomyocytes expressed ASCT2, SNAT1 and SNAT2 but not ASCT1. Oxidative stress significantly enhanced cysteine uptake, which was attenuated by αMeAIB. This was accompanied by significantly enhanced SNAT1 expression, whilst SNAT2 and ASCT2 were unaffected. Incubation with cysteine significantly reduced the oxidative-stress-induced decline in cardiomyocyte glutathione as compared to cells incubated without cysteine or cells incubated with cysteine and αMeAIB. In conclusion, under control conditions SNAT transporters aid in the delivery of cysteine for cardiomyocyte GSH synthesis, whilst oxidative stress increases cardiomyocyte cysteine uptake and stimulates cardiomyocyte SNAT1 expression.
doi:10.1007/s00726-010-0664-6
PMCID: PMC3044454  PMID: 20602128
SNAT1; Oxidative stress; Isolated cardiomyocytes; Glutathione
13.  Cysteine protects freshly isolated cardiomyocytes against oxidative stress by stimulating glutathione peroxidase 
Molecular and cellular biochemistry  2010;343(1-2):125-132.
Cysteine has been implicated in myocardial protection, although this is controversial and constrained by limited knowledge about the effects of cysteine at the cellular level. This study tested the hypothesis that a physiologically relevant dose of l-cysteine could be safely loaded into isolated cardiomyocytes leading to improved protection against oxidative stress. Freshly isolated adult rat ventricular cardiomyocytes were incubated for 2 h at 37°C with (cysteine incubated) or without (control) 0.5 mM cysteine prior to washing and suspension in fresh cysteine-free media. Cysteine incubated cells had higher intracellular cysteine levels compared to controls (9.6 ± 0.78 vs. 6.5 ± 0.65 nmol/mg protein, P < 0.02, n = 6 ± SE). Cell homeostasis indicators were similar in the two groups. Cysteine incubated cells had significantly higher glutathione peroxidase (GPx) activity (1.11 ± 0.23 vs. 0.54 ± 0.1 U/mg protein, P < 0.05, n = 5 ± SE) and significantly greater expression of GPx-1 (5.01 ± 0.48 vs. 3.01 ± 0.25 OD units/mm2, P < 0.05, n = 4 ± SE) compared to controls. Upon exposure to H2O2, cysteine incubated cells generated fewer reactive oxygen species and took longer to show contractile changes and undergo hypercontracture. However, when cells were exposed to H2O2 in the presence of 0.05 mM of the GPx inhibitor mercaptosuccinic acid, this increased the control cells’ susceptibility to H2O2 and completely abolished the cysteine mediated protection. These results suggest a new role for cysteine in myocardial protection involving stimulation of glutathione peroxidase.
doi:10.1007/s11010-010-0506-6
PMCID: PMC2948553  PMID: 20556486
Cardioprotection; Cysteine; Glutathione peroxidase; H2O2
14.  Temperature preconditioning is optimal at 26°C and confers additional protection to hypothermic cardioplegic ischemic arrest 
We have recently shown that brief episodes of hypothermic perfusion interspersed with periods of normothermic perfusion, referred to as temperature preconditioning (TP), are cardioprotective and can be mimicked by consecutive isoproterenol/adenosine treatment. Here we investigate the optimal temperature for TP and whether TP further enhances protection provided by hypothermic ischemia with or without polarized cardioplegic arrest. Three experimental groups of Langendorff-perfused rat hearts were used. In the first group, hearts were subjected to three episodes of hypothermic perfusion at 7, 17, 26 and 32°C during the TP protocol, followed by 30 min normothermic index ischemia and 60 min reperfusion (37°C). Protein kinase A (PKA) activity and cyclic AMP (cAMP) concentrations were measured prior to index ischemia. In the second group, TP (26°C) hearts were subjected to two hours hypothermic index ischemia at 26°C and two hours normothermic reperfusion. In the third group, TP (26°C) hearts or hearts treated with isoproterenol/adenosine (pharmacological simulation of TP) were subjected to four hours hypothermic index ischemia with procaine-induced polarized cardioplegia at 26°C followed by two hours normothermic reperfusion. Hemodynamic function recovery, lactate dehydrogenase release and infarct size were used to assess cardioprotection. TP at 26°C resulted in highest cardioprotection, increased cAMP concentration and PKA activity, while TP at 7°C exacerbated ischemia/reperfusion damage, and had no effect on cAMP concentration or PKA activity. TP at 26°C also protected hearts during hypothermic ischemia with or without polarized cardioplegia. Isoproterenol/adenosine treatment conferred additional protection similar to TP. In conclusion, the study shows that TP-induced cardioprotection is temperature dependent and is optimal at 26°C; TP confers additional protection to hypothermia and polarized cardioplegia; and that the pharmacological treatment based on the mechanism of TP (consecutive isoproterenol/adenosine treatment) is a potential cardioprotective strategy that can be used during heart surgery and transplantation.
doi:10.1258/ebm.2011.010357
PMCID: PMC3125701  PMID: 21606118
heart; ischemia/reperfusion; hypothermia; cardioprotection; preconditioning; cardioplegia

Results 1-14 (14)