Mitochondrial Bioenergetics and Dysfunction in Failing Heart.

Energy insufficiency has been recognized as a key feature of systolic heart failure. Although mitochondria have long been known to sustain myocardial work energy supply, the capacity to therapeutically target mitochondrial bioenergetics dysfunction is hampered by a complex interplay of multiple perturbations that progressively compound causing myocardial failure and collapse. Compared to non-failing human donor hearts, activity rates of complexes I and IV, nicotinamide nucleotide transhydrogenase (NADPH-transhydrogenase, Nnt) and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase are markedly decreased in end-stage heart failure. Diminished REDOX capacity with lower total glutathione and coenzyme Q10 levels are also a feature of chronic left ventricular failure. Decreased enzyme activities in part relate to abundant and highly specific oxidative, nitrosylative, and hyperacetylation modifications. In this brief review we highlight that energy deficiency in end-stage failing human left ventricle predominantly involves concomitantly impaired activities of key electron transport chain and Krebs cycle enzymes rather than altered expression of respective genes or proteins. Augmented oxidative modification of these enzyme subunit structures, and the formation of highly reactive secondary metabolites, implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, which contribute further to progressive decreases in bioenergetic capacity and contractile function in human heart failure.

Posttranslational modifications and dysfunction of mitochondrial enzymes in human heart failure.

Deficiency of energy supply is a major complication contributing to the syndrome of heart failure (HF). Because the concurrent activity profile of mitochondrial bioenergetic enzymes has not been studied collectively in human HF, our aim was to examine the mitochondrial enzyme defects in left ventricular myocardium obtained from explanted end-stage failing hearts. Compared with nonfailing donor hearts, activity rates of complexes I and IV and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase, and aconitase were lower in HF, as determined spectrophotometrically. However, activity rates of complexes II and III and citrate synthase did not differ significantly between the two groups. Protein expression, determined by Western blotting, did not differ between the groups, implying posttranslational perturbation. In the face of diminished total glutathione and coenzyme Q10 levels, oxidative modification was explored as an underlying cause of enzyme dysfunction. Of the three oxidative modifications measured, protein carbonylation was increased significantly by 31% in HF (P < 0.01; n = 18), whereas levels of 4-hydroxynonenal and protein nitration, although elevated, did not differ. Isolation of complexes I and IV and F1FoATP synthase by immunocapture revealed that proteins containing iron-sulphur or heme redox centers were targets of oxidative modification. Energy deficiency in end-stage failing human left ventricle involves impaired activity of key electron transport chain and Krebs cycle enzymes without altered expression of protein levels. Augmented oxidative modification of crucial enzyme subunit structures implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, thus contributing further to reduced bioenergetics in human HF.

Postnatal shifts in ischemic tolerance and cell survival signaling in murine myocardium.

The immature heart is known to be resistant to ischemia-reperfusion (I/R) injury; however, key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the postnatal changes in myocardial tolerance to I/R, including baseline expression of key proteins governing I/R tolerance and their phosphorylation during I/R. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 wk of age, n = 6/group) were assayed for survival signaling/effectors [Akt, p38MAPK, glycogen synthase kinase-3ß (GSK-3ß), heat shock protein 27 (HSP27), connexin-43, hypoxia-inducible factor-1α (HIF-1α), and caveolin-3] and regulators of apoptosis (Bax and Bcl-2) and autophagy (LC3B, Parkin, and Beclin1). The effect of I/R on ventricular function was measured in isolated perfused hearts from immature (4 wk) and adult (12 wk) mice. The neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phospho-inhibition of GSK-3ß; and high expression of caveolin-3, HIF-1α, LC3B, Beclin1, Bax, and Bcl-2. Immature hearts sustained less dysfunction and infarction following I/R than adults. Emergence of I/R intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax, and Bcl-2; increased GSK-3ß, connexin-43, HIF-1α, LC3B, and Bax:Bcl-2; enhanced postischemic HIF-1α, caveolin-3, Bax, and Bcl-2; and greater postischemic GSK-3ß and HSP27 phosphorylation. Neonatal myocardial stress resistance reflects high expression of prosurvival and autophagy proteins and apoptotic regulators. Notably, there is high phosphorylation of GSK-3ß, p38MAPK, and HSP27 and low phosphorylation of Akt (high Akt "reserve"). Subsequent maturation-related reductions in I/R tolerance are associated with reductions in Akt, Bcl-2, LC3B, and Beclin1, despite increased expression and reduced phospho-inhibition of GSK-3ß.

S-Nitrosoglutathione Limits Apoptosis and Reduces Pulmonary Vascular Dysfunction After Bypass.

BACKGROUND: During hypoxia or acidosis, S-nitrosoglutathione (GSNO) has been shown to protect the cardiomyocyte from ischemia-reperfusion injury. In a randomized double-blinded control study of a porcine model of paediatric cardiopulmonary bypass (CPB), we aimed to evaluate the effects of 2 different doses (low and high) of GSNO. METHODS: Pigs weighing 15-20 kg were exposed to CPB with 1 hour of aortic cross-clamp. Prior to and during CPB, animals were randomized to receive low-dose (up to 20 nmol/kg/min) GSNO (n = 8), high-dose (up to 60 nmol/kg/min) GSNO (n = 6), or normal saline (n = 7). Standard cardiac intensive care management was continued for 4 hours post-bypass. RESULTS: There was a reduction in myocyte apoptosis after administration of GSNO (P = .04) with no difference between low- and high-dose GSNO. The low-dose GSNO group had lower pulmonary vascular resistance post-CPB (P = .007). Mitochondrial complex I activity normalized to citrate synthase activity was higher after GSNO compared with control (P = .02), with no difference between low- and high-dose GSNO. CONCLUSIONS: In a porcine model of CPB, intravenous administration of GSNO limits myocardial apoptosis through preservation of mitochondrial complex I activity, and improves pulmonary vascular resistance. There appears to be a dose-dependent effect to this protection.

Diminished NADPH transhydrogenase activity and mitochondrial redox regulation in human failing myocardium.

Although the functional role of nicotinamide nucleotide transhydrogenase (Nnt) remains to be fully elucidated, there is strong evidence that Nnt plays a critical part in mitochondrial metabolism by maintaining a high NADPH-dependent GSH/GSSG ratio, and thus the control of cellular oxidative stress. Using real-time PCR, spectrophotometric and western blotting techniques, we sought to determine the presence, abundance and activity level of Nnt in human heart tissues and to discern whether these are altered in chronic severe heart failure. Left ventricular levels of the NNT gene and protein expression did not differ significantly between the non-failing donor (NF) and heart failure (HF) group. Notably, compared to NF, Nnt activity rates in the HF group were 18% lower, which coincided with significantly higher levels of oxidized glutathione, lower glutathione reductase activity, lower NADPH and a lower GSH/GSSG ratio. In the failing human heart a partial loss of Nnt activity adversely impacts NADPH-dependent enzymes and the capacity to maintain membrane potential, thus contributing to a decline in bioenergetic capacity, redox regulation and antioxidant defense, exacerbating oxidative damage to cellular proteins.

Adaptations in Protein Expression and Regulated Activity of Pyruvate Dehydrogenase Multienzyme Complex in Human Systolic Heart Failure.

Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n = 26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n = 21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1ß subunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 µM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 µM acetyl CoA). In isolated hypertrophic cardiomyocytes in vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways.

Energy deficiency in the failing heart: linking increased reactive oxygen species and disruption of oxidative phosphorylation rate.

Heart failure is a complex syndrome of numerous dysfunctional components which converge to cause chronic progressive failure of ventricular contractile function and maintenance of cardiac output demand. The aim of this brief review is to highlight some of the mounting evidence indicating that augmented superoxide, related reactive oxygen species and other free radicals contribute to the oxidative stress evident during the progression of heart failure. While much of the source of increased reactive oxygen species is mitochondrial, there are other intracellular sources, which together are highly reactive with functional and structural cellular lipids and proteins. Bioenergetic defects limiting ATP synthesis in the failing myocardium relate not only to post-translational modification of electron transport respiratory chain proteins but also to perturbation of Krebs Cycle enzyme-dependent synthesis of NADH. Accumulation of pathological levels of lipid peroxides relate to dysfunction in the intrinsic capacity to clear and renew dysfunctional proteins. This review also features key limitations of human heart failure studies and potential clinical therapies that target the elevated oxidative stress that is a hallmark of human heart failure.

Coenzyme Q10 in cardiovascular disease.

In this review we summarise the current state of knowledge of the therapeutic efficacy and mechanisms of action of CoQ(10) in cardiovascular disease. Our conclusions are: 1. There is promising evidence of a beneficial effect of CoQ(10) when given alone or in addition to standard therapies in hypertension and in heart failure, but less extensive evidence in ischemic heart disease. 2. Large scale multi-centre prospective randomised trials are indicated in all these areas but there are difficulties in funding such trials. 3. Presently, due to the notable absence of clinically significant side effects and likely therapeutic benefit, CoQ(10) can be considered a safe adjunct to standard therapies in cardiovascular disease.

Molecular markers of programmed cell death in donor hearts before transplantation.

BACKGROUND: In this study we investigate whether pro-apoptotic, pro-inflammatory and other early signaling markers indicative of increased propensity for cell death processes were evident in human donor heart allografts immediately before transplantation, and whether there is an association with primary graft failure. METHODS: A prospective study was performed utilizing donor left atrial myocardium collected at the time of implantation of hearts from brain-dead donors (BDD, n = 29). In addition, to explore the potential of donor hearts from donation after circulatory death (DCD), myocardial samples were obtained during transplantation of lungs from DCD donors (n = 6). A comparator reference group (n = 7) consisted of left atrial specimens from patients undergoing mitral valve surgery. RESULTS: Significantly raised levels of caspase-3 specific activity, activated hypoxia inducible factor-1 (HIF-1α) and 8-hydroxy-2'-deoxyguanosine were evident in the transplanted hearts (from BDD) that developed primary graft failure (n = 11). DCD hearts did not differ from BDD with regard to mRNA expression levels of FAS, Bax, IL-6 and caspase-3. Although DCD hearts exhibited lower caspase-3 specific activity and activated hypoxia-inducible factor-1 protein, they had higher levels of mRNA for NF-κB, Bnip3 and caspase-1 mRNA. Increased 8-hydroxy-2'-deoxyguanosine levels reflected greater oxidative stress and reactive oxygen species-related DNA fragmentation. CONCLUSIONS: Our data indicate a significant role of pro-apoptotic and pro-inflammatory activity in allografts that subsequently exhibit primary graft failure. The relatively lower levels of apoptotic and inflammatory activity in DCD hearts suggest they may represent a potentially usable donor cardiac allograft pool. This possibility requires further detailed molecular and clinical research.

Effect of remote ischemic preconditioning on phosphorylated protein signaling in children undergoing tetralogy of Fallot repair: a randomized controlled trial.

BACKGROUND: Our previous randomized controlled trial demonstrated cardiorespiratory protection by remote ischemic preconditioning (RIPC) in children before cardiac surgery. However, the impact of RIPC on myocardial prosurvival intracellular signaling remains unknown in cyanosis. RIPC may augment phosphorylated protein signaling in myocardium and circulating leukocytes during tetralogy of Fallot (ToF) repair. METHODS AND RESULTS: Children (n=40) undergoing ToF repair were double-blind randomized to RIPC (n=11 boys, 9 girls) or control (sham RIPC: n=9 boys, 11 girls). Blood samples were taken before, immediately after, and 24 hours after cardiopulmonary bypass. Resected right ventricular outflow tract muscle and leukocytes were processed for protein expression and mitochondrial respiration. There was no difference in age (7.1 ± 3.4 versus 7.1 ± 3.4 months), weight (7.7 ± 1.8 versus 7.5 ± 1.9 kg), or bypass or aortic cross-clamp times between the groups (control versus RIPC, mean±SD). No differences were seen between the groups for an increase in the ratio of phosphorylated to total protein for protein kinase B, p38 mitogen activated protein kinase, signal transducer and activator of transcription 3, glycogen synthase kinase 3ß, heat shock protein 27, Connexin43, or markers associated with promotion of necrosis (serum cardiac troponin I), apoptosis (Bax, Bcl-2), and autophagy (Parkin, Beclin-1, LC3B). A high proportion of total proteins were in phosphorylated form in control and RIPC myocardium. In leukocytes, mitochondrial respiration and assessed protein levels did not differ between groups. CONCLUSIONS: In patients with cyanotic heart disease, a high proportion of proteins are in phosphorylated form. RIPC does not further enhance phosphorylated protein signaling in myocardium or circulating leukocytes in children undergoing ToF repair. CLINICAL TRIAL REGISTRATION: URL: (http://www.anzctr.org.au/trial_view.aspx?id=335613. Unique identifier: Australian New Zealand Clinical Trials Registry number ACTRN12610000496011.

Remote ischemic preconditioning in cyanosed neonates undergoing cardiopulmonary bypass: a randomized controlled trial.

OBJECTIVE: The myocardial protective effect of remote ischemic preconditioning has been demonstrated in heterogeneous groups of patients undergoing cardiac surgery. No studies have examined this technique in neonates. The present study was performed to examine the remote ischemic preconditioning efficacy in this high-risk patient group. METHODS: A preliminary, randomized, controlled trial was conducted to investigate whether remote ischemic preconditioning in cyanosed neonates undergoing cardiac surgery confers protection against cardiopulmonary bypass. Two groups of neonates undergoing cardiac surgery were recruited for the present study: patients with transposition of the great arteries undergoing the arterial switch procedure and patients with hypoplastic left heart syndrome undergoing the Norwood procedure. The subjects were randomized to the remote ischemic preconditioning or sham control groups. Remote ischemic preconditioning was induced by four 5-minute cycles of lower limb ischemia and reperfusion using a blood pressure cuff. Troponin I and the biomarkers for renal and cerebral injury were measured pre- and postoperatively. RESULTS: A total of 39 neonates were recruited-20 with transposition of the great arteries and 19 with hypoplastic left heart syndrome. Of the 39 neonates, 20 were randomized to remote ischemic preconditioning and 19 to the sham control group. The baseline demographics appeared similar between the randomized groups. The cardiopulmonary bypass and crossclamp times were not significantly different between the 2 groups. The troponin I levels were not significantly different at 6 hours after cardiopulmonary bypass nor were the postoperative inotrope requirements. Markers of renal (neutrophil gelatinase-associated lipocalin) and cerebral injury (S100b, neuron-specific enolase) were not significantly different between the 2 groups. CONCLUSIONS: Our data suggest that remote ischemic preconditioning in hypoxic neonates undergoing cardiopulmonary bypass surgery does not provide myocardial, renal, or neuronal protection. Additional studies are needed to examine the relationships among developmental age, hypoxia, and the molecular mechanisms of ischemic preconditioning.

Oxidation of myofibrillar proteins in human heart failure.

OBJECTIVES: We investigated the incidence and contribution of the oxidation/nitrosylation of tropomyosin and actin to the contractile impairment and cardiomyocyte injury occurring in human end-stage heart failure (HF) as compared with nonfailing donor hearts. BACKGROUND: Although there is growing evidence that augmented intracellular accumulation of reactive oxygen/nitrogen species may play a key role in causing contractile dysfunction, there is a dearth of data regarding their contractile protein targets in human HF. METHODS: In left ventricular (LV) biopsies from explanted failing hearts (New York Heart Association functional class IV; HF group) and nonfailing donor hearts (NF group), carbonylation of actin and tropomyosin, disulphide cross-bridge (DCB) formation, and S-nitrosylation in tropomyosin were assessed, along with plasma troponin I and LV ejection fraction (LVEF). RESULTS: The LV biopsies from the HF group had 2.14 ± 0.23-fold and 2.31 ± 0.46-fold greater levels in actin and tropomyosin carbonylation, respectively, and 1.77 ± 0.45-fold greater levels of high-molecular-weight complexes of tropomyosin due to DCB formation, compared with the NF group. Tropomyosin also underwent S-nitrosylation that was 1.3 ± 0.15-fold higher in the HF group. Notably, actin and tropomyosin carbonylation was significantly correlated with both loss of viability indicated by plasma troponin I and contractile impairment as shown by reduced LVEF. CONCLUSIONS: This study demonstrated that oxidative/nitrosylative changes of actin and tropomyosin are largely increased in human failing hearts. Because these changes are inversely correlated to LVEF, actin and tropomyosin oxidation are likely to contribute to the contractile impairment evident in end-stage HF.

Mitochondrial dysfunction in CD4+ lymphocytes from stavudine-treated HIV patients.

HIV therapy with nucleoside analogue reverse transcriptase inhibitors (NRTI) such as stavudine remains in widespread use in resource-limited nations due to potent efficacy, convenience of formulation and lack of practical alternatives. However, it remains unclear whether adverse side effects with NRTI include reduced mitochondrial respiratory function, particularly in peripheral blood lymphocytes (PBLs). The aim of this study was to determine whether stavudine-based highly active antiretroviral therapy (HAART) is associated with impaired mitochondrial respiratory transport chain function in patient derived CD4+PBLs. CD4+PBLs were isolated from asymptomatic HIV-infected patients treated with stavudine-HAART for 3 months (n=10), HIV-infected patients not on treatment (n=9) and uninfected controls (n=18). The basal mitochondrial oxygen consumption of CD4+PBLs from stavudine-treated patients was reduced relative to that of untreated HIV-infected patients and controls (stavudine treated group, 4.22 (25% 2.16, 75% 8.84); control uninfected, 11.2 (25% 3.95, 75% 16.6); untreated 18.1 (25% 11.8, 75% 37.9)ng oxygen atoms/min/ml). Maximal oxygen consumption (stimulated with the proton ionophore FCCP) in cells from stavudine treated patients was also reduced relative to that of untreated patients and controls (stavudine treated, 24.4+/-10.5; control uninfected, 50.6+/-39.5; untreated, 68.8+/-41.1 ng oxygen atoms/min/ml). Citrate synthase activities, relative mitochondrial volume (by electron microscopy) and mtDNA copy numbers per cell were not different between groups. Therapy with stavudine results in impaired mitochondrial function in CD4+PBLs that does not appear to be due to reduced mitochondrial volume or DNA content and cannot be attributed to infection with HIV.