Olga M. Hernandez

Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3

Coronary artery disease leads to injury and loss of myocardial tissue by deprivation of blood flow (ischemia) and is a major underlying cause of heart failure. Prolonged ischemia causes necrosis and apoptosis of cardiac myocytes and vascular cells; however, the mechanisms of ischemia-mediated cell death are poorly understood. Ischemia is associated with both hypoxia and acidosis due to increased glycolysis and lactic acid production. We recently reported that hypoxia does not induce cardiac myocyte apoptosis in the absence of acidosis. We now report that hypoxia-acidosis-associated cell death is mediated by BNIP3, a member of the Bcl-2 family of apoptosis-regulating proteins. Chronic hypoxia induced the expression and accumulation of BNIP3 mRNA and protein in cardiac myocytes, but acidosis was required to activate the death pathway. Acidosis stabilized BNIP3 protein and increased the association with mitochondria. Cell death by hypoxia-acidosis was blocked by pretreatment with antisense BNIP3 oligonucleotides. The pathway included extensive DNA fragmentation and opening of the mitochondrial permeability transition pore, but no apparent caspase activation. Overexpression of wild-type BNIP3, but not a translocation-defective mutant, activated cardiac myocyte death only when the myocytes were acidic. This pathway may figure significantly in muscle loss during myocardial ischemia.

Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.

Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH](o)). Apoptosis did not occur when [pH](o) was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis-mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25-30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53.

Rapid Activation of Neutral Sphingomyelinase by Hypoxia-Reoxygenation of Cardiac Myocytes

Elevated levels of oxygen free radicals have been implicated in the pathways of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stress responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neutral sphingomyelinase and accumulation of ceramide. Ceramide increased abruptly after reoxygenation, peaking at 10 minutes with 225+/-40% of the control level. Neutral sphingomyelinase activity was induced with similar kinetics, and both activities remained elevated for several hours. c-Jun N-terminal kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreating cells with antioxidants quenched sphingomyelinase activation, ceramide accumulation, and JNK activation. Ceramide did not accumulate in reoxygenated nonmuscle fibroblasts, and JNK was not activated by reoxygenation in these cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramide-mediated activation of JNK.

F110I and R278C Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy Affect Muscle Contraction in Transgenic Mice and Reconstituted Human Cardiac Fibers

We have studied the physiological effects of the troponin T (TnT) F110I and R278C mutations associated with familial hypertrophic cardiomyopathy (FHC) in humans. Three to four-month-old transgenic (Tg) mice expressing F110I-TnT and R278C-TnT did not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. The F110I mutation impaired acute exercise tolerance, whereas R278C did not. Skinned papillary muscle fibers from transgenic mice expressing F110I-TnT demonstrated increased Ca2+ sensitivity of force and ATPase activity, and likewise an increased Ca2+ sensitivity of force was observed in F110I-TnT-reconstituted human cardiac muscle preparations. In contrast, no changes in force or the ATPase-pCa dependencies were observed in transgenic R278C fibers or in human fibers reconstituted with the R278C-TnT mutant. The maximal level of force development was dramatically decreased in both transgenic mice. However, the maximal ATPase was not different (R278C-TnT) or only slightly less (F110I-TnT) than that of non-Tg and WT-Tg littermates. Consequently, their ratios of ATPase/force (energy cost) at all Ca2+ concentrations were dramatically higher compared with non-Tg and WT-Tg fibers. This increase in energy cost most likely results from a decrease in force per myosin cross-bridge, because forcing all cross-bridges into the force generating state by substitution of MgADP for MgATP in maximum contracting solutions resulted in the same increase in maximal force (15%) in all transgenic and non-transgenic preparations. The combination of increased Ca2+ sensitivity and energy cost in the F110I hearts may be responsible for the greater severity of this phenotype compared with the R278C mutation.

The E22K mutation of myosin RLC that causes familial hypertrophic cardiomyopathy increases calcium sensitivity of force and ATPase in transgenic mice

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease caused by mutations in all of the major sarcomeric proteins, including the ventricular myosin regulatory light-chain (RLC). The E22K-RLC mutation has been associated with a rare variant of cardiac hypertrophy defined by mid-left ventricular obstruction due to papillary muscle hypertrophy. This mutation was later found to cause ventricular and septal hypertrophy. We have generated transgenic (Tg) mouse lines of myc-WT (wild type) and myc-E22K mutant of human ventricular RLC and have examined the functional consequences of this FHC mutation in skinned cardiac-muscle preparations. In longitudinal sections of whole mouse hearts stained with hematoxylin and eosin, the E22K-mutant hearts of 13-month-old animals showed signs of inter-ventricular septal hypertrophy and enlarged papillary muscles with no filament disarray. Echo examination did not reveal evidence of cardiac hypertrophy in Tg-E22K mice compared to Tg-WT or Non-Tg hearts. Physiological studies utilizing skinned cardiac-muscle preparations showed an increase by ΔpCa50≥0.1 in Ca2+ sensitivity of myofibrillar ATPase activity and force development in Tg-E22K mice compared with Tg-WT or Non-Tg littermates. Our results suggest that E22K-linked FHC is mediated through Ca2+-dependent events. The FHC-mediated structural perturbations in RLC that affect Ca2+ binding properties of the mutated myocardium are responsible for triggering the abnormal function of the heart that in turn might initiate a hypertrophic process and lead to heart failure.

The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Delta43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Delta43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Delta43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Delta43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Delta43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.

Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

The myosin essential light chain (ELC) is a structural component of the actomyosin cross‐bridge, but its function is poorly understood, especially the role of the cardiac specific N‐terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N‐terminal extension was investigated with the Tg‐Δ43 mouse model, whose myocardium expresses a truncated ELC. Low‐angle X‐ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (~1.5 nm) and no alterations in cross‐bridge mass distribution in Tg‐A57G mice compared to Tg‐WT, expressing the full‐length nonmutated ELC. The truncation mutation showed a 1.3‐fold increase in I1,1/I1,0, indicating a shift of cross‐bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg‐A57G muscle fibers compared to Tg‐WT or Tg‐Δ43. The equilibrium constant for the cross‐bridge force generation step was smallest in Tg‐Δ43. These results support an important role for the N‐terminal ELC extension in prepositioning the cross‐bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross‐bridge properties and lead to pathological phenotypes.—Muthu, P., Wang, L., Yuan, C.‐C., Kazmierczak, K., Huang, W., Hernandez, O. M., Kawai, M., Irving, T. C., Szczesna‐Cordary, D. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction. FASEB J. 25, 4394–4405 (2011). www.fasebj.org