Christopher P. Baines

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-α-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.

Voltage-Dependent Anion Channels are Dispensable for Mitochondrial-Dependent Cell Death

Mitochondria are critically involved in necrotic cell death induced by Ca2+ overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore — a protein complex that spans both the outer and inner mitochondrial membranes — is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix. Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1-, Vdac3-, and Vdac1–Vdac3-null mice exhibited a Ca2+- and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca2+- and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1–Vdac3 and Vdac1–Vdac2–Vdac3. Wild-type and Vdac-deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.

Cell Biology of Ischemia/Reperfusion Injury

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, and peripheral vascular disease, continue to be among the most frequent causes of debilitating disease and death. Tissue injury and/or death occur as a result of the initial ischemic insult, which is determined primarily by the magnitude and duration of the interruption in the blood supply, and then subsequent damage induced by reperfusion. During prolonged ischemia, ATP levels and intracellular pH decrease as a result of anaerobic metabolism and lactate accumulation. As a consequence, ATPase-dependent ion transport mechanisms become dysfunctional, contributing to increased intracellular and mitochondrial calcium levels (calcium overload), cell swelling and rupture, and cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Although oxygen levels are restored upon reperfusion, a surge in the generation of reactive oxygen species occurs and proinflammatory neutrophils infiltrate ischemic tissues to exacerbate ischemic injury. The pathologic events induced by I/R orchestrate the opening of the mitochondrial permeability transition pore, which appears to represent a common end-effector of the pathologic events initiated by I/R. The aim of this treatise is to provide a comprehensive review of the mechanisms underlying the development of I/R injury, from which it should be apparent that a combination of molecular and cellular approaches targeting multiple pathologic processes to limit the extent of I/R injury must be adopted to enhance resistance to cell death and increase regenerative capacity in order to effect long-lasting repair of ischemic tissues.

Protein Kinase Cε Interacts With and Inhibits the Permeability Transition Pore in Cardiac Mitochondria

Abstract— Although functional coupling between protein kinase C&egr; (PKC&egr;) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKC&egr; remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKC&egr; and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKC&egr; with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKC&egr; complex at ≈2%, ≈0.2%, and ≈1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKC&egr; can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKC&egr; resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKC&egr; in mice, which is cardioprotective, greatly increased interaction of PKC&egr; with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKC&egr; did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKC&egr; transgenesis. Collectively, these data demonstrate that PKC&egr; forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKC&egr;-induced cardioprotection.

Mitochondrial PKCε and MAPK Form Signaling Modules in the Murine Heart. Enhanced Mitochondrial PKCε-MAPK Interactions and Differential MAPK Activation in PKCε-Induced Cardioprotection

Although activation of protein kinase C (PKC) epsilon and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCepsilon signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCepsilon and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCepsilon, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCepsilon greatly increased mitochondrial PKCepsilon expression and activity, which was concomitant with increased mitochondrial interaction of PKCepsilon with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCepsilon activity, as the interactions were also observed in mice expressing inactive PKCepsilon. However, although both active and inactive PKCepsilon bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCepsilon but not in mice expressing inactive PKCepsilon. Examination of potential downstream targets of mitochondrial PKCepsilon-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCepsilon forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCepsilon-ERK modules appears to play a role in PKCepsilon-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.

Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure

Loss of cardiac myocytes in heart failure is thought to occur largely through an apoptotic process. Here we show that heart failure can also be precipitated through myocyte necrosis associated with Ca2+ overload. Inducible transgenic mice with enhanced sarcolemmal L-type Ca2+ channel (LTCC) activity showed progressive myocyte necrosis that led to pump dysfunction and premature death, effects that were dramatically enhanced by acute stimulation of beta-adrenergic receptors. Enhanced Ca2+ influx-induced cellular necrosis and cardiomyopathy was prevented with either LTCC blockers or beta-adrenergic receptor antagonists, demonstrating a proximal relationship among beta-adrenergic receptor function, Ca2+ handling, and heart failure progression through necrotic cell loss. Mechanistically, loss of cyclophilin D, a regulator of the mitochondrial permeability transition pore that underpins necrosis, blocked Ca2+ influx-induced necrosis of myocytes, heart failure, and isoproterenol-induced premature death. In contrast, overexpression of the antiapoptotic factor Bcl-2 was ineffective in mitigating heart failure and death associated with excess Ca2+ influx and acute beta-adrenergic receptor stimulation. This paradigm of mitochondrial- and necrosis-dependent heart failure was also observed in other mouse models of disease, which supports the concept that heart failure is a pleiotropic disorder that involves not only apoptosis, but also necrotic loss of myocytes in association with dysregulated Ca2+ handling and beta-adrenergic receptor signaling.

Genetic and pharmacologic inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy

Muscular dystrophies comprise a diverse group of genetic disorders that lead to muscle wasting and, in many instances, premature death. Many mutations that cause muscular dystrophy compromise the support network that connects myofilament proteins within the cell to the basal lamina outside the cell, rendering the sarcolemma more permeable or leaky. Here we show that deletion of the gene encoding cyclophilin D (Ppif) rendered mitochondria largely insensitive to the calcium overload–induced swelling associated with a defective sarcolemma, thus reducing myofiber necrosis in two distinct models of muscular dystrophy. Mice lacking δ-sarcoglycan (Scgd−/− mice) showed markedly less dystrophic disease in both skeletal muscle and heart in the absence of Ppif. Moreover, the premature lethality associated with deletion of Lama2, encoding the α-2 chain of laminin-2, was rescued, as were other indices of dystrophic disease. Treatment with the cyclophilin inhibitor Debio-025 similarly reduced mitochondrial swelling and necrotic disease manifestations in mdx mice, a model of Duchenne muscular dystrophy, and in Scgd−/− mice. Thus, mitochondrial-dependent necrosis represents a prominent disease mechanism in muscular dystrophy, suggesting that inhibition of cyclophilin D could provide a new pharmacologic treatment strategy for these diseases.

New Horizons in Cardioprotection Recommendations From the 2010 National Heart, Lung, and Blood Institute Workshop

Coronary heart disease is the largest major killer of American men and women and accounted for 1 of every 6 deaths in the United States in 2007.1 The annual incidence of myocardial infarction in the United States is estimated to be 935 000, with 610 000 new cases and 325 000 recurrent attacks. Survivors have a much higher chance of suffering from congestive heart failure, arrhythmias, and sudden cardiac death. Prognosis after an acute myocardial ischemic injury is primarily dependent on the amount of myocardium that undergoes irreversible injury.2–4 Large transmural infarcts yield a higher probability of cardiogenic shock, arrhythmias, adverse remodeling, and development of late chronic heart failure. Although it has been known since the early 1970s that the size of a myocardial infarction can be modified by various therapeutic interventions,5 early coronary artery reperfusion by fibrinolysis or percutaneous coronary intervention, including balloon angioplasty with or without stenting, remains the only established intervention capable of consistently reducing infarct size in humans. Although reperfusion has led to significant advances in patient care and reduction in hospital mortality, delays in seeking medical attention and inherent limitations in initiating fibrinolysis or percutaneous coronary intervention dictate that additional substantive improvements in morbidity and mortality can be achieved only with the development of new adjunctive therapies coupled with reperfusion. In addition, reperfusion therapy itself may induce reperfusion injury, a phenomenon that may encompass stunned myocardium, no-reflow phenomenon, and lethal myocardial cell death. If this injury could be prevented or minimized by administration of adjunctive therapy, then the net benefit of reperfusion could be enhanced. The problem of acute ischemic injury and myocardial infarction is not limited to patients with acute coronary artery syndrome. It remains a major problem in cardiac surgery as well. It is well documented that the incidence of myocardial necrosis after surgery, as determined by creatine kinase MB enzyme release and troponin levels, ranges somewhere between 40% and 60%, and, depending on its clinical definition, the incidence of myocardial infarction after coronary artery bypass graft surgery may be as high as 19%. The intermediate and long-term implications are considerable. In a recent retrospective analysis of 18 908 patients who underwent coronary artery bypass graft surgery and in whom long-term follow-up was available, it was shown that myocardial enzyme elevation within the first 24 hours of surgery was associated with increasing mortality over the course of months to years. This study confirms earlier reports that even small enzyme elevations after surgery are associated with worse long-term outcomes.4

The mitochondrial permeability transition pore and ischemia-reperfusion injury.

Mitochondrial dysfunction is an underlying cause of ischemia-reperfusion injury. In particular, ischemic injury induces dramatic increases in mitochondrial permeability, thereby instigating a chain of events that leads to both apoptotic and necrotic cardiomyocyte death. The mitochondrial permeability transition (MPT) pore, a large, non-specific channel that spans the inner mitochondrial membrane, is known to mediate the lethal permeability changes that initiate mitochondrial-driven cardiomyocyte death. The purpose of this review is to focus on the role of the MPT pore in ischemia-reperfusion injury, the mechanisms involved, and, in particular, what we do and do not know regarding the pore’s molecular composition.

Regulation of Mitochondrial Respiratory Chain Biogenesis by Estrogens/Estrogen Receptors and Physiological, Pathological and Pharmacological Implications

There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17beta-estradiol (E(2)) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E(2)-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERalpha and ERbeta and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anti-cancer drug resistance in human breast cancer cells, neuroprotection for Alzheimers disease and Parkinsons disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area. We also suggest a novel conceptual approach that will contribute to innovative regimens for the prevention or treatment of a wide variety of medical complications based on E(2)/ER-mediated MRC biogenesis pathway.