Proteome Biology of Cardiac Mitochondria
Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism, and biogenesis. In addition, there are several other clusters, including proteolysis, protein folding, and reduction/oxidation signaling, which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue– and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles.
Murine Cardiac Mitochondrial Proteome
Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.
Mitochondrial Permeability Transition
Mitochondria play a key role in determining cell fate during exposure to stress. Their role during ischemia/reperfusion is particularly critical because of the conditions that promote both apoptosis by the mitochondrial pathway and necrosis by irreversible damage to mitochondria in association with mitochondrial permeability transition (MPT). MPT is caused by the opening of permeability transition pores in the inner mitochondrial membrane, leading to matrix swelling, outer membrane rupture, release of apoptotic signaling molecules such as cytochrome c from the intermembrane space, and irreversible injury to the mitochondria. During ischemia (the MPT priming phase), factors such as intracellular Ca2+ accumulation, long-chain fatty acid accumulation, and reactive oxygen species progressively increase mitochondrial susceptibility to MPT, increasing the likelihood that MPT will occur on reperfusion (the MPT trigger phase). Because functional cardiac recovery ultimately depends on mitochondrial recovery, cardioprotection by ischemic and pharmacological preconditioning must ultimately involve the prevention of MPT. Investigations into this area are beginning to unravel some of the mechanistic links between cardioprotective signaling and mitochondria.