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Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria.

ABSTRACT: 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.

SUBMITTER: Zhang J

PROVIDER: S-EPMC2799225 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria.

Zhang Jun J Li Xiaohai X Mueller Michael M Wang Yueju Y Zong Chenggong C Deng Ning N Vondriska Thomas M TM Liem David A DA Yang Jeong-In JI Korge Paavo P Honda Henry H Weiss James N JN Apweiler Rolf R Ping Peipei P

Proteomics 20080401 8

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. ...[more]

PMID: 18348319

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Project description: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.

Project description:KEY POINTS:Association of plasma membrane BKCa channels with BK-? subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-? subunits is not established. MitoBKCa -? and the regulatory BK-?1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-?1 (left-shifted voltage dependence of activation, V1/2 = -55 mV, 12 µm matrix Ca2+ ). In BK-?1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+ ) Co-expression of BKCa with the BK-?1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK-?1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. ABSTRACT:Association of the plasma membrane BKCa channel with auxiliary BK-?1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa ) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK-?1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po ) and voltage half-activation (V1/2 = -55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK-?1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -? and its BK-?1 subunit. Mitochondria from the BK-?1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+ ). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK-?1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-?1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-?1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK-?1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.

Dataset Information

Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria.

Publications

Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria.

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