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.

Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions

“…Briefly, cardiac mitochondria were isolated from adult (8-10 week old, ICR strain) mouse hearts in isolation buffer (250mM sucrose, 1mM EGTA, 20mM HEPES, pH7.5) with protease inhibitors cocktail and two different phosphate inhibitors cocktails. For purification, the crude mitochondrial pellet was resuspended in 19% Percoll in isolation buffer, and slowly layered on two layers of 30% and 60% Percoll (v/v) (18). After centrifugation at 10,000g for 15min, mitochondria were collected between the 30% and 60% layers.…”

“…Cardiac mitochondria were isolated from mouse hearts by differential centrifugation according to a standard procedure in our group (18)(19)(20). Briefly, cardiac mitochondria were isolated from adult (8-10 week old, ICR strain) mouse hearts in isolation buffer (250mM sucrose, 1mM EGTA, 20mM HEPES, pH7.5) with protease inhibitors cocktail and two different phosphate inhibitors cocktails.…”

“…Percoll purified mitochondria were washed (3×) prior subjecting them to further analyses. Mitochondrial intactness was measured by cytochrome c oxidase activity (18). All procedures were performed at 4°C.…”

“…In particular, it is unknown whether ischemic injury triggers differential alterations of protein clusters in various sub-mitochondrial compartments, whether changes of mitochondrial subproteomes vary with the severity of injury, and whether these proteomic changes contribute to the subsequent cellular adaptive responses to myocardial ischemic injury. Using functionally validated cardiac mitochondria, our recent investigation (18) provided a comprehensive mitochondrial proteome atlas. On the basis of this mitochondrial protein dataset, we designed a pre-clinical study to carefully address the above questions.…”

Proteomic insights into cardiac cell death and survival

“…This is one area in which proteomics is making significant inroads. The past several years have seen several large-scale studies tackling the mitochondrial proteomes of mice [15,16] and humans [17]. What has emerged from these studies is a portrait of the organelle as the major hub of energy-producing proteins (as expected) that is littered with stress-proteins, cell deathrelated molecules and signaling proteins.…”