Cardiac mitochondrial proteome dynamics with heavy water reveals stable rate of mitochondrial protein synthesis in heart failure despite decline in mitochondrial oxidative capacity

Abstract

We recently developed a method to measure mitochondrial proteome dynamics with heavy water ((2)H2O)-based metabolic labeling and high resolution mass spectrometry. We reported the half-lives and synthesis rates of several proteins in the two cardiac mitochondrial subpopulations, subsarcolemmal and interfibrillar (SSM and IFM), in Sprague Dawley rats. In the present study, we tested the hypothesis that the mitochondrial protein synthesis rate is reduced in heart failure, with possible differential changes in SSM versus IFM. Six to seven week old male Sprague Dawley rats underwent transverse aortic constriction (TAC) and developed moderate heart failure after 22weeks. Heart failure and sham rats of the same age received heavy water (5% in drinking water) for up to 80days. Cardiac SSM and IFM were isolated from both groups and the proteins were separated by 1D gel electrophoresis. Heart failure reduced protein content and increased the turnover rate of several proteins involved in fatty acid oxidation, electron transport chain and ATP synthesis, while it decreased the turnover of other proteins, including pyruvate dehydrogenase subunit in IFM, but not in SSM. Because of these bidirectional changes, the average overall half-life of proteins was not altered by heart failure in both SSM and IFM. The kinetic measurements of individual mitochondrial proteins presented in this study may contribute to a better understanding of the mechanisms responsible for mitochondrial alterations in the failing heart.

Keywords: Cardiac failure; Deuterium; Mitochondria; Proteome dynamics.

Figure 1

Mitochondrial yield for isolated subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) (upper panel) and activity of the citric acid cycle enzyme citrate synthase and the β-oxidation enzyme medium chain acyl-CoA dehydrogenase (MCAD) measured in whole tissue homogenates (n=14/group, * P<0.01, # P<0.001)

Figure 2

State 3 respiration rates in isolated subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) expressed per mg of wet tissue. Values were calculated as the product of the state 3 rate in isolated mitochondria and the mitochondrial yield (n=14/group, * P<0.05, # P<0.005, ^ P<0.000005).

Figure 3

Half-life of proteins in sham and heart failure rats. Each data point represents the half-life of distinct protein calculated using non-linear regression analysis of ²H-labeling of peptides at six time points. The top left panel shows that the protein synthesis rate was slightly, but not significantly slower in IFM than in SSM sham (P=0.08). This trend was not present in animals with heart failure. The bottom panels plot the half-life of protein synthesis for heart failure rats as a function of values in sham animals for SSM (left) and IFM (right).

Figure 4

Effect of the N-terminal end on protein stability. Proteins were grouped based on the N-terminal amino acid of a mature protein. Proteins with the stabilizing amino acids (n=7) have significantly longer half-lives than proteins and destabilizing amino acids (n=40). *P=0.02, **P=0.001.

Figure 5

Comparison of the half-lives of cardiac mitochondrial proteins based on intra-organelle location (left panels) and their metabolic function (right panel) in SSM and IFM from sham and heart failure animals. The data are derived using non-linear regression analysis of peptides ²H-labeling to generate the best-fit curves describing protein turnover (n=6/group), * P<0.05 vs. SSM sham. CAC, citric acid cycle; ETC, electron transfer chain; FAO, fatty acid oxidation. See Table 3 for the list of specific proteins.

Figure 6

Comparison of the half-lives of selected individual cardiac mitochondrial proteins with different metabolic functions in IFM (top panel) and SSM (bottom panel) from sham and heart failure rats. The data are derived from Table 3. 1: Trifunctional protein, 2: carnitinepalmitoyl transferase 1, 3: Dihydropollysine acetyltranferase (a component of pyruvate dehydrogenase complex), 4: Isocitrate Dehydrogenase, 5: NADH Dehydrogenase Flavoprotein, 6: Succinate Dehydrogenase, 7: Citochrome bc, subunit 7, 8: Cytochrome C Oxidase subunit 4 isoform 1, 9: ATP Synthase α, 10: ADP/ATP translocase 2 (n=6/group, ^*P<0.05 vs. sham).