Modulation of mitochondrial proteome and improved mitochondrial function by biventricular pacing of dyssynchronous failing hearts

Abstract

Background: Cardiac resynchronization therapy (CRT) improves chamber mechanoenergetics and morbidity and mortality of patients manifesting heart failure with ventricular dyssynchrony; however, little is known about the molecular changes underlying CRT benefits. We hypothesized that mitochondria may play an important role because of their involvement in energy production.

Methods and results: Mitochondria isolated from the left ventricle in a canine model of dyssynchronous or resynchronized (CRT) heart failure were analyzed by a classical, gel-based, proteomic approach. Two-dimensional gel electrophoresis revealed that 31 mitochondrial proteins where changed when controlling the false discovery rate at 30%. Key enzymes in anaplerotic pathways, such as pyruvate carboxylation and branched-chain amino acid oxidation, were increased. These concerted changes, along with others, suggested that CRT may increase the pool of Krebs cycle intermediates and fuel oxidative phosphorylation. Nearly 50% of observed changes pertained to subunits of the respiratory chain. ATP synthase-beta subunit of complex V was less degraded, and its phosphorylation modulated by CRT was associated with increased formation (2-fold, P=0.004) and specific activity (+20%, P=0.05) of the mature complex. The importance of these modifications was supported by coordinated changes in mitochondrial chaperones and proteases. CRT increased the mitochondrial respiratory control index with tightened coupling when isolated mitochondria were reexposed to substrates for both complex I (glutamate and malate) and complex II (succinate), an effect likely related to ATP synthase subunit modifications and complex quantity and activity.

Conclusions: CRT potently affects both the mitochondrial proteome and the performance associated with improved cardiac function.

Figure 1

Mitochondrial proteome. Representative silver stained 2DE gels on 2 pI ranges: 4 to 7 and 6 to 11. The pI (based on linear distribution) and MW (based on MW markers) values are shown on the top and on the sides, respectively. Proteins are numbered according to Table 2.

Figure 2

Protein changes with CRT. Magnified spot images from CRT and DHF representative gels are presented along with plots of their normalized, background subtracted volume. Protein spots are numbered according to Table 2. Protein names with grey backgrounds are those that increased with CRT.

Figure 3

Characterization of ATP-β posttranslational modifications. A, Distribution of changing ATP-β on the 2DE gels. The unmodified form (marked with a thicker arrow) and fragments are indicated by arrows and accompanied by fold change values. B, Representative dephosphorylation gel for ATP-β. DHF is shown in red (Cy5), CRT in green (Cy3), and the AP-treated pool (AP) in blue (Cy2). C, Representative BN-PAGE/Western blot for ATP-β. Biological replicates are shown (3 sham-operated controls, 5 DHF, and 4 CRT). D, Typical MS/MS profile for the phosphopeptide FT*QAGSEVSALLGR. The MS/MS spectrum is provided, together with a schematic in which the mass values for all b- and y-ions series are reported.

Figure 4

Mitochondrial bioenergetic parameters. Mitochondrial oxygen consumption was measured in the presence of glutamate/ malate (A) and succinate (B) as substrates. Respiration was monitored in basal state, or state 4 (S4); state 3 (S3), induced by the addition of 300 μM ADP; and uncoupled state, induced by the addition of protonophore 2,4-dinitrophenol (10 μM, uncoupled state). C, Respiratory control ratios. D, ADP/oxygen ratios (*P<0.05, **P<0.001 vs CRT, ‡P<0.05, and ‡‡P<0.005 vs shams). E, Representative image of the in-gel ATPase assay (n=3). Specific activity is reported as the density of white PbHPO₄ bands (product) normalized for the amount of complex V as indicated by coomassie blue G250 stain (*P=0.02 vs shams and P=0.05 vs DHF).

Figure 5

Schema of mitochondrial protein changes with CRT. For values, refer to Table 2. The pI indicates observed pI differs >1pH unit from predicted one; MW, observed MW differs more than 10 kDa from predicted one. FAD indicates flavin-adenine dinucleotide; FADH₂, flavin-adenine dinucleotide, reduced form; NAD⁺, oxidized nicotinamide-adenine dinucleotide; P_i, inorganic phosphate; and TCA, tricarboxylic acid. See Table 2 for other abbreviations.