Alterations in skeletal muscle indicators of mitochondrial structure and biogenesis in patients with type 2 diabetes and heart failure: effects of epicatechin rich cocoa

Abstract

(-)-Epicatechin (Epi), a flavanol in cacao stimulates mitochondrial volume and cristae density and protein markers of skeletal muscle (SkM) mitochondrial biogenesis in mice. Type 2 diabetes mellitus (DM2) and heart failure (HF) are diseases associated with defects in SkM mitochondrial structure/function. A study was implemented to assess perturbations and to determine the effects of Epi-rich cocoa in SkM mitochondrial structure and mediators of biogenesis. Five patients with DM2 and stage II/III HF consumed dark chocolate and a beverage containing approximately 100 mg of Epi per day for 3 months. We assessed changes in protein and/or activity levels of oxidative phosphorylation proteins, porin, mitofilin, nNOS, nitric oxide, cGMP, SIRT1, PGC1α, Tfam, and mitochondria volume and cristae abundance by electron microscopy from SkM. Apparent major losses in normal mitochondria structure were observed before treatment. Epi-rich cocoa increased protein and/or activity of mediators of biogenesis and cristae abundance while not changing mitochondrial volume density. Epi-rich cocoa treatment improves SkM mitochondrial structure and in an orchestrated manner, increases molecular markers of mitochondrial biogenesis resulting in enhanced cristae density. Future controlled studies are warranted using Epi-rich cocoa (or pure Epi) to translate improved mitochondrial structure into enhanced cardiac and/or SkM muscle function.

Figure 1

Comparison of changes observed in protein or enzyme activity levels before (b) and after (a) treatment with Epi‐rich cocoa. (A) neuronal‐NOS and phosphorylated‐NOS. Western blots were normalized against S6RP. (B) Changes in skeletal muscle NO production (measured as nitrates/nitrites) and (C) changes in cGMP levels. Significant differences (*p < 0.05) were observed before vs. after treatment by paired t‐test.

Figure 2

Comparison of changes observed in protein levels before (b) and after (a) treatment with Epi‐rich cocoa in: (A) SIRT1, (B) PGC1α (including its acetylated form) and (C); Tfam. PGC1α was immunoprecipitated with an antibody against total PGC1α and the blot probed with an anti‐acetyl‐lysine antibody to detect protein acetylation (a‐). Western blots were normalized against S6RP. Significant changes (*p < 0.05) were observed in all endpoints by paired t‐test.

Figure 3

Comparison of changes observed in protein levels before (b) and after (a) treatment with Epi‐rich cocoa in: (A) Mitofilin and porin and, (B) complex I and complex V. Western blots were normalized against S6RP. Significant changes (*p < 0.05) were observed in all endpoints by paired t‐test.

Figure 4

Microphotographs illustrating representative changes observed in mitochondrial structure of one patient before and after treatment with Epi‐rich cocoa. Plots summarize changes observed in mitochondrial cristae abundance and volume density in all patients before and after Epi‐rich cocoa treatment. *p < 0.05 by paired t‐test.