(-)-Epicatechin induces mitochondrial biogenesis and markers of muscle regeneration in adults with Becker muscular dystrophy

Abstract

Introduction: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD).

Methods: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests.

Results: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states.

Discussion: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.

Keywords: aerobic exercise, Becker muscular dystrophy, epicatechin, mitochondrial biogenesis follistatin.

FIGURE 1

Changes in bioenergetic regulators including LKB1, AMPK, PGC‐1α, and the mitochondrial cristae–associated protein mitofillin after 8 weeks of (−)‐epicatechin treatment by western blot. GAPDH was used as a loading control. Data are mean ± standard error of the mean (n = 6). Significant increases were observed on western blots for LKB1 (A), AMPK (B), PGC‐1α (C), and mitofillin (D). E, Representative western blots. AMPK, 5′‐adenosine monophosphate–activated protein kinase; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; LKB1, liver kinase B1; PGC‐1α, peroxisome proliferator‐activated receptor gamma coactivator‐1alpha

FIGURE 2

Changes in mitochondrial structure–related endpoints with 8 weeks of (−)‐epicatechin treatment. A, Cristae density quantified using imaging software. B and C, Quantitative results derived using electron microscopy. Data are expressed as mean ± standard error of the mean (n = 4). Based on EM imaging analysis (A), nonsignificant increases in mitochondrial volume were observed in three of four subjects (B). C, Significant increases in mitochondria cristae were observed for all four subjects

FIGURE 3

Changes in regulators of skeletal muscle growth regulators and muscle fiber contractile apparatus with 8 weeks of (−)‐epicatechin treatment by western blot. Glyceraldehyde 3‐phosphate dehydrogenase was used as loading control. Data are mean ± standard error of the mean (n = 6). There were significant increases in the western blot–determined growth‐modulating factors follistatin (A), while suppressing myostatin (B). In addition, the structural contractile proteins showed significant increases on western blot, including myosin (C) and actin α1 (D). Representative western blots are shown in E. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; SkM, skeletal muscle

FIGURE 4

Changes in skeletal muscle regeneration–related proteins by western blots with (−)‐epicatechin treatment. Glyceraldehyde 3‐phosphate dehydrogenase was used as loading control. Data are expressed as mean ± standard error of the mean (n = 6).There were significant increases in tissue markers known to participate in the activation of skeletal muscle regeneration including Myf5 (A), MyoD (B), myogenin (C), and MEF2a (D). Representative western blots are shown in E. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; MEF2a, myocyte enhancer factor‐2; Myf5, myogenic regulatory factor‐5; MyoD, myoblast determination protein 1

FIGURE 5

Changes on western blots in skeletal muscle proteins, including dysferlin, utrophin‐NH, and intracellular skeletal muscle creatine kinase, before treatment and after 8 weeks of (−)‐epicatechin. Glyceraldehyde 3‐phosphate dehydrogenase was used as loading control. Data are expressed as mean ± standard error of the mean (n = 6). There were significant increases in dysferlin (A), Utrophin‐NH (B), and intracellular creatine kinase (C). Representative Western blots are shown in D. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; SkM, skeletal muscle

FIGURE 6

Results from graded exercise testing with recumbent cycle ergometer at defined work outputs (measured in watts, X axis) after 8 weeks of (−)‐epicatechin treatment indicating reduced energy cost of exercise and improved muscle energy utilization and respiratory efficiency. A, Heart rate response during graded exercise testing at increasing workloads at baseline and after 8 weeks of treatment. There were statistically significant reductions in exercise‐induced heart rate elevation at workloads of at least 30 W in the 6 subjects studied pre‐ and posttreatment. B, VO₂ per kilogram during graded exercise testing at increasing workloads at baseline and after 8 weeks of treatment. There were statistically significant reductions in VO₂ per kilogram at workloads of at least 30 W. C, Blood lactate levels during graded exercise testing at increasing workloads at baseline and after 8 weeks of treatment. Posttreatment blood lactate levels after treatment were reduced at baseline and significantly throughout the graded exercise test. After treatment, participants demonstrated blood lactate levels that were, on average, 0.47 mmol/L lower than baseline (95% CI, −0.81 to −0.12, P = .009) when controlling for level of work. All data are expressed as mean ± standard error of the mean. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; SkM, skeletal muscle