Restorative effects of (+)-epicatechin in a rodent model of aging induced muscle atrophy: underlying mechanisms

Abstract

Sarcopenia is a progressive and generalized age-related skeletal muscle (SkM) disorder characterized by the accelerated loss of muscle mass (atrophy) and function. SkM atrophy is associated with increased incidence of falls, functional decline, frailty and mortality. In its early stage, SkM atrophy is associated with increased pro-inflammatory cytokine levels and proteasome-mediated protein degradation. These processes also link to the activation of atrophy associated factors and signaling pathways for which, there is a lack of approved pharmacotherapies. The objective of this study, was to characterize the capacity of the flavanol (+)-epicatechin (+Epi) to favorably modulate SkM mass and function in a rat model of aging induced sarcopenia and profile candidate mechanisms. Using 23 month old male Sprague-Dawley rats, an 8 weeks oral administration of the +Epi (1 mg per kg per day in water by gavage) was implemented while control rats only received water. SkM strength (grip), treadmill endurance, muscle mass, myofiber area, creatine kinase, lactate dehydrogenase, troponin, α-actin, tumor necrosis factor (TNF)-α and atrophy related endpoints (follistatin, myostatin, NFκB, MuRF 1, atrogin 1) were quantified in plasma and/or gastrocnemius. We also evaluated effects on insulin growth factor (IGF)-1 levels and downstream signaling (AKT/mTORC1). Treatment of aged rats with +Epi, led to significant increases in front paw grip strength, treadmill time and SkM mass vs. controls as well as beneficial changes in makers of myofiber integrity. Treatment significantly reversed adverse changes in plasma and/or SkM TNF-α, IGF-1, atrophy and protein synthesis related endpoints vs. controls. In conclusion, +Epi has the capacity to reverse sarcopenia associated detrimental changes in regulatory pathways leading to improved SkM mass and function. Given these results and its recognized safety and tolerance profile, +Epi warrants consideration for clinical trials.

Figure 1.

Body weights recorded during aging and treatment. Panel A reports on body weight during the time course of the study. Panel B reports weights before treatment and panel C after treatment. Data are mean ± SEM, n=6/group.

Figure 2.

Front limb strength recorded during aging and +Epi treatment. Panel A reports on limb strength during the time course of the study. Panel B reports strength recorded before +Epi treatment and panel C after treatment. Data are mean ± SEM, n=6/group, *p ≤0.05.

Figure 3.

Treadmill times recorded in control and +Epi treated rats at 23, 24 and 25 months (A). Panel B denotes comparisons within and among groups at 1^st and 2^nd trials. Data are mean ± SEM, n=6/group, *p ≤ 0.05, NS = not significant.

Figure 4.

Creatine Kinase (CK) and lactate dehydrogenase (LDH) enzyme activity in aged rats before and after treadmill testing. Values are reported as fold changes vs. before treadmill testing. CK (A) and LDH (B) were evaluated in control and +Epi treated plasma samples before and after treadmill in the 1^st and 2^nd trials. Data are mean ± SEM, n=6/group, *p values ≤ 0.05.

Figure 5.

Effect of +Epi treatment on gastrocnemius mass. Panels A and B illustrate and report on normalized muscle weight recorded in control and Epi groups. Panel C are representative images from samples stained with hematoxilin and eosin (scale bar = 50 μm). Panel D reports on myofiber cross sectional area measured and (E) on average myofiber size distribution. Data are mean ± SEM, n=6/group, *p values ≤ 0.05.

Figure 6.

Effects of aging and +Epi treatment on plasma and gastrocnemius IGF-1 and pathway related endpoints. Panel A plasma illustrates IGF-1 time course values. Panel B reports post +Epi treatment levels of IGF-1 in gastrocnemius (relative protein levels by Westerns vs. control). Panel C reports post-treatment changes in AKT/mTORC1 protein levels (relative vs. control). Data are mean ± SEM, n=6/group, *p values ≤ 0.05.

Figure 7.

Effects of aging and +Epi treatment on plasma and/or gastrocnemius follistatin and myostatin pathway related endpoints. Panel A illustrates myostatin plasma levels with aging and +Epi treatment. Relative gastrocnemius protein level changes (vs. controls) for follistatin and myostatin following treatment are shown in panel B. Panel C reports post-treatment changes in p-SMAD 2/3 protein levels. Data are mean ± SEM, n=6/group, *p values ≤ 0.05.

Figure 8.

Effects of aging and +Epi treatment on plasma and gastrocnemius TNF-α and NFkB levels. Changes in plasma TNF-α levels are shown in panel A. Panel B reports plasma TNF-α levels before and after +Epi treatment. Panel C reports treatment changes in gastrocnemius relative protein levels (by Westerns) for TNF-α and NFkB while panels D and E reports values quantified by ELISA. Data are mean ± SEM, n=6/group, *p values ≤ 0.05.

Figure 9.

Effects of +Epi treatment on protein degradation endpoints. Panel A reports on relative protein changes for total protein ubiquitylation as detected by Westerns, (B) reports on changes in proteasome 20S levels, (C) on proteasome activity (kit) and (D), on protein degradation rates (kit) before and after +Epi treatment. Arbitrary fluorescent unit (AFU). Data are mean ± SEM, n=6/group, *p values ≤ 0.05.

Figure 10.

Effect of +Epi treatment on regulators of muscle atrophy and structural proteins levels. Panel A reports on relative protein levels changes (by Westerns) for MuRF1 and atrogin 1 while panel B, reports on those for troponin I and α1-actin before and after +Epi treatment. Data are mean ± SEM, n=6/group, *p values ≤ 0.05.