Metabolic Reprogramming Promotes Myogenesis During Aging

Abstract

Sarcopenia is the age-related progressive loss of skeletal muscle mass and strength finally leading to poor physical performance. Impaired myogenesis contributes to the pathogenesis of sarcopenia, while mitochondrial dysfunctions are thought to play a primary role in skeletal muscle loss during aging. Here we studied the link between myogenesis and metabolism. In particular, we analyzed the effect of the metabolic modulator trimetazidine (TMZ) on myogenesis in aging. We show that reprogramming the metabolism by TMZ treatment for 12 consecutive days stimulates myogenic gene expression in skeletal muscle of 22-month-old mice. Our data also reveal that TMZ increases the levels of mitochondrial proteins and stimulates the oxidative metabolism in aged muscles, this finding being in line with our previous observations in cachectic mice. Moreover, we show that, besides TMZ also other types of metabolic modulators (i.e., 5-Aminoimidazole-4-Carboxamide Ribofuranoside-AICAR) can stimulate differentiation of skeletal muscle progenitors in vitro. Overall, our results reveal that reprogramming the metabolism stimulates myogenesis while triggering mitochondrial proteins synthesis in vivo during aging. Together with the previously reported ability of TMZ to increase muscle strength in aged mice, these new data suggest an interesting non-invasive therapeutic strategy which could contribute to improving muscle quality and neuromuscular communication in the elderly, and counteracting sarcopenia.

Keywords: aging; metabolic reprogramming; metabolism; mitochondria; myogenesis; neuromuscular activity; sarcopenia; trimetazidine.

FIGURE 1

Trimetazidine stimulates myogenic and mitochondrial gene expression. (A) Gastrocnemius extracts from untreated aged mice (Ctrl) and TMZ-treated aged mice (TMZ) were assayed for MyoD Myogenin and Desmin protein levels, (B) for the mRNA levels of MyoD, Myogenin, Desmin, MyHC, PGC1α, GLUT4, and CPT1 which were evaluated by qRT-PCR, and (C) for β-ATPase, TFAM, Tom20, PGC1α, and α-SMA protein levels. Protein levels of representative four out of six untreated mice and five out of six TMZ-treated mice are shown. (D) Heart extracts from untreated aged mice (Ctrl) and TMZ-treated aged mice (TMZ) were assayed for PGC1α, β-ATPase, TFAM, and Tom20 protein levels and (E) for CSQ2 and fsTn-I protein levels. Protein levels of representative four out of six untreated mice and 4/5 out of six TMZ-treated mice are shown. (F) Heart RNA extracted from untreated aged mice (Ctrl) and TMZ-treated aged mice (TMZ) was assayed for the mRNA levels of α-MyHC and β/slow-MyHC which were evaluated by qRT-PCR. In all WB density of immunoreactive bands was calculated using the ImageQuant TL software from GE Healthcare Life normalized for α-tubulin or actin used as loading control. Each value indicates the mean ± SEM (reported as percentage of Ctrl) of the densitometric analysis on three independent immunoblots. In all qRT-PCRs, data were normalized to 18S ribosomal RNA used as internal control. Data display the percentage of mRNAs relative to control. Data shown are the mean ± SEM from three experiments each performed in duplicate. For all experiments ^*p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 by Student′s t-test or Mann–Whitney U test as appropriate. Primers used: 18S: Fw-5′-CCCTGCCCTTTGTACACACC-3′ Rv-5′-CGA TCCGAGGGCCTCACTA-3′; MyoD Fw-5′-CCCCGGCGGCAGAATGGCTACG-3′ Rv-5′-GGTCTGGGTTCCCTGTTCTGTG-3′; Myogenin: Fw-5′-GGGCCCCTGGAA GAAAAG-3′ Rv-5′-AGGAGGCGCTGTGGGAGT-3′; Desmin: Fw-5′-GAGGTTGTCAGCGAGGCTAC-3′ Rv-5′-GAAAAGTGGCTGGGTGTGAT-3′; MyHC Fw-5′-CAAG TCATCGGTGTTTGTGG-3′ Rv-5′-TGTCGTACTTGGGAGGGTTC-3′; CTP1: Fw-5′-CCCATGTGCTCCTACCAGAT-3 Rv-5-CCTTGAAGAAGCGACCTTTG-3; PGC1α: Fw-5′-GTCAACAGCAAAAGCCACAA-3 Rv-5′-TCTGGGGTCAGAGGAAGAGA-3′; GLUT-4: Fw-5′-GGCATGGGTTTCCAGTATGT-3′ Rv-5′-GCCCCTCAGTCATTC TCATG-3′; α-MyHC: Fw-5′-AACAACCCATACGACTACGCC-3′ Rv-5′-CAGCATCTTCTGTGCCATCA-3′; β/slow-MyHC Fw-5′-TGCAGCAGTTCTTCAACCAC-3′ Rv-5′-TCGAGGCTTCTGGAAGTTGT-3′; VEGF: Fw-5′-CTGTGCAGGCTGCTGTAACG-3′ Rv-5′-GTTCCCGAAACCCTGAGGAG-3′; MyHC: Fw-5′-TCGTCTCGCTT TGGCAA-3′ Rv-5′-TGGTCGTAATCAGCAGCA-3′.

FIGURE 2

Similarly to TMZ, also AICAR stimulates myogenic differentiation. (A) The mRNA levels of Myogenin and PGC1a were evaluated by quantitative real time PCR in C2C12 myoblasts differentiating for 4 and 18 h with or without 0.5 mM AICAR. The mRNA expression values were normalized to those of 18S ribosomal RNA, used as internal control, and are displayed as folds relative to a plate reference control sample (0 h in differentiation medium, not shown). Data shown are the mean ± SEM from four experiments each performed in triplicate. ^∗∗p ≤ 0.01 by two-tailed Student’s t-test. (B) The mRNA levels of MyoD, Myogenin, and MyHC were evaluated by quantitative real time PCR in primary satellite cells cultured for 24 h and then treated with 0.5 mM AICAR for further 24 h (total 48 h in culture), or cultured for 48 h and then treated with 0.5 mM AICAR for further 4 or 24 h (total 52 or 72 h in culture, respectively). The mRNA expression values were normalized to 18S RNA used as internal control, and are displayed as folds relative to a plate reference control sample (48 h in culture without AICAR treatment). Data shown are the mean ± SEM from three experiments, each performed in triplicate. ^*p < 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.005 by two-tailed Student’s t-test. (C) Satellite cells were cultured for 6 h and then treated with or without 0.5 mM AICAR for 10 h before fixation and co-immunostaining for myogenin (red) and Pax7 (green). The graph represents the quantification of myogenin⁺/Pax7^– , myogenin⁺/Pax7⁺, or myogenin^– /Pax7⁺ cells. Data are the means ± SEM from three experiments and are expressed as the percentage in each category of the total positive cells. For each experiment, at least 10 microscopy fields were analyzed for a total of at least 1000 cells counted for each condition. (D,E) Satellite cells were cultured in 35 mm collagen-coated dishes for 12 h and then treated with or without 0.5 mM AICAR for 22, 40, or 70 h before fixing and immunostaining for MyHC (green). Counterstaining with Hoechst was used to visualize all nuclei (blue). The graph represents the quantification of fusion index at 34 and 52 h. Data are from three independent experiments. Values are expressed as the mean ratio ± SEM of nuclei present in myotubes, containing at least three nuclei, to the total number of nuclei. Asterisks denote significance (^∗∗p < 0.01, ^∗∗∗p < 0.001). Scale bar: 50 μm.