Regulation of mitochondrial biogenesis during myogenesis

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Abstract

Pathways involved in mitochondrial biogenesis associated with myogenic differentiation are poorly defined. Therefore, C2C12 myoblasts were differentiated into multi-nucleated myotubes and parameters/regulators of mitochondrial biogenesis were investigated. Mitochondrial respiration, citrate synthase- and β-hydroxyacyl-CoA dehydrogenase activity as well as protein content of complexes I, II, III and V of the mitochondrial respiratory chain increased 4–8-fold during differentiation. Additionally, an increase in the ratio of myosin heavy chain (MyHC) slow vs MyHC fast protein content was observed. PPAR transcriptional activity and transcript levels of PPAR-α, the PPAR co-activator PGC-1α, mitochondrial transcription factor A and nuclear respiratory factor 1 increased during differentiation while expression levels of PPAR-γ decreased. In conclusion, expression and activity levels of genes known for their regulatory role in skeletal muscle oxidative capabilities parallel the increase in oxidative parameters during the myogenic program. In particular, PGC-1α and PPAR-α may be involved in the regulation of mitochondrial biogenesis during myogenesis.

Introduction

Intrinsically, skeletal muscle is a tissue characterized by a high level of plasticity. The potential to alter muscle size, metabolic properties and/or protein isoform expression gives skeletal muscle the ability to adapt to the different challenges that may be placed upon it (Baldwin and Haddad, 2002). Events triggering skeletal muscle adaptive changes include endurance- and strength training, overload, aging, disuse, starvation, chronic illness and degenerative disorders (Fluck, 2006, Narici et al., 2004, Gosker et al., 2002). Growth and repair (regeneration) of skeletal muscle tissue are inextricably linked to the action of a group of myogenic precursor cells, called satellite cells. Upon activation, these cells proliferate, differentiate and ultimately fuse with existing myofibers (Anderson, 2006). The irreversible transition from the proliferation competent myoblast stage into fused multi-nucleated myotubes is known as myogenic differentiation and recapitulates developmental and regenerative myogenesis (Le Grand and Rudnicki, 2007).

Oxidative phenotype (OXPHEN) of a muscle depends on its oxidative capacity (determined by the activity of mitochondrial enzymes involved in oxidative substrate metabolism) and fiber type composition (e.g. ratio type I oxidative (slow-twitch) vs type IIB/X glycolytic (fast-twitch) fibers). The peroxisome proliferator-activated receptors (PPARs), especially the PPAR-δ and PPAR-α isoforms, and related co-activator molecules as PGC-1α are highly implicated in regulation of skeletal muscle OXPHEN (Luquet et al., 2003, Koves et al., 2005). Accordingly, muscle-specific over-expression of PPAR-δ or PGC-1α in mice potently up-regulated oxidative capabilities of the muscle and mediated a fiber type shift towards an increased OXPHEN (Lin et al., 2002, Wang et al., 2004). Moreover, PGC-1α and the PPARs govern mitochondrial biogenesis through control over other regulatory proteins including nuclear respiratory factor 1 (NRF-1) and the master mitochondrial regulator mitochondrial transcription factor A (Tfam) (Gleyzer et al., 2005, Wu et al., 1999). An improved muscle OXPHEN may be established during the process of myogenesis (i.e. the formation, repair or hypertrophy of muscle), or through alterations within the pre-existing muscle fibers, independent of myogenic differentiation. The regulation of oxidative profile in existing muscle fibers has been described quite extensively in the literature (Lin et al., 2002, Wang et al., 2004, Canto et al., 2007) whereas knowledge regarding the development of muscle oxidative profile and the involvement of key regulators herein during myogenic differentiation is rather limited.

Studying the molecular events governing regulation of mitochondrial biogenesis during myogenesis is of clinical relevance for a number of pathological conditions including chronic inflammatory myopathies and degenerative muscle conditions as e.g. Duchenne's muscular dystrophy (Oexle and Kohlschutter, 2001, Wallace and McNally, 2009), but also in conditions characterized by loss of OXPHEN in skeletal muscle, such as COPD (Gosker et al., 2002).

It has been shown that mitochondria play a mechanistic role not only in myoblast proliferation (Duguez et al., 2004, Rochard et al., 2000), but also in myogenesis by targeting key regulators of myogenic differentiation as myogenin (Rochard et al., 2000) and cellular oncogens as c-myc (Seyer et al., 2006). Moreover, blocking mitochondrial biogenesis potently inhibits myogenic differentiation (Herzberg et al., 1993, Rochard et al., 2000). This demonstrates that mitochondria display a retrograde signaling to regulate their own biogenesis as well as cell cycle progression (Jahnke et al., 2009), which subsequently implies that the investigation of myogenic differentiation is inevitably coupled to studying the process of mitochondrial biogenesis.

Although markers of OXPHEN (including mitochondrial biogenesis) during myogenesis have been described previously (Moyes et al., 1997, Rochard et al., 2000, Duguez et al., 2002, Duguez et al., 2004, Kraft et al., 2006, Jahnke et al., 2009), information about the expression profile of key regulators during the entire myogenic program is incomplete. Therefore, the aim of the present study was to chart the development of multiple aspects of mitochondrial biogenesis during the full length of the differentiation process of skeletal muscle cells in order to increase our understanding of the key regulatory molecules of OXPHEN during myogenesis.

Section snippets

Cell culture

The murine skeletal muscle cell line C2C12 was obtained from the American Type Culture Collection (ATCC CRL1772; Manassas, VA, USA). These cells are able to undergo differentiation into spontaneously contracting myotubes after growth factor withdrawal (Yaffe and Saxel, 1977). Myoblasts were cultured in growth medium (GM) composed of low-glucose Dulbecco's Modified Eagle's medium (DMEM) containing antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) and 9% (v/v) foetal bovine serum (FBS)

Markers of myogenic differentiation

In order to characterize myogenic differentiation of C2C12 cells we compared biochemical and structural markers of differentiation in myoblasts vs differentiating/differentiated myotubes. Results are summarized in Table 2 and are illustrative of good morphological and biochemical differentiation.

Mitochondrial respiration in myoblasts and myotubes

To investigate the development of oxidative capacity during C2C12 myogenesis, we measured mitochondrial O2 consumption in proliferating C2C12 myoblasts and differentiated myotubes (differentiated 7

Discussion

In the present paper we show that multiple key determinants of OXPHEN develop during myogenic differentiation, which coincides with the initiation of mitochondrial biogenesis and increased expression-and activity levels of key regulators of muscle OXPHEN.

Funding

The research of Dr. H. Gosker is supported by a grant from the Netherlands Asthma Foundation (NAF 3.4.05.038). The research of Dr. P. Schrauwen has been made possible by fellowships of the Royal Netherlands Academy of Arts and Sciences. Dr. Langen was sponsored by a VENI grant (VENI 916.56.112). A. Remels is supported by a grant from Numico Research.

Disclosures

Funding sources had no involvement in collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The authors would like to disclose that there are no advisory board affiliations or financial or personal interests in any organization sponsoring the research and accept full responsibility for conduct of the study and the decision to publish the present work. We declare that the manuscript, including related data,

Acknowledgements

The authors like to thank E. Phielix for excellent technical support in measurements of cellular respiration and M. Kelders for technical assistance in QPCR analysis.

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