Contractile Activity-induced Transcriptional Activation of Cytochrome c Involves Sp1 and Is Proportional to Mitochondrial ATP Synthesis in C2C12 Muscle Cells

Abstract

Contractile activity induces adaptations in the expression of genes encoding skeletal muscle mitochondrial proteins; however, the putative signals responsible for these adaptations remain unknown. We used electrical stimulation (5 Hz, 65 V) of C2C12 muscle cells in culture to define some of the mechanisms involved in contractile activity-induced changes in cytochrome c gene expression. Chronic contractile activity (4 days, 3 h/day) augmented cytochrome c mRNA by 1.6-fold above control cells. This was likely mediated by increases in transcriptional activation, because cells transfected with full-length (؊726 base pairs) or minimal (؊66 base pairs) cytochrome c promoter/chloramphenicol acetyltransferase reporter constructs demonstrated contractile activity-induced 1.5-1.7-fold increases in the absence of contractile activity-induced increases in mRNA stability. Transcriptional activation of the ؊726 promoter was abolished when muscle contraction was inhibited at various subcellular locations by pretreatment with either the Na ؉ channel blocker tetrodotoxin, the intracellular Ca 2؉ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetra(acetoxymethyl) ester, or the myosin ATPase inhibitor 2,3-butanedione monoxime. It was further reduced in unstimulated cells when mitochondrial ATP synthesis was impaired using the uncoupler 2,4-dinitrophenol. Because the contractile activity-induced response was evident within the minimal promoter, electromobility shift assays performed within the first intron (؉75 to ؉104 base pairs) containing Sp1 sites revealed an elevated DNA binding in response to contractile activity. This was paralleled by increases in Sp1 protein levels. Sp1 overexpression studies also led to increases in cytochrome c transactivation and mRNA levels. These data suggest that variations in the rate of mitochondrial ATP synthesis are important in determining cytochrome c gene expression in muscle cells and that this is mediated, in part, by Sp1-induced increases in cytochrome c transcription.

Contractile activity-induced mitochondrial biogenesis and mTORC1

“…Before the first bout of contractile activity, DMSO (Sigma) or 1 M rapamycin (BioShop, Toronto, ON, Canada) was added to the media. The protocol for CCA has been described previously in detail (8,42). Briefly, customized six-well plate lids were outfitted with two platinum wires per well and were submerged into the media of plates containing fully differentiated myotubes.…”

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

“…Similar to primary cultures, confluent myoblast cultures of both cell lines can be induced to form myotubes, but only C2C12 myotubes can be induced to contract by electrical stimulation. Methods have been developed to stimulate C2C12 myotubes chronically or intermittently for up to 4 days using stimulus electrodes integrated in standard culture dishes (718,723). This technique allows a more detailed analysis of the interaction between thyroid hormone and contractile activity on muscle gene expression (718).…”

Coordination of metabolic plasticity in skeletal muscle

“…Several studies have demonstrated that changes in the mRNA expression of transcription factors precede the contractile activity-induced regulation of mitochondrial and metabolic plasticity in skeletal muscle (Connor et al, 2001;Irrcher et al, 2003;Michel et al, 1994;Pilegaard et al, 2003;Xia et al, 1997). These include the induction of a family of immediate early genes (c-fos, c-jun), the early growth response gene-1 (Egr-1), specificity protein 1 (Sp1) and nuclear respiratory factor-1 (NRF-1), which respond to both acute and chronic contractile activity (Abu-Shakra et al, 1993;Connor et al, 2001;Irrcher and Hood, 2004;Michel et al, 1994;Murakami et al, 1998;Neufer et al, 1998;Puntschart et al, 1998;Takahashi et al, 1993).…”

“…Several studies have demonstrated that changes in the mRNA expression of transcription factors precede the contractile activity-induced regulation of mitochondrial and metabolic plasticity in skeletal muscle (Connor et al, 2001;Irrcher et al, 2003;Michel et al, 1994;Pilegaard et al, 2003;Xia et al, 1997). These include the induction of a family of immediate early genes (c-fos, c-jun), the early growth response gene-1 (Egr-1), specificity protein 1 (Sp1) and nuclear respiratory factor-1 (NRF-1), which respond to both acute and chronic contractile activity (Abu-Shakra et al, 1993;Connor et al, 2001;Irrcher and Hood, 2004;Michel et al, 1994;Murakami et al, 1998;Neufer et al, 1998;Puntschart et al, 1998;Takahashi et al, 1993). Egr-1 and Sp1 are known to be involved in regulating the transcription of cytochrome c, a nuclear-encoded protein of the electron transport chain (Connor et al, 2001;Freyssenet et al, 2004), while NRF-1 is involved in the transcriptional activation of an even greater diversity of nuclear genes encoding mitochondrial proteins (Kelly and Scarpulla, 2004), including the newly discovered mtDNA transcription factors TFB1M and TFB2M (Gleyzer et al, 2005).…”

“…These include the induction of a family of immediate early genes (c-fos, c-jun), the early growth response gene-1 (Egr-1), specificity protein 1 (Sp1) and nuclear respiratory factor-1 (NRF-1), which respond to both acute and chronic contractile activity (Abu-Shakra et al, 1993;Connor et al, 2001;Irrcher and Hood, 2004;Michel et al, 1994;Murakami et al, 1998;Neufer et al, 1998;Puntschart et al, 1998;Takahashi et al, 1993). Egr-1 and Sp1 are known to be involved in regulating the transcription of cytochrome c, a nuclear-encoded protein of the electron transport chain (Connor et al, 2001;Freyssenet et al, 2004), while NRF-1 is involved in the transcriptional activation of an even greater diversity of nuclear genes encoding mitochondrial proteins (Kelly and Scarpulla, 2004), including the newly discovered mtDNA transcription factors TFB1M and TFB2M (Gleyzer et al, 2005). Acute contractile activity also results in a transient increase in transcriptional co-activator PGC-1␣ promoter activity along with an increase in PGC-1␣ mRNA (Akimoto et al, 2004) and protein (Baar et al, 2002).…”