The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/ calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin A␤ gene-targeted mice, which show a Ϸ50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin A␤؊/؊ and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin A␤؊/؊ mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of genetargeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overloadmediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1-or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.

Non-weight-bearing (NWB) activity [space flight and hindlimb suspension (HS)] results in the loss of soleus muscle mass, a slow-to-fast fiber-type conversion, and decreased beta-myosin heavy chain (beta-MHC) protein and mRNA expression. To identify beta-MHC promoter sequences required for decreased beta-MHC expression in response to HS, we have modified an existing noninvasive hindlimb unweighting model to accommodate the use of (transgenic) mice. After 2 wk of HS, body and muscle (soleus > gastrocnemius > plantaris) weights were decreased as was the proportion of histochemically classified type I fibers in HS soleus muscle. Northern blot analysis revealed decreases in endogenous mRNA representing beta-MHC, slow myosin light chain 1 and 2, and cardiac/slow troponin C, whereas those representing skeletal troponin C, muscle creatine kinase, and glyceraldehyde-3-phosphate dehydrogenase increased. Protein extracts prepared from HS soleus (SS) muscle of mice harboring transgenes comprised of 5.6 or 0.6 kilobase of wild type (wt) mouse beta-MHC promoter (beta 5.6 wt, beta 0.6wt) and those carrying the simultaneous mutation (mut) of the MCAT, C-rich, and beta e3 subregions (beta 5.6mut3, beta 0.6mut3) revealed decreases in chloramphenicol acetyltransferase (CAT) specific activity relative to respective controls. Decreased CAT mRNA was observed for transgene beta 5.6mut3, line 85. Two weeks of the simultaneous imposition of mechanical overload (synergist ablation) and HS (MOV/HS) countermanded the loss in absolute and normalized SS weight but did not decrease beta 0.6wt transgene expression. These transgenic results demonstrate that regulatory sequences within a 600-base pair beta-MHC promoter are sufficient to direct decreased transcription of beta-MHC transgenes after 2 wk of HS.

In adult mouse skeletal muscle, ␤-myosin heavy chain (␤MyHC) gene expression is primarily restricted to slow type I fibers; however, its expression can be induced in fast type II fibers in response to a sustained increase in load-bearing work (mechanical overload [MOV]). Our previous ␤MyHC transgenic and protein-DNA interaction studies have identified an A/T-rich element (␤A/T-rich ؊269/؊258) that is required for slow muscle expression and which potentiates MOV responsiveness of a 293-bp ␤MyHC promoter (␤293wt). Despite the GATA/MEF2-like homology of this element, we found binding of two unknown proteins that were antigenically distinct from GATA and MEF2 isoforms. By using the ␤A/T-rich element as bait in a yeast onehybrid screen of an MOV-plantaris cDNA library, we identified nominal transcription enhancer factor 1 (NTEF-1) as the specific ␤A/T-rich binding factor. Electrophoretic mobility shift assay analysis confirmed that NTEF-1 represents the enriched binding activity obtained only when the ␤A/T-rich element is reacted with MOV-plantaris nuclear extract. Moreover, we show that TEF proteins bind MEF2 elements located in the control region of a select set of muscle genes. In transient-coexpression assays using mouse C2C12 myotubes, TEF proteins transcriptionally activated a 293-bp ␤MyHC promoter devoid of any muscle CAT (MCAT) sites, as well as a minimal thymidine kinase promoter-luciferase reporter gene driven by three tandem copies of the desmin MEF2 or palindromic Mt elements or four tandem ␤A/T-rich elements. These novel findings suggest that in addition to exerting a regulatory effect by binding MCAT elements, TEF proteins likely contribute to regulation of skeletal, cardiac, and smooth muscle gene networks by binding select A/T-rich and MEF2 elements under basal and hypertrophic conditions.

Our previous transgenic analyses revealed that a 600-base pair ␤-myosin heavy chain (␤MyHC) promoter conferred mechanical overload (MOV) and non-weightbearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct ␤MyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair ␤MyHC promoter fused to chloramphenicol acetyltransferase (␤293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the ␤MyHC negative regulatory element (؊332 to ؊300; ␤NRE) within transgene ␤350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the ␤NRE sense strand (d␤NRE-S; ؊332 to ؊311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the ␤NRE sense strand was antigenically distinct from cellular nucleic acidbinding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV crosslinking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated ␤MyHC promoter elements transcriptionally regulate ␤MyHC transgene expression in response to two diverse modes of neuromuscular activity.The sarcomeric myosin heavy chain (MyHC) 1 is a major contractile protein that is encoded by a multigene family constituted by eight members (1). Each member has been shown to be responsive to a complex set of intrinsic and extrinsic signals that collectively regulate their expression in a defined developmental stage-and muscle fiber type-specific pattern. Within the MyHC gene family, the ␤MyHC is the one member whose regulated expression in both cardiac and skeletal muscle has been most extensively studied. Expression of the ␤MyHC gene has been detected in the ventricular myocardium and skeletal muscles comprising the hind limb of fetal mice, whereas in the adult, its expression is primarily restricted to skeletal muscles or muscle regions composed predominately of slow twitch type I fibers (2, 3). In vitro investigations of the control of ␤MyHC gene transcription have led to the identification of a control region located within the proximal promoter (nucleotides Ϫ300 to Ϫ188 of the human ␤MyHC gene) region (4 -7). This control region was found to be composed of three discrete DNA regulatory elements termed MCAT-like, C-rich and ␤e3, which are highly conserved in location and sequence across species (4 -7). The absolute requirement for these elements to obtain high levels of gene expression was demonstrated in transient transfection studies where the independent disruption of any of these conserved elements decreased and/or abolished musclespecific expression of ␤MyHC reporter genes (4 -7).Located just upstream from the ␤MyHC control region is a highly conserved ne...

Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased ␤-myosin heavy chain (␤MyHC) gene expression. Our previous work showed that Sp3 contributes to decreased ␤MyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Pur␣, and Pur␤ proteins mediate repression of ␤MyHC expression under NWB conditions. Binding of Pur␣ or Pur␤ to the single-stranded ␤MyHC distal negative regulatory element-sense strand (d␤NRE-S) element is markedly increased under NWB conditions. Ectopic expression of Pur␣ and Pur␤ decreased ␤MyHC reporter gene expression, while mutation of the d␤NRE-S element increased expression in C2C12 myotubes. The d␤NRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Pur␣ and Pur␤ decreased endogenous Sp3 and Pur protein levels and increased ␤MyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Pur␣, Pur␤, and Sp3, while chromatin immunoprecipitation assays demonstrated Pur␣, Pur␤, and Sp3 binding to the ␤MyHC proximal promoter region harboring the d␤NRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

Mechanical overload leads to hypertrophy, increased type I fiber composition, and beta-myosin heavy chain (beta-MHC) induction in the fast-twitch plantaris muscle. To better understand the mechanism(s) involved in beta-MHC induction, we have examined inducible expression of transgenes carrying the simultaneous mutation of three DNA regulatory subregions [muscle CAT (MCAT), C-rich, and beta e3] in the context of either 5,600-base pair (bp; beta 5.6mut3) or 600-bp (beta 0.6mut3) beta-MHC promoter in overloaded plantaris muscles of transgenic mice. Protein extract from mechanically overloaded plantaris muscle of mice, harboring either mutant transgene beta 5.6mut3 or beta 0.6mut3, showed an unexpected 2.8- to 4.5-fold increase in chloramphenicol acetyltransferase (CAT) specific activity relative to their respective controls. Similar results were obtained with wild-type (wt) beta-MHC transgenes (beta 5.6wt, beta 0.6wt). Histochemical staining for both myofibrillar ATPase and CAT activity and CAT immunohistochemistry revealed a striking increase in type I fibers and that CAT expression was restricted to these fibers in overloaded plantaris muscle of beta 5.6mut3 transgenic mice. Our transgenic data suggest that beta-MHC transgenes, and perhaps the endogenous beta-MHC gene, are induced by mechanical overload via a mechanism(s) that does not exclusively require the MCAT, C-rich, or beta e3 subregions.

To examine the role of the ␤-myosin heavy chain (␤MyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene ␤293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the ␤MyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the ␤MyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene ␤293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.

The DNA regulatory element(s) involved in beta-myosin heavy chain (beta-MHC) induction by the physiological stimulus of mechanical overload have not been identified as yet. To delineate regulatory sequences that are required for mechanical overload induction of the beta-MHC gene, transgenic mouse lines were generated that harbor transgenes containing serial deletions of the human beta-MHC promoter to nucleotides -293 (beta 293), -201 (beta 201), and -141 (beta 141) from the transcription start site (+1). Mechanically overloaded adult plantaris and soleus muscles contained 11- and 1.9-fold increases, respectively, in endogenous beta-MHC-specific mRNA transcripts (Northern blot) compared with sham-operated controls. Expression assays (chloramphenicol acetyltransferase specific activity) revealed that only transgene beta 293 expression was muscle specific in both fetal and adult mice and was induced in the plantaris (10- to 27-fold) and soleus (2- to 2.5-fold) muscles by mechanical overload. Histochemical staining for myosin adenosinetriphosphatase activity revealed a fiber-type transition of type II to type I in the overloaded plantaris and soleus muscles. These transgenic data suggest that sequences located between nucleotides -293 and +120 may be sufficient to regulate the endogenous beta-MHC gene in response to developmental signals and to the physiological signals generated by mechanical overload in fast- and slow-twitch muscles.