Myofiber-specific TEAD1 overexpression drives satellite cell hyperplasia and counters pathological effects of dystrophin deficiency.

When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic 'scaling' of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a 'normal' quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.

TEAD-1 overexpression in the mouse heart promotes an age-dependent heart dysfunction.

TEA domain transcription factor-1 (TEAD-1) is essential for proper heart development and is implicated in cardiac specific gene expression and the hypertrophic response of primary cardiomyocytes to hormonal and mechanical stimuli, and its activity increases in the pressure-overloaded hypertrophied rat heart. To investigate whether TEAD-1 is an in vivo modulator of cardiac specific gene expression and hypertrophy, we developed transgenic mice expressing hemagglutinin-tagged TEAD-1 under the control of the muscle creatine kinase promoter. We show that a sustained increase in TEAD-1 protein leads to an age-dependent dysfunction. Magnetic resonance imaging revealed decreases in cardiac output, stroke volume, ejection fraction, and fractional shortening. Isolated TEAD-1 hearts revealed decreased left ventricular power output that correlated with increased betaMyHC protein. Histological analysis showed altered alignment of cardiomyocytes, septal wall thickening, and fibrosis, although electrocardiography displayed a left axis shift of mean electrical axis. Transcripts representing most members of the fetal heart gene program remained elevated from fetal to adult life. Western blot analyses revealed decreases in p-phospholamban, SERCA2a, p-CX43, p-GSK-3alpha/beta, nuclear beta-catenin, GATA4, NFATc3/c4, and increased NCX1, nuclear DYKR1A, and Pur alpha/beta protein. TEAD-1 mice did not display cardiac hypertrophy. TEAD-1 mice do not tolerate stress as they die over a 4-day period after surgical induction of pressure overload. These data provide the first in vivo evidence that increased TEAD-1 can induce characteristics of cardiac remodeling associated with cardiomyopathy and heart failure.

IGF-I activates the mouse type IIb myosin heavy chain gene.

IGF-I increases skeletal muscle mass, but whether IGF-I increases type IIb myosin heavy chain (MyHC) transcriptional activity is not known. C2C12 myotubes were cultured with or without IGF-I to determine whether IGF-I increases type IIb MyHC promoter activity, and if so, what region of the promoter might IGF-I signaling regulate. At differentiation days 3 and 4, IGF-I increased type IIb MyHC mRNA and mouse 3.0-kb type IIb MyHC promoter activity. Deletion construct studies identified a potential IGF-I-responsive region between 1.25 and 1.2 kb of the type IIb MyHC promoter, which contained an exact 6-bp T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding site at position -1206 to -1201. Site-specific mutation of the putative Tcf/Lef binding site reduced IGF-I-induced 1.3-kb type IIb MyHC promoter activity. To identify potential IGF-I signaling molecules, the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002 were both found to markedly attenuate IGF-I activation of the 1.3-kb type IIb MyHC promoter. Downstream signaling of IGF-I can phosphorylate and inactivate GSK-3beta, thereby enhancing beta-catenin protein. The GSK-3beta inhibitor, LiCl, dramatically enhanced IGF-I induction of the 1.3-kb type IIb MyHC promoter, and constitutively active GSK-3beta attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity. Finally, IGF-I increased nuclear beta-catenin protein, and small interfering RNA knockdown of beta-catenin attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity and type IIb MyHC mRNA. In summary, IGF-I stimulation of C2C12 myotubes increases mouse type IIb MyHC promoter activity, likely through signaling of PI3K, GSK-3beta, beta-catenin, and a Tcf/Lef binding site at -1,206 to -1,201 bp in the promoter.

Overexpression of TEAD-1 in transgenic mouse striated muscles produces a slower skeletal muscle contractile phenotype.

TEA domain (TEAD) transcription factors serve important functional roles during embryonic development and in striated muscle gene expression. Our previous work has implicated a role for TEAD-1 in the fast-to-slow fiber-type transition in response to mechanical overload. To investigate whether TEAD-1 is a modulator of slow muscle gene expression in vivo, we developed transgenic mice expressing hemagglutinin (HA)-tagged TEAD-1 under the control of the muscle creatine kinase promoter. We show that striated muscle-restricted HA-TEAD-1 expression induced a transition toward a slow muscle contractile protein phenotype, slower shortening velocity (Vmax), and longer contraction and relaxation times in adult fast twitch extensor digitalis longus muscle. Notably, HA-TEAD-1 overexpression resulted in an unexpected activation of GSK-3alpha/beta and decreased nuclear beta-catenin and NFATc1/c3 protein. These effects could be reversed in vivo by mechanical overload, which decreased muscle creatine kinase-driven TEAD-1 transgene expression, and in cultured satellite cells by TEAD-1-specific small interfering RNA. These novel in vivo data support a role for TEAD-1 in modulating slow muscle gene expression.

Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy.

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 Abeta gene-targeted mice, which show a approximately 50% 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 Abeta-/- 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 Abeta-/- 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 gene-targeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overload-mediated 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.

Divergence in species and regulatory role of beta -myosin heavy chain proximal promoter muscle-CAT elements.

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair beta-myosin heavy chain (beta MyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized beta MyHC proximal promoter elements contribute to NWB regulation.