New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways.

Serum response factor (SRF) is a crucial transcriptional factor for muscle-specific gene expression. We investigated SRF function in adult skeletal muscles, using mice with a postmitotic myofiber-targeted disruption of the SRF gene. Mutant mice displayed severe skeletal muscle mass reductions due to a postnatal muscle growth defect resulting in highly hypotrophic adult myofibers. SRF-depleted myofibers also failed to regenerate following injury. Muscles lacking SRF had very low levels of muscle creatine kinase and skeletal alpha-actin (SKA) transcripts and displayed other alterations to the gene expression program, indicating an overall immaturity of mutant muscles. This loss of SKA expression, together with a decrease in beta-tropomyosin expression, contributed to myofiber growth defects, as suggested by the extensive sarcomere disorganization found in mutant muscles. However, we observed a downregulation of interleukin 4 (IL-4) and insulin-like growth factor 1 (IGF-1) expression in mutant myofibers which could also account for their defective growth and regeneration. Indeed, our demonstration of SRF binding to interleukin 4 and IGF-1 promoters in vivo suggests a new crucial role for SRF in pathways involved in muscle growth and regeneration.

Mosaic inactivation of the serum response factor gene in the myocardium induces focal lesions and heart failure.

BACKGROUND AND AIMS: Regional alterations in ventricular mechanical functions are a primary determinant for the risk of myocardial injuries in various cardiomyopathies. The serum response factor (SRF) is a transcription factor regulating contractile and cytoskeletal genes and may play an important role in the remodelling of myocardium at the cellular level. METHODS: Using Desmin-Cre transgenic mice, we generated a model of mosaic inactivation of a floxed-Srf allele in the heart to analyze the consequence of regional alterations of SRF-mediated functions in the myocardium. RESULTS: Two types of cardiomyocytes co-existed in the Desmin-Cre:Sf/Sf mice. Cardiomyocytes lacking SRF became thin and elongated while cardiomyocytes containing SRF became hypertrophic. Several physiological contractile genes were down-regulated while skeletal alpha-actin was induced in SRF positive area only. Mutants developed heart failure associated with the presence of focal lesions in the myocardium, and died before month 11. CONCLUSIONS: Juxtaposition of functional SRF wild-type and failing SRF mutant cardiomyocytes generates deleterious heterogeneity in the myocardium. Our results show that SRF contributes to the capacity of cardiomyocytes to remodel their shape and contractile functions in response to their local environment; suggesting that it may play a role in pathologies involving regional alterations of ventricular mechanics in the heart.

Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.

Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes.

Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart.

BACKGROUND: Serum response factor (SRF) is a cardiac transcription factor involved in cell growth and differentiation. We have shown, using the Cre/loxP system, that cardiac-specific disruption of SRF gene in the embryonic heart results in lethal cardiac defects. The role of SRF in adult heart is unknown. METHODS AND RESULTS: We disrupted SRF in the adult heart using a heart-specific tamoxifen-inducible Cre recombinase. This disruption led to impaired left ventricular function with reduced contractility, subsequently progressing to dilated cardiomyopathy, as demonstrated by serial echocardiography, including tissue Doppler imaging. The cytoarchitecture of cardiomyocytes was altered in the intercalated disks. All mutant mice died from heart failure 10 weeks after treatment. These functional and structural defects were preceded by early alterations in the cardiac gene expression program: major decreases in mRNA levels for cardiac alpha-actin, muscle creatine kinase, and calcium-handling genes. CONCLUSIONS: SRF is crucial for adult cardiac function and integrity. We suggest that the rapid progression to heart failure in SRF mutant mice results primarily from decreased expression of proteins involved in force generation and transmission, low levels of polymerized actin, and changes in cytoarchitecture, without hypertrophic compensation. These cardiac-specific SRF-deficient mice have the morphological and clinical features of acquired dilated cardiomyopathy in humans and may therefore be used as an inducible model of this disorder.

Locally expressed IGF1 propeptide improves mouse heart function in induced dilated cardiomyopathy by blocking myocardial fibrosis and SRF-dependent CTGF induction.

Cardiac fibrosis is critically involved in the adverse remodeling accompanying dilated cardiomyopathies (DCMs), which leads to cardiac dysfunction and heart failure (HF). Connective tissue growth factor (CTGF), a profibrotic cytokine, plays a key role in this deleterious process. Some beneficial effects of IGF1 on cardiomyopathy have been described, but its potential role in improving DCM is less well characterized. We investigated the consequences of expressing a cardiac-specific transgene encoding locally acting IGF1 propeptide (muscle-produced IGF1; mIGF1) on disease progression in a mouse model of DCM [cardiac-specific and inducible serum response factor (SRF) gene disruption] that mimics some forms of human DCM. Cardiac-specific mIGF1 expression substantially extended the lifespan of SRF mutant mice, markedly improved cardiac functions, and delayed both DCM and HF. These protective effects were accompanied by an overall improvement in cardiomyocyte architecture and a massive reduction of myocardial fibrosis with a concomitant amelioration of inflammation. At least some of the beneficial effects of mIGF1 transgene expression were due to mIGF1 counteracting the strong increase in CTGF expression within cardiomyocytes caused by SRF deficiency, resulting in the blockade of fibroblast proliferation and related myocardial fibrosis. These findings demonstrate that SRF plays a key role in the modulation of cardiac fibrosis through repression of cardiomyocyte CTGF expression in a paracrine fashion. They also explain how impaired SRF function observed in human HF promotes fibrosis and adverse cardiac remodeling. Locally acting mIGF1 efficiently protects the myocardium from these adverse processes, and might thus represent a therapeutic avenue to counter DCM.

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy.

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.

The essential function for serum response factor in T-cell development reflects its specific coupling to extracellular signal-regulated kinase signaling.

Serum response factor (SRF) recruits members of two families of signal-regulated coactivators, the extracellular signal-regulated kinase (ERK)-regulated ternary complex factors (TCFs) and the actin-regulated myocardin-related transcription factors (MRTFs), to its target genes through its DNA-binding domain. Whether coactivator association is required for SRF function in vivo and whether particular SRF functions reflect specific coupling to one or the other signal pathway have remained largely unexplored. We show that SRF is essential for thymocyte positive selection and thymic T(reg) and NK T-cell development but dispensable for early thymocyte development and negative selection. Expression of wild-type SRF, or mutants lacking the N-terminal phosphorylation sites or C-terminal transcriptional activation domain, restores positive selection in SRF null thymocytes. In contrast, SRF.V194E, which cannot recruit TCF or MRTF family members, is inactive, although it is recruited to target genes. Fusion of a TCF C-terminal activation domain to SRF.V194E effectively restores ERK-dependent single-positive (SP) thymocyte development. The resulting SP thymocytes exhibit normal surface marker expression and proliferation following T-cell receptor cross-linking. Thus, ERK signaling through the TCF pathway to SRF is necessary and sufficient for SRF function in thymocyte positive selection.

Premature aging in skeletal muscle lacking serum response factor.

Aging is associated with a progressive loss of muscle mass, increased adiposity and fibrosis that leads to sarcopenia. At the molecular level, muscle aging is known to alter the expression of a variety of genes but very little is known about the molecular effectors involved. SRF (Serum Response Factor) is a crucial transcription factor for muscle-specific gene expression and for post-natal skeletal muscle growth. To assess its role in adult skeletal muscle physiology, we developed a post-mitotic myofiber-specific and tamoxifen-inducible SRF knockout model. Five months after SRF loss, no obvious muscle phenotype was observed suggesting that SRF is not crucial for myofiber maintenance. However, mutant mice progressively developed IIB myofiber-specific atrophy accompanied by a metabolic switch towards a more oxidative phenotype, muscular lipid accumulation, sarcomere disorganization and fibrosis. After injury, mutant muscles exhibited an altered regeneration process, showing smaller regenerated fibers and persistent fibrosis. All of these features are strongly reminiscent of abnormalities encountered in aging skeletal muscle. Interestingly, we also observed an important age associated decrease in SRF expression in mice and human muscles. Altogether, these results suggest that a naturally occurring SRF down-regulation precedes and contributes to the muscle aging process. Indeed, triggering SRF loss in the muscles of mutant mice results in an accelerated aging process.

Delayed liver regeneration in mice lacking liver serum response factor.

Various immediate early genes (IEGs) upregulated during the early process of liver regeneration are transcriptional targets of the serum response factor (SRF). We show here that the expression of SRF is rapidly induced in rodent liver after partial hepatectomy. Because the inactivation of the SRF gene in mice is embryonic lethal, the in vivo role of SRF in liver regeneration after partial hepatectomy was analyzed in mutant mice conditionally deleted for SRF in the liver. We demonstrate that SRF is not an essential factor for liver ontogenesis. However, adult mutant mice show impaired liver regeneration after partial hepatectomy, associated with a blunted upregulation of various SRF target IEGs. In conclusion, our work suggests that SRF is an early response transcription factor that may contribute to the initial phases of liver regeneration through its activation of IEGs.

Conditional inactivation of the murine serum response factor in the pancreas leads to severe pancreatitis.

The Serum Response Factor (SRF) is widely expressed transcription factor acting at the confluence of multiple signaling pathways and has been implicated in the control of differentiation, growth, and cell death. In the present study, we found that SRF is expressed in the developing and adult pancreas. To explore the possible role of SRF in this organ, we have generated mutant mice with conditional disruption of the Srf gene. Such mutants presented normal development of both the exocrine and endocrine pancreas indicating that SRF is dispensable for pancreas ontogenesis. However, after weaning, these mice developed profound morphological alterations of the exocrine pancreas, which were reminiscent of severe pancreatitis. In these mice, massive acinar injury, Nuclear Factor Kappa B activation and proinflammatory cytokines release led to complete destruction of the exocrine pancreas and its replacement by adipose tissue. Despite these changes, the organization and function of the endocrine islets of Langerhans remained well-preserved. This new animal model of spontaneous pancreatitis could prove a valuable tool to gain further insight into the physiopathology of this disease.

High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.

Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration.

Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters.

Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.