No abstract

Set7 associates with the MyoD transcription factor to enhance expression of genes required for muscle differentiation.

Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin-glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.

In vivo protein kinases A and G (PKA and PKG) coordinately phosphorylate a broad range of substrates to mediate their various physiological effects. The functions of many of these substrates have yet to be defined genetically. Herein we show a role for smoothelin-like protein 1 (SMTNL1), a novel in vivo target of PKG/PKA, in mediating vascular adaptations to exercise. Aortas from smtnl1 ؊/؊ mice exhibited strikingly enhanced vasorelaxation before exercise, similar in extent to that achieved after endurance training of wild-type littermates. Additionally, contractile responses to ␣-adrenergic agonists were greatly attenuated. Immunological studies showed SMTNL1 is expressed in smooth muscle and type 2a striated muscle fibers. Consistent with a role in adaptations to exercise, smtnl1 ؊/؊ mice also exhibited increased type 2a fibers before training and better performance after forced endurance training compared smtnl1 ؉/؉ mice. Furthermore, exercise was found to reduce expression of SMTNL1, particularly in female mice. In both muscle types, SMTNL1 is phosphorylated at Ser-301 in response to adrenergic signals. In vitro SMTNL1 suppresses myosin phosphatase activity through a substrate-directed effect, which is relieved by Ser-301 phosphorylation. Our findings suggest roles for SMTNL1 in cGMP/cAMP-mediated adaptations to exercise through mechanisms involving direct modulation of contractile activity.The contractile state of smooth muscle (SM) 2 is largely governed by phosphorylation of myosin regulatory light chain (LC20), which in turn is regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase, SMPP1M (1). In SM, both MLCK and SMPP1M are regulated, thereby enabling homeostatic control of contractile activity (2). Exquisite control is necessary because of the essential roles of SM in many physiological processes, such as maintenance of blood pressure. In most SMs, release of cyclic nucleotides (cGMP/cAMP), in response to hormonal or neuronal stimulation, promote relaxation either by lowering intracellular [Ca 2ϩ ] or desensitizing the muscle to Ca 2ϩ by inhibiting MLCK and/or activating SMPP1M (2).Targeted deletions of both PKA and PKG in mice produce profound phenotypes, underscoring the importance of these kinases in many physiological processes (3, 4). In vivo, both kinases are known to selectively target a discrete number of substrates and current thinking suggests that selective targeting is the means by which these broadly acting enzymes bring about coordinated physiological responses (5). A few groups have begun to test this hypothesis by selectively deleting PKA/PKG targets in mice. Schlossmann et al. (6) demonstrated a major role for IRAG (inositol 1,4,5-trisphosphate receptor 1 IP3R1-associated protein, with exon 12 deleted by removing the 1,4,5-trisphosphate receptor binding domain) in PKG-mediated regulation of [Ca 2ϩ ] in SM. Disruption of IRAG resulted in a selective loss of signaling response. Other PKG/PKA-mediated responses were largely intact, contrasting wi...

This study reports a new mechanism of cAMP mediated relaxation of Ca2+sensitized force, in smooth muscle (SM) through Epac, a GTP exchange factor for the small GTPase Rap1 which results in suppression of RhoA activity. We find that Epac selective cAMP analogue, 8‐pCPT‐2′‐O‐Me‐cAMP (007), significantly reduced agonist‐induced contractile force, in both intact and permeabilized vascular, gut and airway SM. Responses to 007 were independent of PKA and PKG. Activation of Epac resulted in increased Rap1·GTP accompanied by a significant decrease in RhoA activity and reductions in phosphorylation of RLC20 and MLCP. Transcriptional regulation of SM α‐actin and SM22, known to be regulated by RhoA, was also significantly decreased by activation of Epac. Forskolin, the phosphodiesterase inhibitor IBMX and isoproterenol significantly increased Rap1·GTP in rat aortic SM cells. Over‐expression of wild‐type Epac but not dominant negative Epac1R279E increased Rap1 activation after 007 stimulation. LPA‐induced activation of RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts. All together, our findings show a novel signaling mechanism whereby activation of Epac via cAMP results in PKA independent, Rap1 dependent Ca2+ desensitization of force in SM.

No abstract

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle ␣-actin (SM␣〈) or smooth musclemyosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SM␣〈-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca 2؉ transients, expressed functional Ca 2؉ channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

Neppl et al. identify a long noncoding RNA named Chronos whose expression increases with age and decreases in Akt-mediated growth. Inhibition of Chronos induces myofiber hypertrophy in vitro and in vivo, in part, through the epigenetic modulation of Bmp7 signaling.