Myocardin and microRNA‐1 modulate bladder activity through connexin 43 expression during post‐natal development

Overactive bladder (OAB) is a pervasive clinical problem involving alterations in both neurogenic and myogenic activity. While there has been some progress in understanding neurogenic inputs to OAB, the mechanisms controlling myogenic bladder activity are unclear. We report the involvement of myocardin (MYOCD) and microRNA‐1 (miR‐1) in the regulation of connexin 43 (GJA1), a major gap junction in bladder smooth muscle, and the collective role of these molecules during post‐natal bladder development. Wild‐type (WT) mouse bladders showed normal development from early post‐natal to adult including increases in bladder capacity and maintenance of normal sensitivity to cholinergic agents concurrent with down‐regulation of MYOCD and several smooth muscle cell (SMC) contractile genes. Myocardin heterozygous‐knockout mice exhibited reduced expression of Myocd mRNA and several SMC contractile genes concurrent with bladder SMC hypersensitivity that was mediated by gap junctions. In both cultured rat bladder SMC and in vivo bladders, MYOCD down‐regulated GJA1 expression through miR‐1 up‐regulation. Interestingly, adult myocardin heterozygous‐knockout mice showed normal increases in bladder and body weight but lower bladder capacity compared to WT mice. These results suggest that MYOCD down‐regulates GJA1 expression via miR‐1 up‐regulation, thereby contributing to maintenance of normal sensitivity and development of bladder capacity. J. Cell. Physiol. 228: 1819–1826, 2013. © 2013 Wiley Periodicals, Inc.     

Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein,HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.     

Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.