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Indiana University-Purdue University Indianapolis (IUPUI) / There are many clinical diseases involving abnormal differentiation of smooth muscle, such as atherosclerosis, hypertension and asthma. In these diseases, one important pathological process is the disruption of the balance between differentiation and proliferation of smooth muscle cells. Serum Response Factor (SRF) has been shown to be a key regulator of smooth muscle differentiation, proliferation and migration through its interaction with various accessory proteins. Myocardin Related Transcrition Factors (MRTFs) are important co-activators of SRF that induce smooth muscle differentiation. Elucidating the mechanism of how MRTFs and SRF discriminate between genes required to regulate smooth muscle differentiation and those regulating proliferation will be a significant step toward finding a cure for these diseases. We hypothesized that SWI/SNF ATPdependent chromatin remodeling complexes, containing Brg1 and Brm, may play a role in this process. Results from western blotting and quantitative reverse transcription - polymerase chain reaction (qRT-PCR) analysis demonstrated that expression of dominant negative Brg1 or knockdown of Brg1 with silence ribonucleic acid (siRNA) attenuated expression of SRF/MRTF dependent smooth muscle-specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and that Brg1 directly binds MRTFs, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated SRF binding and the ability of MRTFs to increase SRF binding to the promoters of smooth muscle-specific genes, but not proliferation-related early response genes. The above data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in differentially regulating expression of SRF/MRTF-dependent genes through controlling the accessibility of SRF/MRTF to their target gene promoters. To examine the role of SWI/SNF in smooth muscle cells in vivo, we have generated mice harboring a smooth muscle-specific knockout of Brg1. Preliminary analysis of these mice revealed defects in gastrointestinal (GI) development, including a significantly shorter gut in Brg1 knockout mice. These data suggest that Brg1-containing SWI/SNF complexes play an important role in the development of the GI tract.

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Muscular dystrophy is a disease characterized by skeletal muscle weakness and wasting, but little is known of alterations in the vascular system that occur with this disease. The culprit in many muscular dystrophies is a defective dystrophin-glycoprotein complex (DGC). The DGC is a group of transmembrane proteins that connects the cytoskeleton of muscle cells to the extracellular matrix; it plays a role in mechanotransduction and the maintenance of structural integrity of these cells, and includes the proteins dystrophin and sarcoglycan-delta. The absence of these proteins results in severe muscular dystrophies in humans, and thus knockout mice lacking the genes encoding for dystrophin (mdx mice) and sarcoglycan-delta (sgcd-/- mice) were studied to detect any vascular alterations that occur as a result of a defective DGC. Acute biaxial biomechanical data were obtained through pressure-diameter and axial force-length tests on common carotid arteries of mdx, sgcd-/-, and wild-type mice in the active and passive smooth muscle state. Functional response to the vasoreactive compounds phenylephrine, carbamylcholine chloride, and sodium nitroprusside was also tested. We found significant biomechanical differences between the knockout and wild-type mouse arteries: the mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries also having increased circumferential stresses, and the knockout arteries generated increased axial loads and stresses in the axial force-length tests. The mdx and sgcd-/- arteries also differed from the wild-type in that their ‘homeostatic’ axial stretch, at which the axial force remains constant upon pressurization, was significantly decreased. We conclude that the loss of DGC proteins does trigger changes in vascular smooth muscle cells or their interactions with the extracellular matrix, yet that the altered vascular system was able to adapt and function without the DGC. Knowledge of alterations to the vascular system (and adaptations to these changes) of patients with muscular dystrophy could help physicians customize their treatment to extend and enhance their lives, especially as medical advances extend the lifespan of these patients and they begin to suffer from diseases such as hypertension and atherosclerosis that affect the normal aging population.