The alpha(1)-subunit of the cardiac/vascular Ca(2+) channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca(2+) entry. It contains the binding sites for multiple classes of drugs collectively known as Ca(2+) antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alpha(1C)-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences and identified the alpha(1C)-subunit gene transcription start site. The rat alpha(1C)-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha(1C)-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca(2+) channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha(1C)-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.
Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGI␣ was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGI␣ in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964 -1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3-to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration. Nitric oxide (NO)1 is the most important endogenous vasodilator known (1-5). NO causes vasodilatation by stimulating soluble guanylate cyclase in VSMCs to increase intracellular cGMP levels. Cyclic GMP, in turn, binds and activates cyclic GMP-dependent protein kinase I (PKGI), its principal effector in vascular smooth muscle cells (6 -9). Mice lacking the gene for PKGI develop systemic hypertension, and their vessels fail to relax normally (9,10). Mouse models also demonstrate that PKGI is important in the regulation of vascular remodeling and thrombosis (9). The intracellular substrates of PKGI and the mechanisms by which PKGI regulates these substrates to alter vascular smooth muscle cell responses is, therefore, of central importance to vascular biology in health and disease.A number of laboratories have sought proteins capable of interacting with PKGI isoforms (PKGI-interacting proteins, cGMP-dependent protein kinase-anchoring proteins, cGMP-dependent protein kinase-interacting proteins (GKIPs)). In many instances, PKG both directly binds to and phosphorylates such proteins, suggesting that one way to identify PKG substrates and to unravel PKG signaling pathways is to identify PKGbinding partners. Nearly a decade ago, using gel overlay and immunoprecipitation approaches, the interm...
Therapy with VAD normalizes iNOS expression in association with diminished cardiomyocyte apoptosis in the failing heart. Further work is required to define whether a causal relationship exists between iNOS and cardiomyocyte apoptosis.
Despite recent advances in medical therapy, mortality remains high following the diagnosis of heart failure (HF). Cardiac transplantation is still the standard surgical treatment option for highly selected patients with severe end-stage HF; however, it is only available to a small percentage of patients. The small number of available donor hearts is an inherent limitation on the ability of cardiac transplantation to greatly impact the management of advanced HF. The increased incidence and prevalence of HF in an ever aging and medically complex population has paved the way for alternative surgical and device treatment strategies. Some of these treatment options include ventricular reduction/remodeling surgery, mitral valve repair, mechanical ventricular assist device implantation, implantable cardioverter-defibrillators, and cardiac resynchronization therapy. Several recent trials have demonstrated the effectiveness of these therapies with regard to improvement in primary cardiac end points, HF symptoms, and survival. Surgical and device techniques are usually combined with optimal medical management of HF. The total cost and actual cost-effectiveness of employing these new therapeutic modalities in a growing population of HF patients remains to be determined.
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