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beta-Adrenergic receptor (betaAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with betaAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca(2+)-release channel, which enhances Ca(2+) release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca(2+)-ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in betaAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic betaAR signaling.

Calcineurin, a calcium/calmodulin-regulated protein phosphatase, modulates gene expression in cardiac and skeletal muscles during development and in remodeling responses such as cardiac hypertrophy that are evoked by environmental stresses or disease. Recently, we identified two genes encoding proteins (MCIP1 and MCIP2) that are enriched in striated muscles and that interact with calcineurin to inhibit its enzymatic activity. In the present study, we show that expression of MCIP1 is regulated by calcineurin activity in hearts of mice with cardiac hypertrophy, as well as in cultured skeletal myotubes. In contrast, expression of MCIP2 in the heart is not altered by activated calcineurin but responds to thyroid hormone, which has no effect on MCIP1. A approximately 900-bp intragenic segment located between exons 3 and 4 of the MCIP1 gene functions as an alternative promoter that responds to calcineurin. This region includes a dense cluster of 15 consensus binding sites for NF-AT transcription factors. Because MCIP proteins can inhibit calcineurin, these results suggest that MCIP1 participates in a negative feedback circuit to diminish potentially deleterious effects of unrestrained calcineurin activity in cardiac and skeletal myocytes. Inhibitory effects of MCIP2 on calcineurin activity may be pertinent to gene switching events driven by thyroid hormone in striated muscles. The full text of this article is available at http://www. circresaha.org.

A hallmark of vascular lesions is the phenotypic modulation of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a more primitive, proliferative phenotype with a more fetal pattern of gene expression. Using subtraction hybridization to identify genes that may regulate this transition, we cloned a novel gene named EVEC, an acronym for its expression in the embryonic vasculature and the presence of Ca2+ binding epidermal growth factor-like repeats contained in the predicted protein structure. Although these repeats are characteristic of the extracellular matrix proteins, fibrillin, fibulin, and the latent transforming growth factor-beta binding proteins, EVEC most closely resembles the H411 and T16/S1-5 gene products, the latter of which are believed to regulate DNA synthesis in quiescent fibroblasts. Using in situ hybridization, we demonstrated that EVEC is expressed predominantly in the VSMCs of developing arteries in E11.5 through E16.5 mouse embryos. Lower levels of expression are also observed in endothelial cells, perichondrium, intestine, and mesenchyme of the face and kidney. EVEC mRNA expression is dramatically downregulated in adult arteries, except in the uterus, where cyclic angiogenesis continues; however, EVEC expression is reactivated in 2 independent rodent models of vascular injury. EVEC mRNA is observed in cellular elements of atherosclerotic plaques of LDL receptor-deficient, human apolipoprotein B transgenic mice and in VSMCs of the media and neointima of balloon-injured rat carotid arteries. These data suggest that EVEC may play an important role in the regulation of vascular growth and maturation during development and in lesions of injured vessels.

Left ventricular hypertrophy (LVH) is a compensatory mechanism to cope with pressure overload. Recently, a calcineurin pathway mediating LVH and its prevention by cyclosporine was reported. We examined whether calcineurin mediates LVH due to pressure overload in mice. Pressure overload was induced by aortic banding in 53 mice (32 treated with cyclosporine [25 mg. kg-1. d-1], 21 treated with vehicle). There were 17 sham-operated mice (9 treated with vehicle, 8 treated with cyclosporine). At 3 weeks after surgery, LV weight to body weight was greater in the nontreatment banded group (4.39+/-0. 16 mg/g) than in the cyclosporine-treated banded group (3.95+/-0.14 mg/g, P<0.05), with both groups being greater compared with the entire group of sham-operated mice (3.02+/-0.04 mg/g). The pressure gradient between the ascending and abdominal aorta was not different between the cyclosporine-treated (49.6+/-6.1 mm Hg) and nontreatment groups (48.7+/-4.6 mm Hg). Although LV systolic pressure was lower in the cyclosporine-treated banded animals, LV systolic wall stress was similar in the nontreatment banded group and in the cyclosporine-treated group. However, LV dP/dt was lower (P=0.05) in the cyclosporine-treated banded group (4774+/-656 mm Hg/s) than in the nontreatment banded group (6604+/-516 mm Hg/s). During the protocol, 23 of 32 mice in the cyclosporine-treated group and 9 of 21 mice in the nontreatment group died. All deaths occurred within 10 days after surgery. Deaths caused by heart failure were 7.2-fold higher (P<0.05) in the cyclosporine-treated group, whereas deaths due to other causes were not different between the 2 groups. In addition, LV function of mice was assessed at 48 hours after banding; LV ejection fraction measured with echocardiography was lower (P<0.05) in the cyclosporine-treated banded group (66+/-3.0%) than in the nontreatment banded group (79+/-1.5%), whereas LV systolic wall stresses were similar. Calcineurin phosphatase activity was depressed similarly in both cyclosporine-treated groups compared with both nontreatment groups. Thus, cyclosporine could attenuate, but not prevent, LVH at the expense of inhibiting an important compensatory mechanism in response to pressure overload, resulting in reduced LV wall stress and function and increased susceptibility to decompensation and heart failure.

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase calcineurin as a cellular target for a variety of Ca2+-dependent signaling pathways culminating in left ventricular hypertrophy (LVH). Most of the recent experimental support for this hypothesis is derived from in vitro studies or in vivo studies in transgenic mice expressing activated calcineurin or mutant sarcomeric proteins. The aim of the present study was to test whether calcineurin inhibitors, cyclosporin A (CsA) and FK 506, prevent pressure-overload LVH using 2 standard rat models: (1) the spontaneously hypertensive rat (SHR) and (2) aortic banding. The major new findings are 2-fold. First, in SHR, LVH (left ventricular weight to body weight ratio) was unaffected by a dose of CsA (5 mg. kg-1. d-1) that was sufficient to raise blood pressure and to inhibit calcineurin-mediated transcriptional activation in skeletal muscle. Second, in rats with aortic banding, LVH was unaffected by FK 506 (0.3 mg. kg-1. d-1) or even higher doses of CsA (10 and 20 mg. kg-1. d-1) that were sufficient to inhibit 90% of total calcineurin phosphatase activity in the hypertrophied myocardium. In the latter experiments, CsA blocked neither the elevated left ventricular end-diastolic pressures, a measure of diastolic function, nor the induction in atrial natriuretic peptide mRNA in the hypertrophic ventricles. Thus, in numerous experiments, systemic administration of potent calcineurin inhibitors did not prevent the development of LVH in 2 classic models of pressure-overload hypertrophy. These results demonstrate that pressure-overload hypertrophy can arise through calcineurin-independent pathways.

In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.

The objective of the present study was to examine the molecular mechanisms whereby angiotensin II (Ang II) stimulates smooth muscle (SM) alpha-actin expression in rat aortic smooth muscle cells (SMCs). Nuclear run-on analysis and transfection studies indicated that the effects of Ang II on SM alpha-actin were mediated at least in part at the transcriptional level. Transfection of various rat SM alpha-actin promoter/chloramphenicol acetyltransferase (CAT) constructs into SMCs demonstrated that the first 155 bp of the SM alpha-actin promoter was sufficient to confer maximal Ang II responsiveness, conferring an approximately 4-fold increase in reporter activities in these SMCs compared with vehicle-treated SMCs. Mutation of either of two highly conserved CArG elements, designated A (-62) and B (-112), completely abolished Ang II-induced increases in reporter activity, whereas mutation of a homeodomain-like binding sequence at -145 (ATTA) reduced reporter activity by half. Results of EMSAs showed that nuclear extracts from Ang II-treated SMCs exhibited enhanced binding activity of serum response factor (SRF) to the CArG elements and of a homeodomain factor, MHox, to the ATTA element. Northern analyses showed that Ang II also stimulated marked increases in MHox mRNA levels. Western analyses demonstrated that Ang II-induced increases in SRF binding were not due to increased SRF protein expression. Recombinant MHox markedly enhanced binding activity of SRF in EMSAs. Finally, MHox overexpression transactivated a SM alpha-actin promoter/CAT reporter construct by approximately 3.5-fold in transient cotransfection studies. These results provide evidence for involvement of a homeodomain transcription factor, MHox, in Ang II-mediated stimulation of SM alpha-actin via a CArG/SRF-dependent mechanism.

Background: Retinoids have been used in the successful treatment of a variety of human hyperproliferative diseases. Their role in smooth muscle cell (SMC) growth control, however, has not been clearly established. The present study was designed to assess the retinoid receptor mRNA expression profile in SMCs and to determine whether retinoids exert a growth-inhibitory effect in these cells., Methods and Results: Five of the six retinoid receptors were expressed in both cultured SMCs and aorta as determined by Northern blotting or reverse transcriptase-polymerase chain reaction. Receptor activity was demonstrated in SMCs with the use of a reporter assay with a retinoid receptor DNA binding sequence linked to a chloramphenicol acetyltransferase reporter gene. DNA synthesis and cell proliferation assays were performed to show that all-trans retinoic acid (atRA) antagonized platelet-derived growth factor-BB and serum-stimulated SMC growth. Growth inhibition was distal to early growth-signaling events because induction of c-fos, c-jun, and egr-1 mRNA was unaffected by atRA. However, with an activated protein-1-linked chloramphenicol acetyltransferase reporter, atRA was shown to inhibit the activity of activated protein-1-dependent transcription in a transient transfection assay., Conclusions: These results establish the presence of functional retinoid receptors in SMCs and document the growth-inhibitory action of atRA on these cells. Retinoid compounds, already in clinical use as antiproliferative agents for nonvascular indications, should be assessed further in in vivo models of intimal disease.

Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel's response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.

SM22 alpha is a calponin-related protein that is expressed specifically in adult smooth muscle. To begin to define the mechanisms that regulate the establishment of the smooth muscle lineage, we analyzed the expression pattern of the SM22 alpha gene during mouse embryogenesis. In situ hybridization demonstrated that SM22 alpha transcripts were first expressed in vascular smooth muscle cells at about embryonic day (E) 9.5 and thereafter continued to be expressed in all smooth muscle cells into adulthood. In contrast to its smooth muscle specificity in adult tissues, SM22 alpha was expressed transiently in the heart between E8.0 and E12.5 and in skeletal muscle cells in the myotomal compartment of the somites between E9.5 and E12.5. The expression of SM22 alpha in smooth muscle cells, as well as early cardiac and skeletal muscle cells, suggests that there may be commonalities between the regulatory programs that direct muscle-specific gene expression in these three myogenic cell types.

Despite intense interest in understanding the differentiation of vascular smooth muscle, very little is known about the cellular and molecular mechanisms that control differentiation of this cell type. Progress in this field has been hampered by the lack of an inducible in vitro system for study of the early steps of smooth muscle differentiation. In this study, we describe a model system in which multipotential mouse P19 embryonal carcinoma cells (P19s) can be induced to express multiple characteristics of differentiated smooth muscle. Treatment of P19s with retinoic acid was associated with profound changes in cell morphology and with the appearance at high frequency of smooth muscle alpha-actin-positive cells that were absent or present at extremely low frequency in parental P19s. A clonal line derived from retinoic acid-treated P19s (9E11G) stably expressed multiple characteristics of differentiated smooth muscle, including smooth muscle-specific isoforms of alpha-actin and myosin heavy chain, as well as functional responses to the contractile agonists phenylephrine, angiotensin II, ATP, bradykinin, histamine, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Additionally, 9E11G cells expressed transcripts for MHox, a muscle homeobox gene expressed in smooth, cardiac, and skeletal muscles, but not the skeletal muscle-specific regulatory factors, MyoD and myogenin. Results demonstrate that retinoic acid treatment of multipotential P19 cells is associated with formation of cell lines that stably express multiple properties of differentiated smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

We cloned a portion of the mouse smooth muscle myosin heavy chain (SM-MHC) cDNA and analyzed its mRNA expression in adult tissues, several cell lines, and developing mouse embryos to determine the suitability of the SM-MHC promoter as a tool for identifying smooth muscle-specific transcription factors and to define the spatial and temporal pattern of smooth muscle differentiation during mouse development. RNase protection assays showed SM-MHC mRNA in adult aorta, intestine, lung, stomach, and uterus, with little or no signal in brain, heart, kidney, liver, skeletal muscle, spleen, and testes. From an analysis of 14 different cell lines, including endothelial cells, fibroblasts, and rhabdomyosarcomas, we failed to detect any SM-MHC mRNA; all of the cell lines induced to differentiate also showed no detectable SM-MHC. In situ hybridization of staged mouse embryos first revealed SM-MHC transcripts in the early developing aorta at 10.5 days post coitum (dpc). No hybridization signal was demonstrated beyond the aorta and its arches until 12.5 to 13.5 dpc, when SM-MHC mRNA appeared in smooth muscle cells (SMCs) of the developing gut and lungs as well as peripheral blood vessels. By 17.5 dpc, SM-MHC transcripts had accumulated in esophagus, bladder, and ureters. Except for blood vessels, no SM-MHC transcripts were ever observed in developing brain, heart, or skeletal muscle. These results indicate that smooth muscle myogenesis begins by 10.5 days of embryonic development in the mouse and establish SM-MHC as a highly specific marker for the SMC lineage. The SM-MHC promoter should therefore serve as a useful model for defining the mechanisms that govern SMC transcription during development and disease.

The two striated muscle cell types, skeletal and cardiac muscle, express overlapping sets of muscle-specific genes. Activation of muscle-specific transcription in skeletal muscle is controlled by the MyoD family of regulatory factors, which are expressed exclusively in skeletal muscle. Members of the MyoD family share homology within a basic helix-loop-helix (HLH) motif that mediates DNA binding and dimerization and form heterodimers with widely expressed HLH proteins, referred to as E proteins. Although many of the genes that are regulated by members of the MyoD family are also expressed in cardiac muscle, known members of the MyoD family have never been detected in cardiac muscle, suggesting that cardiac myocytes either express unique cell type-specific HLH proteins or rely on a distinct regulatory strategy for activation of cardiac muscle transcription. This review will summarize current knowledge of the mechanisms through which the MyoD family activates skeletal muscle transcription and will consider potential mechanisms that may regulate gene expression in the heart.