This study tested the hypothesis that nitric oxide (NO) and atrial natriuretic peptide (ANP) can attenuate the effects of adrenergic agonists on the growth of cardiac myocytes and fibroblasts. In ventricular cells cultured from neonatal rat heart, ANP and the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) caused concentration-dependent decreases in the norepinephrine (NE)-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts. In myocytes, the NO synthase inhibitor NG-monomethyl-L-arginine potentiated NE-stimulated [3H]leucine incorporation. In both cell types, ANP and SNAP increased intracellular cGMP levels, and their growth-suppressing effects were mimicked by the cGMP analogue 8-bromo-cGMP. Furthermore, in myocytes, 8-bromo-cGMP attenuated the alpha1-adrenergic receptor-stimulated increases in c-fos. Likewise, ANP and 8-bromo-cGMP attenuated the alpha1-adrenergic receptor- stimulated increase in prepro-ANP mRNA and the alpha1-adrenergic receptor-stimulated decrease in sarcoplasmic reticulum calcium ATPase mRNA. The L-type Ca2+ channel blockers verapamil and nifedipine inhibited NE-stimulated incorporation of [3H]leucine in myocytes and [3H]thymidine in fibroblasts, and these effects were not additive with those of ANP, SNAP, or 8-bromo-cGMP. In myocytes, the Ca2+ channel agonist BAY K8644 caused an increase in [3H]leucine incorporation which was inhibited by ANP. These findings indicate that NO and ANP can attenuate the effects of NE on the growth of cardiac myocytes and fibroblasts, most likely by a cGMP-mediated inhibition of NE-stimulated Ca2+ influx.

IntroductionMononuclear cell infiltration and local cytokine elaboration are hallmarks of inflammatory and immunologic heart diseases. To test the hypothesis that cytokines can modulate cardiac myocyte growth and phenotype, myocytes cultured from neonatal rat hearts were exposed to IL-1.3, an inflammatory cytokine prevalent in myocardial inflammation.IL-1p (2 ng/ml, 24 h) increased [3H]leucine incorporation by 30±4% (P < 0.001, n = 29) and net cellular protein content by 20±4% (P < 0.001, n = 27), but had no effect on DNA synthesis. Northern hybridization showed that IL-1j3 increased prepro-atrial natriuretic factor (ANF) mRNA (5.8+1.5-fold, P < 0.01, n = 13) and (8-myosin heavy chain ((3-MHC) mRNA (> 10-fold, n = 4), and decreased mRNA levels for sarcoplasmic reticulum Ca2+-ATPase (SERCA2) (-46±7%; P < 0.001; n = 11), calcium release channel (CRC) (-65±11%, P < 0.001, n = 8) and voltagedependent calcium channel (VDCC) (-53±7%, P < 0.001,, an inhibitor of nitric oxide (NO) synthesis, did not inhibit the IL-1i-induced protein synthesis or changes in mRNA levels. In ventricular myocardium obtained from adult rats treated with lipopolysaccharide (4 mg/kg intraperitoneally 18 h) to stimulate systemic cytokine production, there were changes in the mRNA levels for l-MHC (6±1-fold, P < 0.01, n = 4), SERCA2 (-65±4%, P < 0.0001, n = 4), CRC (-67±5%, P < 0.001, n = 4), and VDCC (-58±5%, P < 0.001; n = 4) that were qualitatively similar to those observed in cultured production by upregulating expression of inducible nitric oxide synthase in cardiac myocytes (8, 9) and could therefore act via cGMP, which has been shown to modulate growth in vascular smooth muscle cells (10). Thus, it is possible that cytokines contribute to the structural and functional alterations of the myocardium observed in states associated with myocardial inflammation.We hypothesized that exposure to inflammatory cytokines would cause myocyte hypertrophy associated with reexpression of fetal genes and downregulation of adult muscle-specific genes involved in maintaining calcium homeostasis. To test this hypothesis, cultured neonatal rat cardiac myocytes were exposed to IL-1,3, an inflammatory cytokine prevalent in myocarditis (11, 12). We examined the effect of IL-1,3 on protein synthesis, DNA synthesis, the mRNA levels for three fetal genes (prepro-atrial natriuretic factor, ANF; ,3-myosin heavy chain, f-MHC; and skeletal a-actin, a-SkA), and the mRNA levels for three calcium regulatory genes (sarcoplasmic reticulum Ca2+-ATPase, SERCA2; calcium release channel, CRC; and voltage-dependent calcium channel, VDCC). The effect of NGmonomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase (8), was determined to test the role of NO. To address the pathophysiologic relevance of the in vitro observations, we also examined the phenotypic changes induced in vivo in myocardium obtained from adult rats treated with LPS, a potent stimulus for elaborating several inflammatory cytokines (13). 2-d-old Sprague-Dawley rats killed by decapitation. ...

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases.

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