Abstract
Maintenance of skeletal and cardiac muscle structure and function requires precise control of the synthesis, assembly, and turnover of contractile proteins of the sarcomere. Abnormalities in accumulation of sarcomere proteins are responsible for a variety of myopathies. However, the mechanisms that mediate turnover of these long-lived proteins remain poorly defined. We show that muscle RING finger 1 (MuRF1) and MuRF3 act as E3 ubiquitin ligases that cooperate with the E2 ubiquitin–conjugating enzymes UbcH5a, -b, and -c to mediate the degradation of β/slow myosin heavy chain (β/slow MHC) and MHCIIa via the ubiquitin proteasome system (UPS) in vivo and in vitro. Accordingly, mice deficient for MuRF1 and MuRF3 develop a skeletal muscle myopathy and hypertrophic cardiomyopathy characterized by subsarcolemmal MHC accumulation, myofiber fragmentation, and diminished muscle performance. These findings identify MuRF1 and MuRF3 as key E3 ubiquitin ligases for the UPS-dependent turnover of sarcomeric proteins and reveal a potential basis for myosin storage myopathies.
Authors
Jens Fielitz, Mi-Sung Kim, John M. Shelton, Shuaib Latif, Jeffrey A. Spencer, David J. Glass, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson
Abstract
Noonan syndrome (NS) is an autosomal dominant disorder characterized by a wide spectrum of defects, which most frequently include proportionate short stature, craniofacial anomalies, and congenital heart disease (CHD). NS is the most common nonchromosomal cause of CHD, and 80%–90% of NS patients have cardiac involvement. Mutations within the protein tyrosine phosphatase Src homology region 2, phosphatase 2 (SHP2) are responsible for approximately 50% of the cases of NS with cardiac involvement. To understand the developmental stage– and cell type–specific consequences of the NS SHP2 gain-of-function mutation, Q79R, we generated transgenic mice in which the mutated protein was expressed during gestation or following birth in cardiomyocytes. Q79R SHP2 embryonic hearts showed altered cardiomyocyte cell cycling, ventricular noncompaction, and ventricular septal defects, while, in the postnatal cardiomyocyte, Q79R SHP2 expression was completely benign. Fetal expression of Q79R led to the specific activation of the ERK1/2 pathway, and breeding of the Q79R transgenics into ERK1/2-null backgrounds confirmed the pathway’s necessity and sufficiency in mediating mutant SHP2’s effects. Our data establish the developmental stage–specific effects of Q79R cardiac expression in NS; show that ablation of subsequent ERK1/2 activation prevents the development of cardiac abnormalities; and suggest that ERK1/2 modulation could have important implications for developing therapeutic strategies in CHD.
Authors
Tomoki Nakamura, Melissa Colbert, Maike Krenz, Jeffery D. Molkentin, Harvey S. Hahn, Gerald W. Dorn II, Jeffrey Robbins
Abstract
Liver X receptors (LXRs) α and β are transcriptional regulators of cholesterol homeostasis and potential targets for the development of antiatherosclerosis drugs. However, the specific roles of individual LXR isotypes in atherosclerosis and the pharmacological effects of synthetic agonists remain unclear. Previous work has shown that mice lacking LXRα accumulate cholesterol in the liver but not in peripheral tissues. In striking contrast, we demonstrate here that LXRα–/–apoE–/– mice exhibit extreme cholesterol accumulation in peripheral tissues, a dramatic increase in whole-body cholesterol burden, and accelerated atherosclerosis. The phenotype of these mice suggests that the level of LXR pathway activation in macrophages achieved by LXRβ and endogenous ligand is unable to maintain homeostasis in the setting of hypercholesterolemia. Surprisingly, however, a highly efficacious synthetic agonist was able to compensate for the loss of LXRα. Treatment of LXRα–/–apoE–/– mice with synthetic LXR ligand ameliorates the cholesterol overload phenotype and reduces atherosclerosis. These observations indicate that LXRα has an essential role in maintaining peripheral cholesterol homeostasis in the context of hypercholesterolemia and provide in vivo support for drug development strategies targeting LXRβ.
Authors
Michelle N. Bradley, Cynthia Hong, Mingyi Chen, Sean B. Joseph, Damien C. Wilpitz, Xuping Wang, Aldons J. Lusis, Allan Collins, Willa A. Hseuh, Jon L. Collins, Rajendra K. Tangirala, Peter Tontonoz
Abstract
Macrophage ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), and ABCG1 have been shown to promote cholesterol efflux to extracellular acceptors in vitro and influence atherosclerosis in mice, but their roles in mediating reverse cholesterol transport (RCT) from macrophages in vivo are unknown. Using an assay of macrophage RCT in mice, we found that primary macrophages lacking ABCA1 had a significant reduction in macrophage RCT in vivo, demonstrating the importance of ABCA1 in promoting macrophage RCT, however substantial residual RCT exists in the absence of macrophage ABCA1. Using primary macrophages deficient in SR-BI expression, we found that macrophage SR-BI, which was shown to promote cholesterol efflux in vitro, does not contribute to macrophage RCT in vivo. To investigate whether macrophage ABCG1 is involved in macrophage RCT in vivo, we used ABCG1-overexpressing, -knockdown, and -knockout macrophages. We show that increased macrophage ABCG1 expression significantly promoted while knockdown or knockout of macrophage ABCG1 expression significantly reduced macrophage RCT in vivo. Finally, we show that there was a greater decrease in macrophage RCT from cells where both ABCA1 and ABCG1 expression were knocked down than from ABCG1-knockdown cells. These results demonstrate that ABCA1 and ABCG1, but not SR-BI, promote macrophage RCT in vivo and are additive in their effects.
Authors
Xun Wang, Heidi L. Collins, Mollie Ranalletta, Ilia V. Fuki, Jeffrey T. Billheimer, George H. Rothblat, Alan R. Tall, Daniel J. Rader
Abstract
The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT1A), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT1A leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.
Authors
Sandrine Billet, Sabine Bardin, Sonia Verp, Véronique Baudrie, Annie Michaud, Sophie Conchon, Martine Muffat-Joly, Brigitte Escoubet, Evelyne Souil, Ghislaine Hamard, Kenneth E. Bernstein, Jean Marie Gasc, Jean-Luc Elghozi, Pierre Corvol, Eric Clauser
Abstract
Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1β (NRG-1β, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1β/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1β induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1β upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1β/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.
Authors
Yoshikazu Nakaoka, Keigo Nishida, Masahiro Narimatsu, Atsunori Kamiya, Takashi Minami, Hirofumi Sawa, Katsuya Okawa, Yasushi Fujio, Tatsuya Koyama, Makiko Maeda, Manami Sone, Satoru Yamasaki, Yuji Arai, Gou Young Koh, Tatsuhiko Kodama, Hisao Hirota, Kinya Otsu, Toshio Hirano, Naoki Mochizuki
Abstract
The anterior heart field (AHF), which contributes to the outflow tract and right ventricle of the heart, is defined in part by expression of the LIM homeobox transcription factor Isl-1. The importance of Isl-1–positive cells in cardiac development and homeostasis is underscored by the finding that these cells are required for cardiac development and act as cardiac stem/progenitor cells within the postnatal heart. However, the molecular pathways regulating these cells’ expansion and differentiation are poorly understood. We show that Isl-1–positive AHF progenitor cells in mice were responsive to Wnt/β-catenin signaling, and these responsive cells contributed to the outflow tract and right ventricle of the heart. Loss of Wnt/β-catenin signaling in the AHF caused defective outflow tract and right ventricular development with a decrease in Isl-1–positive progenitors and loss of FGF signaling. Conversely, Wnt gain of function in these cells led to expansion of Isl-1–positive progenitors with a concomitant increase in FGF signaling through activation of a specific set of FGF ligands including FGF3, FGF10, FGF16, and FGF20. These data reveal what we believe to be a novel Wnt-FGF signaling axis required for expansion of Isl-1–positive AHF progenitors and suggest future therapies to increase the number and function of these cells for cardiac regeneration.
Authors
Ethan David Cohen, Zhishan Wang, John J. Lepore, Min Min Lu, Makoto M. Taketo, Douglas J. Epstein, Edward E. Morrisey
Abstract
ST2 is an IL-1 receptor family member with transmembrane (ST2L) and soluble (sST2) isoforms. sST2 is a mechanically induced cardiomyocyte protein, and serum sST2 levels predict outcome in patients with acute myocardial infarction or chronic heart failure. Recently, IL-33 was identified as a functional ligand of ST2L, allowing exploration of the role of ST2 in myocardium. We found that IL-33 was a biomechanically induced protein predominantly synthesized by cardiac fibroblasts. IL-33 markedly antagonized angiotensin II– and phenylephrine-induced cardiomyocyte hypertrophy. Although IL-33 activated NF-κB, it inhibited angiotensin II– and phenylephrine-induced phosphorylation of inhibitor of NF-κBα (IκBα) and NF-κB nuclear binding activity. sST2 blocked antihypertrophic effects of IL-33, indicating that sST2 functions in myocardium as a soluble decoy receptor. Following pressure overload by transverse aortic constriction (TAC), ST2–/– mice had more left ventricular hypertrophy, more chamber dilation, reduced fractional shortening, more fibrosis, and impaired survival compared with WT littermates. Furthermore, recombinant IL-33 treatment reduced hypertrophy and fibrosis and improved survival after TAC in WT mice, but not in ST2–/– littermates. Thus, IL-33/ST2 signaling is a mechanically activated, cardioprotective fibroblast-cardiomyocyte paracrine system, which we believe to be novel. IL-33 may have therapeutic potential for beneficially regulating the myocardial response to overload.
Authors
Shoji Sanada, Daihiko Hakuno, Luke J. Higgins, Eric R. Schreiter, Andrew N.J. McKenzie, Richard T. Lee
Abstract
Mutations in LMNA, which encodes nuclear Lamins A and C cause diseases affecting various organs, including the heart. We have determined the effects of an Lmna H222P mutation on signaling pathways involved in the development of cardiomyopathy in a knockin mouse model of autosomal dominant Emery-Dreifuss muscular dystrophy. Analysis of genome-wide expression profiles in hearts using Affymetrix GeneChips showed statistically significant differences in expression of genes in the MAPK pathways at the incipience of the development of clinical disease. Using real-time PCR, we showed that activation of MAPK pathways preceded clinical signs or detectable molecular markers of cardiomyopathy. In heart tissue and isolated cardiomyocytes, there was activation of MAPK cascades and downstream targets, implicated previously in the pathogenesis of cardiomyopathy. Expression of H222P Lamin A in cultured cells activated MAPKs and downstream target genes. Activation of MAPK signaling by mutant A-type lamins could be a cornerstone in the development of heart disease in autosomal dominant Emery-Dreifuss muscular dystrophy.
Authors
Antoine Muchir, Paul Pavlidis, Valérie Decostre, Alan J. Herron, Takuro Arimura, Gisèle Bonne, Howard J. Worman
Abstract
Studies with isolated membrane fractions have shown that calmodulin (CaM) inhibits the activity of cardiac muscle cell Ca2+ release channel ryanodine receptor 2 (RyR2). To determine the physiological importance of CaM regulation of RyR2, we generated a mouse with 3 amino acid substitutions (RyR2-W3587A/L3591D/F3603A) in exon 75 of the Ryr2 gene, which encodes the CaM-binding site of RyR2. Homozygous mutant mice showed an increased ratio of heart weight to body weight, greatly reduced fractional shortening of the left ventricle, and lethality at 9–16 days of age. Biochemical analysis of hearts of 7- and 10-day-old homozygous mutant mice indicated an impaired CaM inhibition of RyR2 at micromolar Ca2+ concentrations, reduction in RyR2 protein levels and sarcoplasmic reticulum Ca2+ sequestration, and upregulation of genes and/or proteins associated with class II histone deacetylase/myocyte enhancer factor-2 and calcineurin signaling pathways. Sustained Ca2+ transients, often displaying repeated periods of incomplete Ca2+ removal, were observed in homozygous cardiomyocytes. Taken together, the data indicate that impaired CaM inhibition of RyR2, associated with defective sarcoplasmic reticulum Ca2+ release and altered gene expression, leads to cardiac hypertrophy and early death.
Authors
Naohiro Yamaguchi, Nobuyuki Takahashi, Le Xu, Oliver Smithies, Gerhard Meissner
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ISSN: 0021-9738 (print), 1558-8238 (online)