Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), α-skeletal actin (1.7-fold), and β-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7–10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.
Rita Holtwick, Martin van Eickels, Boris V. Skryabin, Hideo A. Baba, Alexander Bubikat, Frank Begrow, Michael D. Schneider, David L. Garbers, Michaela Kuhn
Scott P. Heximer, Russell H. Knutsen, Xiaoguang Sun, Kevin M. Kaltenbronn, Man-Hee Rhee, Ning Peng, Antonio Oliveira-dos-Santos, Josef M. Penninger, Anthony J. Muslin, Thomas H. Steinberg, J. Michael Wyss, Robert P. Mecham, Kendall J. Blumer
The chemokine receptor CX3CR1 is a proinflammatory leukocyte receptor specific for the chemokine fractalkine (FKN or CX3CL1). In two retrospective studies, CX3CR1 has been implicated in the pathogenesis of atherosclerotic cardiovascular disease (CVD) based on statistical association of a common receptor variant named CX3CR1-M280 with lower prevalence of atherosclerosis, coronary endothelial dysfunction, and acute coronary syndromes. However, the general significance of CX3CR1-M280 and its putative mechanism of action have not previously been defined. Here we show that FKN-dependent cell-cell adhesion under conditions of physiologic shear is severely reduced in cells expressing CX3CR1-M280. This was associated with marked reduction in the kinetics of FKN binding as well as reduced FKN-induced chemotaxis of primary leukocytes from donors homozygous for CX3CR1-M280. We also show that CX3CR1-M280 is independently associated with a lower risk of CVD (adjusted odds ratio, 0.60, P = 0.008) in the Offspring Cohort of the Framingham Heart Study, a long-term prospective study of the risks and natural history of this disease. These data provide mechanism-based and consistent epidemiologic evidence that CX3CR1 may be involved in the pathogenesis of CVD in humans, possibly by supporting leukocyte entry into the coronary artery wall. Moreover, they suggest that CX3CR1-M280 is a genetic risk factor for CVD.
David H. McDermott, Alan M. Fong, Qiong Yang, Joan M. Sechler, L. Adrienne Cupples, Maya N. Merrell, Peter W.F. Wilson, Ralph B. D’Agostino, Christopher J. O’Donnell, Dhavalkumar D. Patel, Philip M. Murphy
Cellular proliferation, migration, and expression of extracellular matrix proteins and MMPs contribute to neointimal formation upon vascular injury. Wild-type mice undergoing arterial endothelial denudation displayed striking upregulation of receptor for advanced glycation end products (RAGE) in the injured vessel, particularly in activated smooth muscle cells of the expanding neointima. In parallel, two of RAGE’s signal transducing ligands, advanced glycation end products (AGEs) and S100/calgranulins, demonstrated increased deposition/expression in the injured vessel wall. Blockade of RAGE, employing soluble truncated receptor or antibodies, or in homozygous RAGE null mice, resulted in significantly decreased neointimal expansion after arterial injury and decreased smooth muscle cell proliferation, migration, and expression of extracellular matrix proteins. A critical role for smooth muscle cell RAGE signaling was demonstrated in mice bearing a transgene encoding a RAGE cytosolic tail-deletion mutant, specifically in smooth muscle cells, driven by the SM22α promoter. Upon arterial injury, neointimal expansion was strikingly suppressed compared with that observed in wild-type littermates. Taken together, these data highlight key roles for RAGE in modulating smooth muscle cell properties after injury and suggest that RAGE is a logical target for suppression of untoward neointimal expansion consequent to arterial injury.
Taichi Sakaguchi, Shi Fang Yan, Shi Du Yan, Dmitri Belov, Ling Ling Rong, Monica Sousa, Martin Andrassy, Steven P. Marso, Stephan Duda, Bernd Arnold, Birgit Liliensiek, Peter P. Nawroth, David M. Stern, Ann Marie Schmidt, Yoshifumi Naka
Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis.
Chao-Ling Yang, Jordan Angell, Rose Mitchell, David H. Ellison
Cardiac hypertrophy is a common response to pressure overload and is associated with increased mortality. Mechanical stress in the heart can result in the integrin-mediated activation of focal adhesion kinase and the subsequent recruitment of the Grb2 adapter molecule. Grb2, in turn, can activate MAPK cascades via an interaction with the Ras guanine nucleotide exchange factor SOS and with other signaling intermediates. We analyzed the role of the Grb2 adapter protein and p38 MAPK in cardiac hypertrophy. Mice with haploinsufficiency of the Grb2 gene (Grb2+/– mice) appear normal at birth but have defective T cell signaling. In response to pressure overload, cardiac p38 MAPK and JNK activation was inhibited and cardiac hypertrophy and fibrosis was blocked in Grb2+/– mice. Next, transgenic mice with cardiac-specific expression of dominant negative forms of p38α (DN-p38α) and p38β (DN-p38β) MAPK were examined. DN-p38α and DN-p38β mice developed cardiac hypertrophy but were resistant to cardiac fibrosis in response to pressure overload. These results establish that Grb2 action is essential for cardiac hypertrophy and fibrosis in response to pressure overload, and that different signaling pathways downstream of Grb2 regulate fibrosis, fetal gene induction, and cardiomyocyte growth.
Shaosong Zhang, Carla Weinheimer, Michael Courtois, Attila Kovacs, Cindy E. Zhang, Alec M. Cheng, Yibin Wang, Anthony J. Muslin
Jens Mogensen, Toru Kubo, Mauricio Duque, William Uribe, Anthony Shaw, Ross Murphy, Juan R. Gimeno, Perry Elliott, William J. McKenna
Cardiac hypertrophy, either compensated or decompensated, is associated with cardiomyocyte contractile dysfunction from depressed sarcoplasmic reticulum (SR) Ca2+ cycling. Normalization of Ca2+ cycling by ablation or inhibition of the SR inhibitor phospholamban (PLN) has prevented cardiac failure in experimental dilated cardiomyopathy and is a promising therapeutic approach for human heart failure. However, the potential benefits of restoring SR function on primary cardiac hypertrophy, a common antecedent of human heart failure, are unknown. We therefore tested the efficacy of PLN ablation to correct hypertrophy and contractile dysfunction in two well-characterized and highly relevant genetic mouse models of hypertrophy and cardiac failure, Gαq overexpression and human familial hypertrophic cardiomyopathy mutant myosin binding protein C (MyBP-CMUT) expression. In both models, PLN ablation normalized the characteristically prolonged cardiomyocyte Ca2+ transients and enhanced unloaded fractional shortening with no change in SR Ca2+ pump content. However, there was no parallel improvement in in vivo cardiac function or hypertrophy in either model. Likewise, the activation of JNK and calcineurin associated with Gαq overexpression was not affected. Thus, PLN ablation normalized contractility in isolated myocytes, but failed to rescue the cardiomyopathic phenotype elicited by activation of the Gαq pathway or MyBP-C mutations.
Qiujing Song, Albrecht G. Schmidt, Harvey S. Hahn, Andrew N. Carr, Beate Frank, Luke Pater, Mike Gerst, Karen Young, Brian D. Hoit, Bradley K. McConnell, Kobra Haghighi, Christine E. Seidman, Jonathan G. Seidman, Gerald W. Dorn II, Evangelia G. Kranias
In human disease and experimental animal models, depressed Ca2+ handling in failing cardiomyocytes is widely attributed to impaired sarcoplasmic reticulum (SR) function. In mice, disruption of the PLN gene encoding phospholamban (PLN) or expression of dominant-negative PLN mutants enhances SR and cardiac function, but effects of PLN mutations in humans are unknown. Here, a T116G point mutation, substituting a termination codon for Leu-39 (L39stop), was identified in two families with hereditary heart failure. The heterozygous individuals exhibited hypertrophy without diminished contractile performance. Strikingly, both individuals homozygous for L39stop developed dilated cardiomyopathy and heart failure, requiring cardiac transplantation at ages 16 and 27. An over 50% reduction in PLN mRNA and no detectable PLN protein were noted in one explanted heart. The expression of recombinant PLN-L39stop in human embryonic kidney (HEK) 293 cells and adult rat cardiomyocytes showed no PLN inhibition of SR Ca2+-ATPase and the virtual absence of stable PLN expression; where PLN was expressed, it was misrouted to the cytosol or plasma membrane. These findings describe a naturally-occurring loss-of-function human PLN mutation (PLN null). In contrast to reported benefits of PLN ablation in mouse heart failure, humans lacking PLN develop lethal dilated cardiomyopathy.
Kobra Haghighi, Fotis Kolokathis, Luke Pater, Roy A. Lynch, Michio Asahi, Anthony O. Gramolini, Guo-Chang Fan, Dimitris Tsiapras, Harvey S. Hahn, Stamatis Adamopoulos, Stephen B. Liggett, Gerald W. Dorn II, David H. MacLennan, Dimitrios T. Kremastinos, Evangelia G. Kranias
Elevation of lung capillary pressure causes exocytosis of the leukocyte adhesion receptor P-selectin in endothelial cells (ECs), indicating that lung ECs generate a proinflammatory response to pressure-induced stress. To define underlying mechanisms, we followed the EC signaling sequence leading to P-selectin exocytosis through application of real-time, in situ fluorescence microscopy in lung capillaries. Pressure elevation increased the amplitude of cytosolic Ca2+ oscillations that triggered increases in the amplitude of mitochondrial Ca2+ oscillations and in reactive oxygen species (ROS) production. Responses to blockers of the Ca2+ oscillations and of mitochondrial electron transport indicated that the ROS production was Ca2+ dependent and of mitochondrial origin. A new proinflammatory mechanism was revealed in that pressure-induced exocytosis of P-selectin was inhibited by both antioxidants and mitochondrial inhibitors, indicating that the exocytosis was driven by mitochondrial ROS. In this signaling pathway mitochondria coupled pressure-induced Ca2+ oscillations to the production of ROS that in turn acted as diffusible messengers to activate P-selectin exocytosis. These findings implicate mitochondrial mechanisms in the lung’s proinflammatory response to pressure elevation and identify mitochondrial ROS as critical to P-selectin exocytosis in lung capillary ECs.
Hideo Ichimura, Kaushik Parthasarathi, Sadiqa Quadri, Andrew C. Issekutz, Jahar Bhattacharya