The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, the mechanism by which this occurs is not clearly defined. Here, we tested in mice the hypothesis that CD36, a class B scavenger receptor expressed on macrophages, has a role in this process. Using both in vivo and in vitro migration assays, we found that oxidized LDL (oxLDL), but not native LDL, inhibited migration of WT mouse macrophages but not CD36-deficient cells. We further observed a crucial role for CD36 in modulating the in vitro migratory response of human peripheral blood monocyte–derived macrophages to oxLDL. oxLDL also induced rapid spreading and actin polymerization in CD36-sufficient but not CD36-deficient mouse macrophages in vitro. The underlying mechanism was dependent on oxLDL-mediated CD36 signaling, which resulted in sustained activation of focal adhesion kinase (FAK) and inactivation of Src homology 2–containing phosphotyrosine phosphatase (SHP-2). The latter was due to NADPH oxidase–mediated ROS generation, resulting in oxidative inactivation of critical cysteine residues in the SHP-2–active site. Macrophage migration in the presence of oxLDL was restored by both antioxidants and NADPH oxidase inhibitors, which restored the dynamic activation of FAK. We conclude therefore that CD36 signaling in response to oxLDL alters cytoskeletal dynamics to enhance macrophage spreading, inhibiting migration. This may induce trapping of macrophages in the arterial intima and promote atherosclerosis.
Young Mi Park, Maria Febbraio, Roy L. Silverstein
Transcriptional upregulation of the proapoptotic BCL2 family protein NIX limits red blood cell formation and can cause heart failure by inducing cell death, but the requisite molecular events are poorly defined. Here, we show complementary mechanisms for NIX-mediated cell death involving direct and ER/sarcoplasmic reticulum–mediated (ER/SR-mediated) mitochondria disruption. Endogenous cardiac NIX and recombinant NIX localize both to the mitochondria and to the ER/SR. In genetic mouse models, cardiomyocyte ER/SR calcium stores are proportional to the level of expressed NIX. Whereas Nix ablation was protective in a mouse model of apoptotic cardiomyopathy, genetic correction of the decreased SR calcium content of Nix-null mice restored sensitivity to cell death and reestablished cardiomyopathy. Nix mutants specific to ER/SR or mitochondria activated caspases and were equally lethal, but only ER/SR-Nix caused loss of the mitochondrial membrane potential. These results establish a new function for NIX as an integrator of transcriptional and calcium-mediated signals for programmed cell death.
Abhinav Diwan, Scot J. Matkovich, Qunying Yuan, Wen Zhao, Atsuko Yatani, Joan Heller Brown, Jeffery D. Molkentin, Evangelia G. Kranias, Gerald W. Dorn II
In human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca2+ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca2+ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca2+-sensitizing agent EMD 57033 and prevented by myofilament Ca2+ desensitization with blebbistatin. Ca2+ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca2+ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca2+ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.
Franz Baudenbacher, Tilmann Schober, Jose Renato Pinto, Veniamin Y. Sidorov, Fredrick Hilliard, R. John Solaro, James D. Potter, Björn C. Knollmann
The response of cardiomyocytes to biomechanical stress can determine the pathophysiology of hypertrophic cardiac disease, and targeting the pathways regulating these responses is a therapeutic goal. However, little is known about how biomechanical stress is sensed by the cardiomyocyte sarcomere to transduce intracellular hypertrophic signals or how the dysfunction of these pathways may lead to disease. Here, we found that four-and-a-half LIM domains 1 (FHL1) is part of a complex within the cardiomyocyte sarcomere that senses the biomechanical stress–induced responses important for cardiac hypertrophy. Mice lacking Fhl1 displayed a blunted hypertrophic response and a beneficial functional response to pressure overload induced by transverse aortic constriction. A link to the Gαq (Gq) signaling pathway was also observed, as Fhl1 deficiency prevented the cardiomyopathy observed in Gq transgenic mice. Mechanistic studies demonstrated that FHL1 plays an important role in the mechanism of pathological hypertrophy by sensing biomechanical stress responses via the N2B stretch sensor domain of titin and initiating changes in the titin- and MAPK-mediated responses important for sarcomere extensibility and intracellular signaling. These studies shed light on the physiological regulation of the sarcomere in response to hypertrophic stress.
Farah Sheikh, Anna Raskin, Pao-Hsien Chu, Stephan Lange, Andrea A. Domenighetti, Ming Zheng, Xingqun Liang, Tong Zhang, Toshitaka Yajima, Yusu Gu, Nancy D. Dalton, Sushil K. Mahata, Gerald W. Dorn II, Joan Heller-Brown, Kirk L. Peterson, Jeffrey H. Omens, Andrew D. McCulloch, Ju Chen
Plasma HDL levels are inversely related to the incidence of atherosclerotic disease. Some of the atheroprotective effects of HDL are likely mediated via preservation of EC function. Whether the beneficial effects of HDL on ECs depend on its involvement in cholesterol efflux via the ATP-binding cassette transporters ABCA1 and ABCG1, which promote efflux of cholesterol and oxysterols from macrophages, has not been investigated. To address this, we assessed endothelial function in Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– mice fed either a high-cholesterol diet (HCD) or a Western diet (WTD). Non-atherosclerotic arteries from HCD-fed Abcg1–/– and Abca1–/–Abcg1–/– mice exhibited a marked decrease in endothelium-dependent vasorelaxation, while Abca1–/– mice had a milder defect. In addition, eNOS activity was reduced in aortic homogenates generated from Abcg1–/– mice fed either a HCD or a WTD, and this correlated with decreased levels of the active dimeric form of eNOS. More detailed analysis indicated that ABCG1 was expressed primarily in ECs, and that these cells accumulated the oxysterol 7-ketocholesterol (7-KC) when Abcg1–/– mice were fed a WTD. Consistent with these data, ABCG1 had a major role in promoting efflux of cholesterol and 7-KC in cultured human aortic ECs (HAECs). Furthermore, HDL treatment of HAECs prevented 7-KC–induced ROS production and active eNOS dimer disruption in an ABCG1-dependent manner. Our data suggest that ABCG1 and HDL maintain EC function in HCD-fed mice by promoting efflux of cholesterol and 7-oxysterols and preserving active eNOS dimer levels.
Naoki Terasaka, Shuiqing Yu, Laurent Yvan-Charvet, Nan Wang, Nino Mzhavia, Read Langlois, Tamara Pagler, Rong Li, Carrie L. Welch, Ira J. Goldberg, Alan R. Tall
Histone deacetylase (HDAC) inhibitors show remarkable therapeutic potential for a variety of disorders, including cancer, neurological disease, and cardiac hypertrophy. However, the specific HDAC isoforms that mediate their actions are unclear, as are the physiological and pathological functions of individual HDACs in vivo. To explore the role of Hdac3 in the heart, we generated mice with a conditional Hdac3 null allele. Although global deletion of Hdac3 resulted in lethality by E9.5, mice with a cardiac-specific deletion of Hdac3 survived until 3–4 months of age. At this time, they showed massive cardiac hypertrophy and upregulation of genes associated with fatty acid uptake, fatty acid oxidation, and electron transport/oxidative phosphorylation accompanied by fatty acid–induced myocardial lipid accumulation and elevated triglyceride levels. These abnormalities in cardiac metabolism can be attributed to excessive activity of the nuclear receptor PPARα. The phenotype associated with cardiac-specific Hdac3 gene deletion differs from that of all other Hdac gene mutations. These findings reveal a unique role for Hdac3 in maintenance of cardiac function and regulation of myocardial energy metabolism.
Rusty L. Montgomery, Matthew J. Potthoff, Michael Haberland, Xiaoxia Qi, Satoshi Matsuzaki, Kenneth M. Humphries, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson
Apelin and its cognate G protein–coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II–mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II–mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.
Hyung J. Chun, Ziad A. Ali, Yoko Kojima, Ramendra K. Kundu, Ahmad Y. Sheikh, Rani Agrawal, Lixin Zheng, Nicholas J. Leeper, Nathan E. Pearl, Andrew J. Patterson, Joshua P. Anderson, Philip S. Tsao, Michael J. Lenardo, Euan A. Ashley, Thomas Quertermous
Emerging metabolomic tools have created the opportunity to establish metabolic signatures of myocardial injury. We applied a mass spectrometry–based metabolite profiling platform to 36 patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of planned myocardial infarction (PMI). Serial blood samples were obtained before and at various intervals after PMI, with patients undergoing elective diagnostic coronary angiography and patients with spontaneous myocardial infarction (SMI) serving as negative and positive controls, respectively. We identified changes in circulating levels of metabolites participating in pyrimidine metabolism, the tricarboxylic acid cycle and its upstream contributors, and the pentose phosphate pathway. Alterations in levels of multiple metabolites were detected as early as 10 minutes after PMI in an initial derivation group and were validated in a second, independent group of PMI patients. A PMI-derived metabolic signature consisting of aconitic acid, hypoxanthine, trimethylamine N-oxide, and threonine differentiated patients with SMI from those undergoing diagnostic coronary angiography with high accuracy, and coronary sinus sampling distinguished cardiac-derived from peripheral metabolic changes. Our results identify a role for metabolic profiling in the early detection of myocardial injury and suggest that similar approaches may be used for detection or prediction of other disease states.
Gregory D. Lewis, Ru Wei, Emerson Liu, Elaine Yang, Xu Shi, Maryann Martinovic, Laurie Farrell, Aarti Asnani, Marcoli Cyrille, Arvind Ramanathan, Oded Shaham, Gabriel Berriz, Patricia A. Lowry, Igor F. Palacios, Murat Taşan, Frederick P. Roth, Jiangyong Min, Christian Baumgartner, Hasmik Keshishian, Terri Addona, Vamsi K. Mootha, Anthony Rosenzweig, Steven A. Carr, Michael A. Fifer, Marc S. Sabatine, Robert E. Gerszten
β3-adrenergic receptor (β3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β3-AR in the human atrium. Unexpectedly, β3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (ICa,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The ICa,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β3-AR agonists, SR58611 and BRL37344, and a β3-AR partial agonist, CGP12177, stimulated ICa,L in HAMs with nanomolar potency and a 60%–90% efficacy compared with isoprenaline. The β3-AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β3-AR antagonist L-748,337, β1-/β2-AR antagonist nadolol, and β1-/β2-/β3-AR antagonist bupranolol were used to confirm the involvement of β3-ARs (and not β1-/β2-ARs) in these effects. The β3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked ICa,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.
V. Arvydas Skeberdis, Vida Gendvilienė, Danguolė Zablockaitė, Rimantas Treinys, Regina Mačianskienė, Andrius Bogdelis, Jonas Jurevičius, Rodolphe Fischmeister
Hewang Li, Ines Armando, Peiying Yu, Crisanto Escano, Susette C. Mueller, Laureano Asico, Annabelle Pascua, Quansheng Lu, Xiaoyan Wang, Van Anthony M. Villar, John E. Jones, Zheng Wang, Ammasi Periasamy, Yuen-Sum Lau, Patricio Soares-da-Silva, Karen Creswell, Gaétan Guillemette, David R. Sibley, Gilbert Eisner, John J. Gildea, Robin A. Felder, Pedro A. Jose