Abstract
The role of cardiocytes in physiologic removal of apoptotic cells and the subsequent effect of surface binding by anti-SSA/Ro and -SSB/La antibodies was addressed. Initial experiments evaluated induction of apoptosis by extrinsic and intrinsic pathways. Nuclear injury and the translocation of SSA/Ro and SSB/La antigens to the fetal cardiocyte plasma membrane were common downstream events of Fas and TNF receptor ligation, requiring caspase activation. As assessed by phase-contrast and confirmed by confocal microscopy, coculturing of healthy cardiocytes with cardiocytes rendered apoptotic via extrinsic pathways revealed a clearance mechanism that to our knowledge has not previously been described. Cultured fetal cardiocytes expressed phosphatidylserine receptors (PSRs), as did cardiac tissue from a fetus with congenital heart block (CHB) and an age-matched control. Phagocytic uptake was blocked by anti-PSR antibodies and was significantly inhibited following preincubation of apoptotic cardiocytes with chicken and murine anti-SSA/Ro and -SSB/La antibodies, with IgG from an anti-SSA/Ro– and -SSB/La–positive mother of a CHB child, but not with anti–HLA class I antibody. In a murine model, anti-Ro60 bound and inhibited uptake of apoptotic cardiocytes from wild-type but not Ro60-knockout mice. Our results suggest that resident cardiocytes participate in physiologic clearance of apoptotic cardiocytes but that clearance is inhibited by opsonization via maternal autoantibodies, resulting in accumulation of apoptotic cells, promoting inflammation and subsequent scarring.
Authors
Robert M. Clancy, Petra J. Neufing, Ping Zheng, Marguerita O’Mahony, Falk Nimmerjahn, Tom P. Gordon, Jill P. Buyon
Abstract
Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II–elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with α1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.
Authors
Junlan Yao, Lucinda A. Davies, Jason D. Howard, Scott K. Adney, Philip J. Welsby, Nancy Howell, Robert M. Carey, Roger J. Colbran, Paula Q. Barrett
Abstract
Neurofibromatosis type I (NF1; also known as von Recklinghausen’s disease) is a common autosomal-dominant condition primarily affecting neural crest–derived tissues. The disease gene, NF1, encodes neurofibromin, a protein of over 2,800 amino acids that contains a 216–amino acid domain with Ras–GTPase-activating protein (Ras-GAP) activity. Potential therapies for NF1 currently in development and being tested in clinical trials are designed to modify NF1 Ras-GAP activity or target downstream effectors of Ras signaling. Mice lacking the murine homolog (Nf1) have mid-gestation lethal cardiovascular defects due to a requirement for neurofibromin in embryonic endothelium. We sought to determine whether the GAP activity of neurofibromin is sufficient to rescue complete loss of function or whether other as yet unidentified functions of neurofibromin might also exist. Using cre-inducible ubiquitous and tissue-specific expression, we demonstrate that the isolated GAP-related domain (GRD) rescued cardiovascular development in Nf1–/– embryos, but overgrowth of neural crest–derived tissues persisted, leading to perinatal lethality. These results suggest that neurofibromin may possess activities outside of the GRD that modulate neural crest homeostasis and that therapeutic approaches solely aimed at targeting Ras activity may not be sufficient to treat tumors of neural crest origin in NF1.
Authors
Fraz A. Ismat, Junwang Xu, Min Min Lu, Jonathan A. Epstein
Abstract
ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc–protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.
Authors
Johannes-Peter Stasch, Peter M. Schmidt, Pavel I. Nedvetsky, Tatiana Y. Nedvetskaya, Arun Kumar H.S., Sabine Meurer, Martin Deile, Ashraf Taye, Andreas Knorr, Harald Lapp, Helmut Müller, Yagmur Turgay, Christiane Rothkegel, Adrian Tersteegen, Barbara Kemp-Harper, Werner Müller-Esterl, Harald H.H.W. Schmidt
Abstract
Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca2+ storage protein essential for SR Ca2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca2+-binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca2+ leak, resulting in premature spontaneous SR Ca2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca2+ storage, lack of Casq2 also causes increased diastolic SR Ca2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.
Authors
Björn C. Knollmann, Nagesh Chopra, Thinn Hlaing, Brandy Akin, Tao Yang, Kristen Ettensohn, Barbara E.C. Knollmann, Kenneth D. Horton, Neil J. Weissman, Izabela Holinstat, Wei Zhang, Dan M. Roden, Larry R. Jones, Clara Franzini-Armstrong, Karl Pfeifer
Abstract
The carboxypeptidase ACE2 is a homologue of angiotensin-converting enzyme (ACE). To clarify the physiological roles of ACE2, we generated mice with targeted disruption of the Ace2 gene. ACE2-deficient mice were viable, fertile, and lacked any gross structural abnormalities. We found normal cardiac dimensions and function in ACE2-deficient animals with mixed or inbred genetic backgrounds. On the C57BL/6 background, ACE2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ACE2 had no effect on baseline blood pressures in 129/SvEv mice. After acute Ang II infusion, plasma concentrations of Ang II increased almost 3-fold higher in ACE2-deficient mice than in controls. In a model of Ang II–dependent hypertension, blood pressures were substantially higher in the ACE2-deficient mice than in WT. Severe hypertension in ACE2-deficient mice was associated with exaggerated accumulation of Ang II in the kidney, as determined by MALDI-TOF mass spectrometry. Although the absence of functional ACE2 causes enhanced susceptibility to Ang II–induced hypertension, we found no evidence for a role of ACE2 in the regulation of cardiac structure or function. Our data suggest that ACE2 is a functional component of the renin-angiotensin system, metabolizing Ang II and thereby contributing to regulation of blood pressure.
Authors
Susan B. Gurley, Alicia Allred, Thu H. Le, Robert Griffiths, Lan Mao, Nisha Philip, Timothy A. Haystead, Mary Donoghue, Roger E. Breitbart, Susan L. Acton, Howard A. Rockman, Thomas M. Coffman
Abstract
Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit+ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit+ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.
Authors
Shafie Fazel, Massimo Cimini, Liwen Chen, Shuhong Li, Denis Angoulvant, Paul Fedak, Subodh Verma, Richard D. Weisel, Armand Keating, Ren-Ke Li
Abstract
Class IIa histone deacetylases (HDACs) regulate a variety of cellular processes, including cardiac growth, bone development, and specification of skeletal muscle fiber type. Multiple serine/threonine kinases control the subcellular localization of these HDACs by phosphorylation of common serine residues, but whether certain class IIa HDACs respond selectively to specific kinases has not been determined. Here we show that calcium/calmodulin-dependent kinase II (CaMKII) signals specifically to HDAC4 by binding to a unique docking site that is absent in other class IIa HDACs. Phosphorylation of HDAC4 by CaMKII promotes nuclear export and prevents nuclear import of HDAC4, with consequent derepression of HDAC target genes. In cardiomyocytes, CaMKII phosphorylation of HDAC4 results in hypertrophic growth, which can be blocked by a signal-resistant HDAC4 mutant. These findings reveal a central role for HDAC4 in CaMKII signaling pathways and have implications for the control of gene expression by calcium signaling in a variety of cell types.
Authors
Johannes Backs, Kunhua Song, Svetlana Bezprozvannaya, Shurong Chang, Eric N. Olson
Abstract
Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/β-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.
Authors
Eduardo Garcia-Gras, Raffaella Lombardi, Michael J. Giocondo, James T. Willerson, Michael D. Schneider, Dirar S. Khoury, Ali J. Marian
Abstract
Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor–knockout/reporter gene–knock-in (A2BAR-knockout/reporter gene–knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-α, and a consequent downregulation of IκB-α are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.
Authors
Dan Yang, Ying Zhang, Hao G. Nguyen, Milka Koupenova, Anil K. Chauhan, Maria Makitalo, Matthew R. Jones, Cynthia St. Hilaire, David C. Seldin, Paul Toselli, Edward Lamperti, Barbara M. Schreiber, Haralambos Gavras, Denisa D. Wagner, Katya Ravid
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ISSN: 0021-9738 (print), 1558-8238 (online)