In ventricular fibrillation (VF), the principal cause of sudden cardiac death, waves of electrical excitation break up into turbulent and incoherent fragments. The causes of this breakup have been intensely debated. Breakup can be caused by fixed anatomical properties of the tissue, such as the biventricular geometry and the inherent anisotropy of cardiac conduction. However, wavebreak can also be caused purely by instabilities in wave conduction that arise from ion channel dynamics, which represent potential targets for drug action. To study the interaction between these two wave-breaking mechanisms, we used a physiologically based mathematical model of the ventricular cell, together with a realistic three-dimensional computer model of cardiac anatomy, including the distribution of fiber angles throughout the myocardium. We find that dynamical instabilities remain a major cause of the wavebreak that drives VF, even in an anatomically realistic heart. With cell physiology in its usual operating regime, dynamics and anatomical features interact to promote wavebreak and VF. However, if dynamical instability is reduced, for example by modeling of certain pharmacologic interventions, electrical waves do not break up into fibrillation, despite anatomical complexity. Thus, interventions that promote dynamical wave stability show promise as an antifibrillatory strategy in this more realistic setting.
Fagen Xie, Zhilin Qu, Junzhong Yang, Ali Baher, James N. Weiss, Alan Garfinkel
Ablation or inhibition of phospholamban (PLN) has favorable effects in several genetic murine dilated cardiomyopathies, and we showed previously that a pseudophosphorylated form of PLN mutant (S16EPLN) successfully prevented progressive heart failure in cardiomyopathic hamsters. In this study, the effects of PLN inhibition were examined in rats with heart failure after myocardial infarction (MI), a model of acquired disease. S16EPLN was delivered into failing hearts 5 weeks after MI by transcoronary gene transfer using a recombinant adeno-associated virus (rAAV) vector. In treated (MI-S16EPLN, n = 16) and control (MI-saline, n = 18) groups, infarct sizes were closely matched and the left ventricle was similarly depressed and dilated before gene transfer. At 2 and 6 months after gene transfer, MI-S16EPLN rats showed an increase in left ventricular (LV) ejection fraction and a much smaller rise in LV end-diastolic volume, compared with progressive deterioration of LV size and function in MI-saline rats. Hemodynamic measurements at 6 months showed lower LV end-diastolic pressures, with enhanced LV function (contractility and relaxation), lowered LV mass and myocyte size, and less fibrosis in MI-S16EPLN rats. Thus, PLN inhibition by in vivo rAAV gene transfer is an effective strategy for the chronic treatment of an acquired form of established heart failure.
Yoshitaka Iwanaga, Masahiko Hoshijima, Yusu Gu, Mitsuo Iwatate, Thomas Dieterle, Yasuhiro Ikeda, Moto-o Date, Jacqueline Chrast, Masunori Matsuzaki, Kirk L. Peterson, Kenneth R. Chien, John Ross Jr.
Use of long-term constitutive expression of VEGF for therapeutic angiogenesis may be limited by the growth of abnormal blood vessels and hemangiomas. We investigated the relationship between VEGF dosage and the morphology and function of newly formed blood vessels by implanting retrovirally transduced myoblasts that constitutively express VEGF164 into muscles of adult mice. Reducing VEGF dosage by decreasing the total number of VEGF myoblasts implanted did not prevent vascular abnormalities. However, when clonal populations of myoblasts homogeneously expressing different levels of VEGF were implanted, a threshold between normal and aberrant angiogenesis was found. Clonal myoblasts that expressed low to medium levels of VEGF induced growth of stable, pericyte-coated capillaries of uniform size that were not leaky and became VEGF independent, as shown by treatment with the potent VEGF blocker VEGF-TrapR1R2. In contrast, clones that expressed high levels of VEGF induced hemangiomas. Remarkably, when different clonal populations were mixed, even a small proportion of cells with high production of VEGF was sufficient to cause hemangioma growth. These results show for the first time to our knowledge that the key determinant of whether VEGF-induced angiogenesis is normal or aberrant is the microenvironmental amount of growth factor secreted, rather than the overall dose. Long-term continuous delivery of VEGF, when maintained below a threshold microenvironmental level, can lead to normal angiogenesis without other exogenous growth factors.
Clare R. Ozawa, Andrea Banfi, Nicole L. Glazer, Gavin Thurston, Matthew L. Springer, Peggy E. Kraft, Donald M. McDonald, Helen M. Blau
HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S1P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S1P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S1P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S1P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.
Jerzy-Roch Nofer, Markus van der Giet, Markus Tölle, Iza Wolinska, Karin von Wnuck Lipinski, Hideo A. Baba, Uwe J. Tietge, Axel Gödecke, Isao Ishii, Burkhard Kleuser, Michael Schäfers, Manfred Fobker, Walter Zidek, Gerd Assmann, Jerold Chun, Bodo Levkau
HDL and its associated apo, APOE, inhibit S-phase entry of murine aortic smooth muscle cells. We report here that the antimitogenic effect of APOE maps to the N-terminal receptor–binding domain, that APOE and its N-terminal domain inhibit activation of the cyclin A promoter, and that these effects involve both pocket protein–dependent and independent pathways. These antimitogenic effects closely resemble those seen in response to activation of the prostacyclin receptor IP. Indeed, we found that HDL and APOE suppress aortic smooth muscle cell cycle progression by stimulating Cox-2 expression, leading to prostacyclin synthesis and an IP-dependent inhibition of the cyclin A gene. Similar results were detected in human aortic smooth muscle cells and in vivo using mice overexpressing APOE. Our results identify the Cox-2 gene as a target of APOE signaling, link HDL and APOE to IP action, and describe a potential new basis for the cardioprotective effect of HDL and APOE.
Devashish Kothapalli, Ilia Fuki, Kamilah Ali, Sheryl A. Stewart, Liang Zhao, Ron Yahil, David Kwiatkowski, Elizabeth A. Hawthorne, Garret A. FitzGerald, Michael C. Phillips, Sissel Lund-Katz, Ellen Puré, Daniel J. Rader, Richard K. Assoian
Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C–deficient (Lmna–/–) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna–/– myocytes show reduced shortening with normal baseline and peak amplitude of Ca2+ transients. Lmna–/– LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna–/– cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARγ expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna–/– mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna–/– cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.
Vesna Nikolova, Christiana Leimena, Aisling C. McMahon, Ju Chiat Tan, Suchitra Chandar, Dilesh Jogia, Scott H. Kesteven, Jan Michalicek, Robyn Otway, Fons Verheyen, Stephen Rainer, Colin L. Stewart, David Martin, Michael P. Feneley, Diane Fatkin
Prior studies have shown that PI3Ks play a necessary but incompletely defined role in platelet activation. One potential effector for PI3K is the serine/threonine kinase, Akt, whose contribution to platelet activation was explored here. Two isoforms of Akt were detected in mouse platelets, with expression of Akt2 being greater than Akt1. Deletion of the gene encoding Akt2 impaired platelet aggregation, fibrinogen binding, and granule secretion, especially in response to low concentrations of agonists that activate the Gq-coupled receptors for thrombin and thromboxane A2. Loss of Akt2 also impaired arterial thrombus formation and stability in vivo, despite having little effect on platelet responses to collagen and ADP. In contrast, reducing Akt1 expression had no effect except when Akt2 was also deleted. Activation of Akt by thrombin was abolished by deletion of Gαq but was relatively unaffected by deletion of Gαi2, which abolished Akt activation by ADP. From these results we conclude that Akt2 is a necessary component of PI3K-dependent signaling downstream of Gq-coupled receptors, promoting thrombus growth and stability in part by supporting secretion. The contribution of Akt1 is less evident except in the setting in which Akt2 is absent.
Donna Woulfe, Hong Jiang, Alicia Morgans, Robert Monks, Morris Birnbaum, Lawrence F. Brass
Recent studies suggest that mitochondrial aldehyde dehydrogenase (ALDH-2) plays a central role in the process of nitroglycerin (glyceryl trinitrate, GTN) biotransformation in vivo and that its inhibition accounts for mechanism-based tolerance in vitro. The extent to which ALDH-2 contributes to GTN tolerance (impaired relaxation to GTN) and cross-tolerance (impaired endothelium-dependent relaxation) in vivo remain to be elucidated. Rats were treated for three days with GTN. Infusions were accompanied by decreases in vascular ALDH-2 activity, GTN biotransformation, and cGMP-dependent kinase (cGK-I) activity. Further, whereas in control vessels, multiple inhibitors and substrates of ALDH-2 reduced both GTN-stimulation of cGKI and GTN-induced vasodilation, these agents had little effect on tolerant vessels. A state of functional tolerance (in the GTN/cGMP pathway) was recapitulated in cultured endothelial cells by knocking down mitochondrial DNA (ρ0 cells). In addition, GTN increased the production of reactive oxygen species (ROS) by mitochondria, and these increases were associated with impaired relaxation to acetylcholine. Finally, antioxidants/reductants decreased mitochondrial ROS production and restored ALDH-2 activity. These observations suggest that nitrate tolerance is mediated, at least in significant part, by inhibition of vascular ALDH-2 and that mitochondrial ROS contribute to this inhibition. Thus, GTN tolerance may be viewed as a metabolic syndrome characterized by mitochondrial dysfunction.
Karsten Sydow, Andreas Daiber, Matthias Oelze, Zhiqiang Chen, Michael August, Maria Wendt, Volker Ullrich, Alexander Mülsch, Eberhard Schulz, John F. Keaney Jr., Jonathan S. Stamler, Thomas Münzel
This study tests the hypothesis that P2X1 receptors mediate pressure-induced afferent arteriolar autoregulatory responses. Afferent arterioles from rats and P2X1 KO mice were examined using the juxtamedullary nephron technique. Arteriolar diameter was measured in response to step increases in renal perfusion pressure (RPP). Autoregulatory adjustments in diameter were measured before and during P2X receptor blockade with NF279 or A1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Acute papillectomy or furosemide perfusion was performed to interrupt distal tubular fluid flow past the macula densa, thus minimizing tubuloglomerular feedback–dependent influences on afferent arteriolar function. Under control conditions, arteriolar diameter decreased by 17% and 29% at RPP of 130 and 160 mmHg, respectively. Blockade of P2X1 receptors with NF279 blocked pressure-mediated vasoconstriction, reflecting an attenuated autoregulatory response. The A1 receptor blocker DPCPX did not alter autoregulatory behavior or the response to ATP. Deletion of P2X1 receptors in KO mice significantly blunted autoregulatory responses induced by an increase in RPP, and this response was not further impaired by papillectomy or furosemide. WT control mice exhibited typical RPP-dependent vasoconstriction that was significantly attenuated by papillectomy. These data provide compelling new evidence indicating that tubuloglomerular feedback signals are coupled to autoregulatory preglomerular vasoconstriction through ATP-mediated activation of P2X1 receptors.
Edward W. Inscho, Anthony K. Cook, John D. Imig, Catherine Vial, Richard J. Evans
Osteopontin (OPN) is expressed in atherosclerotic lesions, particularly in diabetic patients. To determine the role of OPN in atherogenesis, ApoE–/–OPN+/+, ApoE–/–OPN+/–, and ApoE–/–OPN–/– mice were infused with Ang II, inducing vascular OPN expression and accelerating atherosclerosis. Compared with ApoE–/–OPN+/+ mice, ApoE–/–OPN+/– and ApoE–/–OPN–/– mice developed less Ang II–accelerated atherosclerosis. ApoE–/– mice transplanted with bone marrow derived from ApoE–/–OPN–/– mice had less Ang II–induced atherosclerosis compared with animals receiving ApoE–/–OPN+/+ cells. Aortae from Ang II–infused ApoE–/–OPN–/– mice expressed less CD68, C-C-chemokine receptor 2, and VCAM-1. In response to intraperitoneal thioglycollate, recruitment of leukocytes in OPN–/– mice was impaired, and OPN–/– leukocytes exhibited decreased basal and MCP-1–directed migration. Furthermore, macrophage viability in atherosclerotic lesions from Ang II–infused ApoE–/–OPN–/– mice was decreased. Finally, Ang II–induced abdominal aortic aneurysm formation in ApoE–/–OPN–/– mice was reduced and associated with decreased MMP-2 and MMP-9 activity. These data suggest an important role for leukocyte-derived OPN in mediating Ang II–accelerated atherosclerosis and aneurysm formation.
Dennis Bruemmer, Alan R. Collins, Grace Noh, Wei Wang, Mary Territo, Sarah Arias-Magallona, Michael C. Fishbein, Florian Blaschke, Ulrich Kintscher, Kristof Graf, Ronald E. Law, Willa A. Hsueh