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Yeong-Hoon Choi,
Christof Stamm,
Peter E Hammer,
Kevin F Kwaku,
Jennifer J Marler,
Ingeborg Friehs, Mara Jones,
Christine M Rader,
Nathalie Roy,
Mau-Thek Eddy,
John K Triedman,
Edward P Walsh,
Francis X McGowan,
Pedro J del Nido,
Douglas B Cowan
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ABSTRACT: In children, interruption of cardiac atrioventricular (AV) electrical conduction can result from congenital defects, surgical interventions, and maternal autoimmune diseases during pregnancy. Complete AV conduction block is typically treated by implanting an electronic pacemaker device, although long-term pacing therapy in pediatric patients has significant complications. As a first step toward developing a substitute treatment, we implanted engineered tissue constructs in rat hearts to create an alternative AV conduction pathway. We found that skeletal muscle-derived cells in the constructs exhibited sustained electrical coupling through persistent expression and function of gap junction proteins. Using fluorescence in situ hybridization and polymerase chain reaction analyses, myogenic cells in the constructs were shown to survive in the AV groove of implanted hearts for the duration of the animal's natural life. Perfusion of hearts with fluorescently labeled lec-tin demonstrated that implanted tissues became vascularized and immunostaining verified the presence of proteins important in electromechanical integration of myogenic cells with surrounding re-cipient rat cardiomyocytes. Finally, using optical mapping and electrophysiological analyses, we provide evidence of permanent AV conduction through the implant in one-third of recipient animals. Our experiments provide a proof-of-principle that engineered tissue constructs can function as an electrical conduit and, ultimately, may offer a substitute treatment to conventional pacing therapy.
American Journal Of Pathology 08/2006; 169(1):72-85. · 4.89 Impact Factor
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ABSTRACT: Necrosis and apoptosis differentially contribute to myocardial injury. Determination of the contribution of these processes in ischemia-reperfusion injury would allow for the preservation of myocardial tissue. Necrosis and apoptosis were investigated in Langendorff-perfused rabbit hearts (n = 47) subjected to 0 (Control group), 5 (GI-5), 10 (GI-10), 15 (GI-15), 20 (GI-20), 25 (GI-25), and 30 min (GI-30) of global ischemia (GI) and 120 min of reperfusion. Myocardial injury was determined by triphenyltetrazolium chloride (TTC) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), bax, bcl2, poly(ADP)ribose polymerase (PARP) cleavage, caspase-3, -8, and -9 cleavage and activity, Fas ligand (FasL), and Fas-activated death domain (FADD). The contribution of apoptosis was determined separately (n = 42) using irreversible caspase-3, -8, and -9 inhibitors. Left ventricular peak developed pressure (LVPDP) and systolic shortening (SS) were significantly decreased and infarct size and TUNEL-positive cells were significantly increased (P < 0.05 vs. Control group) at GI-20, GI-25, and GI-30. Proapoptotic bax, PARP cleavage, and caspase-3 and -9 cleavage and activity were apparent at GI-5 to GI-30. Fas, FADD, and caspase-8 cleavage and activity were unaltered. Irreversible inhibition of caspase-3 and -9 activity significantly decreased (P < 0.05) infarct size at GI-25 and GI-30 but had no effect on LVPDP or SS. Myocardial injury results from a significant increase in both necrosis and apoptosis (P < 0.05 vs. Control group) evident by TUNEL, TTC staining, and caspase activity at GI-20. Intrinsic proapoptotic activation is evident early during ischemia but does not significantly contribute to infarct size before GI-25. The contribution of necrosis to infarct size at GI-20, GI-25, and GI-30 is significantly greater than that of apoptosis. Apoptosis is significantly decreased by caspase inhibition during early reperfusion, but this protection does not improve immediate postischemic functional recovery.
AJP Heart and Circulatory Physiology 06/2004; 286(5):H1923-35. · 3.71 Impact Factor
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ABSTRACT: Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals.
American Journal Of Pathology 05/2004; 164(4):1211-23. · 4.89 Impact Factor
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ABSTRACT: We hypothesized that the alterations in vasomotor tone and adaptive remodeling responses that occur in the circulation because of hypoxia were dependent on changes in cell to cell communication through regulation of gap junction protein expression and function. Consequently, we studied the amount, distribution, and permeability of the principal vascular smooth muscle cell (VSMC) gap junction protein, connexin43, in rat aortic cultures exposed to oxygen partial pressures of 150 or 15 mm Hg.
Immunohistochemical staining, immunoblot assays, and Northern blot analyses demonstrated that connexin43 expression was reversibly increased in hypoxic cultures. As a result, hypoxic cells exhibited greater intercellular communication as determined by fluorescence recovery after photobleaching experiments. Using a fluorogenic substrate, hypoxic VSMCs showed increased reactive oxygen species generation, which could be prevented by the glutathione peroxidase mimic ebselen and the mitochondrial complex I inhibitor rotenone but not with the redox-sensitive thiol pyrrolidine dithiocarbamate. The rise in connexin43 expression attributable to hypoxia could be attenuated by ebselen and rotenone treatment. Interestingly, the previously reported induction of connexin43 expression by tensile stretch was also contingent on oxidative activity.
Hypoxia and stretch increased gap junctional intercellular communication in VSMCs attributable to enhanced connexin43 expression initiated by reactive oxygen species formation.
Arteriosclerosis Thrombosis and Vascular Biology 11/2003; 23(10):1754-60. · 6.37 Impact Factor
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ABSTRACT: Smooth muscle cell migration is critical to neointimal formation after arterial injury. The purpose of this study was to elucidate the regulation and functional significance of cell-cell adhesion via adherens junctions during this process.
Using balloon catheter injury of rat carotid artery, we showed that neointimal formation is accompanied by dramatic but transient upregulation of intimal N-cadherin and associated catenins, proteins that mediate adhesion at adherens junctions. Upregulation was demonstrated by immunofluorescence microscopy and by immunoblotting, and it coincided with evidence of phenotypic modulation of smooth muscle cells. Similar upregulation was observed when postconfluent cultures of porcine aortic smooth muscle cells were subjected to linear denuding injuries. Furthermore, treatment of wounded cultures with a blocking antibody against the extracellular domain of the N-cadherin protein significantly suppressed the repair of wounds.
N-cadherin and associated proteins are dynamically regulated during neointimal formation and provide evidence that this regulation is important for migratory repair. Therefore, N-cadherin may provide a novel target for therapies that are directed toward intimal proliferative disorders, including restenosis and vascular bypass graft failure.
Arteriosclerosis Thrombosis and Vascular Biology 01/2003; 22(12):1972-7. · 6.37 Impact Factor
