Faculty Bibliography

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction

Lethal cardiac arrhythmias are a hallmark of the hereditary Long QT syndrome (LQTS), a disease produced by mutations of cardiac ion channels [1]. Often these arrhythmias are stress-induced, suggesting a relationship between beta-adrenergic activation of adenylate cyclase and cAMP-dependent alteration of one or more of the ion channels involved in LQTS. Second messengers modulate ion channel activity either by direct interaction or through intermediary kinases and phosphatases. Here we show that the second messenger cAMP regulates the K(+) channel mutated in the LQT2 form of LQTS, HERG [2], both directly and indirectly. Activation of cAMP-dependent protein kinase (PKA) causes phosphorylation of HERG accompanied by a rapid reduction in current amplitude, acceleration of voltage-dependent deactivation, and depolarizing shift in voltage-dependent activation. In a parallel pathway, cAMP directly binds to the HERG protein with the opposing effect of a hyperpolarizing shift in voltage-dependent activation. The summation of cAMP-mediated effects is a net diminution of the effective current, but when HERG is complexed with with the K(+) channel accessory proteins MiRP1 or minK, the stimulatory effects of cAMP are favored. These findings provide a direct link between stress and arrhythmia by a unique mechanism where a single second messenger exerts complex regulation of an ion channel via two distinct pathways

Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G(i)-signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G(i)-coupled receptor (Ro1), we provide evidence that increased G(i) signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G(i) signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G(i) signaling in causing cardiac pathology

HERG(1) K(+) channel mutations are responsible for one form of dominantly inherited long QT syndrome (LQT). Some LQT mutations exert a dominant negative effect on wild-type current expression. To investigate mechanisms of dominant-negative behavior, we co-expressed wild-type HERG with the A561V mutant in mammalian cells. Transfection with various cDNA ratios produced HERG K(+) current densities that approached a predicted binomial distribution where mutant and wild-type subunits co-assemble in a tetramer with nearly complete dominance. Using C terminus myc-tagged wild-type HERG we specifically followed the mutant's effect on full-length wild-type HERG protein expression. Co-expression with A561V reduced the abundance of full-length wild-type HERG protein comparable to the current reduction. Reduction of wild-type protein was due to decreased synthesis and increased turnover. Conditions facilitating protein folding (growth at 30 degrees C, or in 10% glycerol) resulted in partial rescue from the dominant effect, as did the 26 S proteosome inhibitor ALLN. Thus, for A561V, dominant negative effects result from assembly of wild-type subunits with mutant very early in production leading to rapid recognition of mutant channels and targeting for proteolysis. These results establish protein misfolding, cellular proofreading, and bystander involvement as contributing mechanisms for dominant effects in LQT2

Gap junction channels composed of connexin43 (Cx43) are essential for normal heart formation and function. We studied the potential role of the Wnt family of secreted polypeptides as regulators of Cx43 expression and gap junction channel function in dissociated myocytes and intact hearts. Neonatal rat cardiomyocytes responded to Li(+), which mimics Wnt signaling, by accumulating the effector protein beta-catenin and by inducing Cx43 mRNA and protein markedly. Induction of Cx43 expression was also observed in cardiomyocytes cocultured with Rat-2 fibroblasts or N2A neuroblastoma cells programmed to secrete bioactive Wnt-1. By transfecting a Cx43 promoter-reporter gene construct into cardiomyocytes, we demonstrated that the inductive effect of Wnt signaling was transcriptionally mediated. Enhanced expression of Cx43 increased cardiomyocyte cell coupling, as determined by Lucifer Yellow dye transfer and by calcium wave propagation. Conversely, in a transgenic cardiomyopathic mouse model that exhibits ventricular arrhythmias and gap junctional remodeling, beta-catenin and Cx43 expression were downregulated concordantly. In response to Wnt signaling, the accumulating Cx43 colocalized with beta-catenin in the junctional membrane; moreover, forced expression of Cx43 in cardiomyocytes reduced the transactivation potential of beta-catenin. These findings demonstrate that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling in the heart, and they support the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart

Action potential duration is prolonged in many forms of heart disease, often as a result of reductions in Ca(2+)-independent transient outward K(+) currents (ie, I(to)). To examine the effects of a primary reduction in I(to) current in the heart, transgenic mice were generated that express a dominant-negative N-terminal fragment of the K(v)4.2 pore-forming potassium channel subunit under the control of the mouse alpha-myosin heavy chain promoter. Two of 6 founders died suddenly, and only 1 mouse successfully transmitted the transgene in mendelian fashion. Electrophysiological analysis at 2 to 4 weeks of age demonstrated that I(to) density was specifically reduced and action potential durations were prolonged in a subset of transgenic myocytes. The heterogeneous reduction in I(to) was accompanied by significant prolongation of monophasic action potentials. In vivo hemodynamic studies at this age revealed significant elevations in the mean arterial pressure, peak systolic ventricular pressures, and +/-dP/dt, indicative of enhanced contractility. Surprisingly, by 10 to 12 weeks of age, transgenic mice developed clinical and hemodynamic evidence of congestive heart failure. Failing transgenic hearts displayed molecular and cellular remodeling, with evidence of hypertrophy, chamber dilatation, and interstitial fibrosis, and individual myocytes showed sharp reductions in I(to) and I(K1) densities, action potential duration prolongation, and increased cell capacitance. Our results confirm that K(v)4.2 subunits contribute to I(to) in the mouse and demonstrate that manipulation of cardiac excitability may secondarily influence contractile performance

To control G protein signaling in vivo, we have modified G protein-coupled receptors to respond exclusively to synthetic small molecule agonists and not to their natural agonist(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). A prototype RASSL (Ro1) based on the Gi-coupled K opioid receptor was expressed in transgenic mice under the control of the tetracycline transactivator (tet) system. Activation of Ro1 expressed in the heart decreased heart rate by up to 80%, an expected effect of increased Gi signaling. Maximal heart rate changes occurred in less than 1 min, demonstrating the speed of this inducible signaling system. This Ro1-mediated slowing of heart rate was also subject to desensitization, which lasted more than 24 h. Both the initial effect on heart rate and the desensitization occurred, even though Ro1 is derived from a human opioid receptor not normally involved in heart rate control. In addition, the tet system was used to induce Ro1 expression in hepatocytes and salivary gland, where Gi signaling is known to control physiologic events such as proliferation and secretion. These studies demonstrate that a RASSL can be inducibly expressed in several mouse tissues and used in vivo to activate G protein signaling in a controllable fashion

Cell loss contributes to the pathogenesis of many inherited and acquired human diseases. We have developed a system to conditionally ablate cells of any lineage and developmental stage in the mouse by regulated expression of the diphtheria toxin A (DTA) gene by using tetracycline-responsive promoters. As an example of this approach, we targeted expression of DTA to the hearts of adult mice to model structural abnormalities commonly observed in human cardiomyopathies. Induction of DTA expression resulted in cell loss, fibrosis, and chamber dilatation. As in many human cardiomyopathies, transgenic mice developed spontaneous arrhythmias in vivo, and programmed electrical stimulation of isolated-perfused transgenic hearts demonstrated a strikingly high incidence of spontaneous and inducible ventricular tachycardia. Affected mice showed marked perturbations of cardiac gap junction channel expression and localization, including a subset with disorganized epicardial activation patterns as revealed by optical action potential mapping. These studies provide important insights into mechanisms of arrhythmogenesis and suggest that conditional lineage ablation may have wide applicability for studies of disease pathogenesis