CAP2 in cardiac conduction, sudden cardiac death and eye development.

Sudden cardiac death kills 180,000 to 450,000 Americans annually, predominantly males. A locus that confers a risk for sudden cardiac death, cardiac conduction disease, and a newly described developmental disorder (6p22 syndrome) is located at 6p22. One gene at 6p22 is CAP2, which encodes a cytoskeletal protein that regulates actin dynamics. To determine the role of CAP2 in vivo, we generated knockout (KO) mice. cap2(-)/cap2(-) males were underrepresented at weaning and ~70% died by 12 weeks of age, but cap2(-)/cap2(-) females survived at close to the expected levels and lived normal life spans. CAP2 knockouts resembled patients with 6p22 syndrome in that mice were smaller and they developed microphthalmia and cardiac disease. The cardiac disease included cardiac conduction disease (CCD) and, after six months of age, dilated cardiomyopathy (DCM), most noticeably in the males. To address the mechanisms underlying these phenotypes, we used Cre-mediated recombination to knock out CAP2 in cardiomyocytes. We found that the mice developed CCD, leading to sudden cardiac death from complete heart block, but no longer developed DCM or the other phenotypes, including sex bias. These studies establish a direct role for CAP2 and actin dynamics in sudden cardiac death and cardiac conduction disease.

Cardiac melanocytes influence atrial reactive oxygen species involved with electrical and structural remodeling in mice.

Cardiac melanocyte-like cells (CMLCs) contribute to atrial arrhythmias when missing the melanin synthesis enzyme dopachrome tautomerase (Dct). While scavenging reactive oxygen species (ROS) in Dct-null mice partially suppressed atrial arrhythmias, it remains unclear if CMLCs influence atrial ROS and structure or if the electrical response of CMLCs to ROS differs from that of atrial myocytes. This study is designed to determine if CMLCs contribute to overall atrial oxidative stress or structural remodeling, and if ROS affects the electrophysiology of CMLCs differently than atrial myocytes. Immunohistochemical analysis showed higher expression of the oxidative marker 8-hydroxy-2'-deoxyguanosine in Dct-null atria versus Dct-heterozygous (Dct-het) atria. Exposing isolated CMLCs from Dct-het and Dct-null mice to hydrogen peroxide increased superoxide anion more in Dct-null CMLCs. Trichrome staining showed increased fibrosis in Dct-null atria, and treating Dct-null mice with the ROS scavenger Tempol reduced atrial fibrosis. Action potential recordings from atrial myocytes and isolated Dct-het and Dct-null CMLCs in response to hydrogen peroxide showed that the EC50 for action potential duration (APD) prolongation of Dct-null CMLCs was 8.2 ± 1.7 µmol/L versus 16.8 ± 2.0 µmol/L for Dct-het CMLCs, 19.9 ± 2.1 µmol/L for Dct-null atrial myocytes, and 20.5 ± 1.9 µmol/L for Dct-het atrial myocytes. However, APD90 was longer in CMLCs versus atrial myocytes in response to hydrogen peroxide. Hydrogen peroxide also induced more afterdepolarizations in CMLCs compared to atrial myocytes. These studies suggest that Dct within CMLCs contributes to atrial ROS balance and remodeling. ROS prolongs APD to a greater extent and induces afterdepolarizations more frequently in CMLCs than in atrial myocytes.

GATA-Binding Factor 6 Contributes to Atrioventricular Node Development and Function.

BACKGROUND: Several transcription factors regulate cardiac conduction system (CCS) development and function but the role of each in specifying distinct CCS components remains unclear. GATA-binding factor 6 (GATA6) is a zinc-finger transcription factor that is critical for patterning the cardiovascular system. However, the role of GATA6 in the embryonic heart and CCS has never been shown. METHODS AND RESULTS: We report that Gata6 is expressed abundantly in the proximal CCS during midgestation in mice. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 induces loss of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the compact atrioventricular node with some retention of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the atrioventricular bundle, but has no significant effect on the connexin-40-positive bundle branches. Furthermore, myocardial-specific deletion of the carboxyl zinc-finger of Gata6 alters atrioventricular conduction in postnatal life as assessed by surface and invasive electrophysiological evaluation, as well as decreasing the number of ventricular myocytes and inducing compensatory myocyte hypertrophy. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 is also associated with downregulation of the transcriptional repressor ID2 and the cardiac sodium-calcium exchanger NCX1 in the proximal CCS, where GATA6 transactivates both of these factors. Finally, carboxyl zinc-finger deletion of Gata6 reduces cell-cycle exit of TBX3+ myocytes in the developing atrioventricular bundle during the period of atrioventricular node specification, which results in fewer TBX3+ cells in the proximal CCS of mature mutant mice. CONCLUSIONS: GATA6 contributes to the development and postnatal function of the murine atrioventricular node by promoting cell-cycle exit of specified cardiomyocytes toward a conduction system lineage.

TBX5 drives Scn5a expression to regulate cardiac conduction system function.

Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Nav1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease.

Atrial fibrillation in the elderly: the potential contribution of reactive oxygen species.

Atrial fibrillation (AF) is the most commonly encountered cardiac arrhythmia, and is a significant source of healthcare expenditures throughout the world. It is an arrhythmia with a very clearly defined predisposition for individuals of advanced age, and this fact has led to intense study of the mechanistic links between aging and AF. By promoting oxidative damage to multiple subcellular and cellular structures, reactive oxygen species (ROS) have been shown to induce the intra- and extra-cellular changes necessary to promote the pathogenesis of AF. In addition, the generation and accumulation of ROS have been intimately linked to the cellular processes which underlie aging. This review begins with an overview of AF pathophysiology, and introduces the critical structures which, when damaged, predispose an otherwise healthy atrium to AF. The available evidence that ROS can lead to damage of these critical structures is then reviewed. Finally, the evidence linking the process of aging to the pathogenesis of AF is discussed.

Regulatable atrial natriuretic peptide gene therapy for hypertension.

Hypertension (HTN) is a disease that begins with dysfunctional renal-sodium excretion and progresses to a syndrome of highly elevated systolic, diastolic, and mean arterial pressures. Inadequacies in the therapy of HTN have led to the investigation of the gene therapy of this disease by using systemic overproduction of vasodilatory peptides, such as atrial natriuretic peptide (ANP). However, gene-therapy approaches to HTN using ANP are limited by the need for long-term ANP gene expression and, most important, control of ANP gene expression. Here, we introduce a helper-dependent adenoviral vector carrying the mifepristone (Mfp)-inducible gene-regulatory system to control in vivo ANP expression. In the BPH/2 mouse model of HTN, Mfp-inducible ANP expression was seen for a period of >120 days after administration of vector. Physiological effects of ANP, including decreased systolic blood pressure, increased urinary cGMP output, and decreases in heart weight as a percentage of body weight were also under the control of Mfp. Given these capabilities, this vector represents a paradigm for the gene therapy of HTN.

Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.