QRS interval on the electrocardiogram reflects ventricular depolarization and conduction time, and is a risk factor for mortality, sudden death, and heart failure. We performed a genome-wide association meta-analysis in 40,407 European-descent individuals from 14 studies, with further genotyping in 7170 additional Europeans, and identified 22 loci associated with QRS duration (P < 5 × 10−8). These loci map in or near genes in pathways with established roles in ventricular conduction such as sodium channels, transcription factors, and calcium-handling proteins, but also point to novel biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a gene at our most significant locus, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. These findings extend our current knowledge of ventricular depolarization and conduction.

Circulating platelets contain high concentrations of TGF-␤1 in their ␣-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-␤1 may confound measurement of plasma TGF-␤1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-␤1 levels in blood collected from C57Bl/6 mice by the standard retrobulbar technique were much higher than those obtained when prostaglandin E 1 was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-␤1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-␤1 because of megakaryocytespecific disruption of their TGF-␤1 gene (Tgfb1 flox ). Tgfb1 flox mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-␤1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-␤1 contributes to plasma levels of TGF-␤1; and that platelet TGF-␤1 contributes to the pathologic cardiac changes that occur in response to aortic constriction. (Blood.

Rationale Post-translational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathologic gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood. Objective To establish whether specific connexin43 phosphorylation events influence gap junction expression and pathologic remodeling. Methods and Results We generated Cx43 germline knock-in mice in which serines 325/328/330 were replaced with phosphomimetic glutamic acids (S3E) or non-phosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathologic gap junction remodeling (GJR) and displayed diminished susceptibility to the induction of ventricular arrhythmias. Conversely, the S3A mice showed deleterious effects on cardiac gap junction formation and function, developed electrical remodeling and were highly susceptible to inducible arrhythmias. Conclusions These data demonstrate a mechanistic link between post-translational phosphorylation of Cx43 and gap junction formation, remodeling and arrhythmic susceptibility.

Gap junction channels are required for normal cardiac impulse propagation, and gap junction remodeling is associated with enhanced arrhythmic risk. Oculodentodigital dysplasia (ODDD) is a multisystem syndrome due to mutations in the connexin43 (Cx43) gap junction channel gene. To determine the effects of a human connexin channelopathy on cardiac electrophysiology and arrhythmogenesis, we generated a murine model of ODDD by introducing the disease-causing I130T mutant allele into the mouse genome. Cx43 abundance was markedly reduced in mutant hearts with preferential loss of phosphorylated forms that interfered with trafficking and assembly of gap junctions in the junctional membrane. Dual whole-cell patch-clamp studies showed significantly lower junctional conductance between neonatal cell pairs from mutant hearts, and optical mapping of isolated-perfused hearts with voltage-sensitive dyes demonstrated significant slowing of conduction velocity. Programmed electrical stimulation revealed a markedly increased susceptibility to spontaneous and inducible ventricular tachyarrhythmias. In summary, our data demonstrate that the I130T mutation interferes with Cx43 posttranslational processing, resulting in diminished cell-cell coupling, slowing of impulse propagation, and a proarrhythmic substrate.arrhythmia ͉ connexin43 ͉ transgenic ͉ channel ͉ mouse

Abstract-Pressure overload is a common pathological insult to the heart and the resulting hypertrophy is an independent risk factor for sudden cardiac death. Gap junction remodeling (GJR) has been described in hypertrophied hearts; however, a detailed understanding of the remodeling process and its effects on impulse propagation is lacking. Moreover, there has been little progress developing therapeutic strategies to diminish GJR. Accordingly, transverse aortic banding (TAC) was performed in mice to determine the effects of progressive pathological hypertrophy on connexin (Cx)43 expression, posttranslational phosphorylation, gap junction assembly, and impulse propagation. Within 2 weeks after TAC, total and phospho-Cx43 abundance was reduced and incorporation of Cx43 into gap junctional plaques was markedly diminished. These molecular changes were associated with progressive slowing of impulse propagation, as determined by optical mapping with voltage-sensitive dyes. Treatment with the aldosterone receptor antagonist spironolactone, which has been shown to diminish sudden arrhythmic death in clinical trials, was examined for its effects on GJR. We found that spironolactone blunted the development of GJR and also potently reversed established GJR, both at the molecular and functional levels, without diminishing the extent of hypertrophy. These data suggest a potential mechanism for some of the salutary electrophysiological and clinical effects of mineralocorticoid antagonists in myopathic hearts.

Recent progresses in signal transduction have revealed that beta-catenin signaling controls embryonic development, tumorigenesis, cell shape, and polarity. The role of this pathway in myocyte shape regulation during cardiac hypertrophy and failure is, however, not clearly defined. Since homozygous knockout of beta-catenin is embryonically lethal, we have deleted beta-catenin genes specifically in the heart of adult mice by crossing loxP-flanked beta-catenin mice with transgenic mice expressing tamoxifen-activated MerCreMer protein (MCM) driven by the alpha-myosin heavy chain promoter. Administration of tamoxifen to homozygous loxP-flanked beta-catenin mice positive for MCM induces the deletion of beta-catenin only in cardiomyocytes. Immunolabeling with beta-catenin antibody demonstrates that 90% of cardiomyocytes completely lose their beta-catenin expression but maintain normal rod-shaped morphology. The intercalated disk of cardiomyocytes lacking beta-catenin is morphologically unremarkable with normal distribution of vinculin, N-cadherin, desmoplakin, ZO-1, connexin43, and alpha-, gamma-, and p120 catenins. The expression level of these proteins, except that of gamma-catenin, is also similar in tamoxifen-treated and control mice with both homozygous loxP-flanked beta-catenin genes and the MCM transgene. Western blot analyses reveal that gamma-catenin increases in the heart of beta-catenin knockout mice compared with controls. Confocal microscopy also demonstrates that gamma-catenin has significantly increased in the intercalated disk of cardiomyocytes lacking beta-catenin. Echocardiographic data indicate that the knockout mice maintain normal ventricular geometry and cardiac function. The results suggest that upregulation of gamma-catenin can compensate for the loss of beta-catenin in cardiomyocytes to maintain normal cardiac structure and function.

In addition to its role in cell adhesion, β-catenin is an important signaling molecule in the Wnt/ Wingless signaling pathway. Recent studies have indicated that β-catenin is stabilized by hypertrophic stimuli and may regulate cardiac hypertrophic responses. To explore the role and requirement of β-catenin in cardiac development and hypertrophy, we deleted the β-catenin gene specifically in cardiac myocytes by crossing loxP-floxed β-catenin mice with transgenic mice expressing a Cre recombinase under the control of the α-myosin heavy chain promoter. No homozygous β-catenin deleted mice were born alive and died before embryonic day 14.5, indicating significant and irreplaceable roles of β-catenin in embryonic heart development. Heterozygous β-catenin deleted mice, however, demonstrated no structural and functional abnormality. The response of heterozygous β-catenin deleted mice to transverse aortic constriction, however, was significantly attenuated with decreased heart weight and heart weight/body weight ratio compared to controls with intact β-catenin genes. Hemodynamic analysis revealed that there was no difference in cardiac function between wild type and heterozygous β-catenin deleted mice. On the other hand, the expression of fetal genes, β-myosin heavy chain, atrial and brain natriuretic peptides was significantly higher in heterozygous β-catenin deleted mice when compared to wild type β-catenin mice. These results suggest that the cytoplasmic level of β-catenin modulates hypertrophic response and fetal gene reprogramming after pressure overload.

Two related ER oxidation 1 (ERO1) proteins, ERO1α and ERO1β, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1α and ERO1β. The peak amplitude of calcium transients in homozygous Ero1α mutant adult cardiomyocytes was reduced to 42.0 ± 2.2% (n=10, P ≤ 0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of β adrenergic blockade (P ≤ 0.01). In addition, mice lacking ERO1α were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31 ± 0.02 in the mutant (n=20) vs. 0.23 ± 0.03 in the wild type (n=18); P ≤ 0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.