Faculty Bibliography

Skeletal muscle development involves the differentiation of myoblasts into myotubes; likely involving connexins (Cx) and the gap junctional exchange of secondary messengers and metabolites. Some patients with oculodentodigital dysplasia (ODDD), a rare primarily autosomal dominant disease caused by mutations in the gene encoding Cx43, become less ambulatory with aging and exhibit symptoms that may reflect defects in skeletal muscle development, maintenance, and repair. In this study, the role of Cx43 in skeletal muscle development was examined in differentiation-competent L6 myoblasts and two ODDD-linked Cx43-mutant mouse lines. Undifferentiated L6 myoblasts exhibited high levels of Cx43-based gap junctions which were inhibited by the co-expression of I130T and G60S mutants. Upon myotube formation, Cx43 was rapidly down-regulated and gap junctions were lost but myotubes continued to enlarge. Cx43-mutant mice heterozygous for the I130T mutation exhibited similar body weight, tibial length and muscle fiber size as littermate controls. While mutant mice heterozygous for the G60S mutation also had similar tibial length their overall size and muscle fibre diameter were significantly smaller. Overall, Cx43 regulation may be important for initial myoblast differentiation but not linked to myotube growth and while our mutant mouse studies suggest that some ODDD patients are predicted to have smaller muscle fibres, this appears to be dependent on the site of the Cx43 mutation or other unknown etiologies

Two related ER oxidation 1 (ERO1) proteins, ERO1alpha and ERO1beta, 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 ERO1alpha and ERO1beta. The peak amplitude of calcium transients in homozygous Ero1alpha 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 beta adrenergic blockade (P</=0.01). In addition, mice lacking ERO1alpha 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.-Chin, K. T., Kang, G., Qu, J., Gardner, L. B., Coetzee, W. A., Zito, E., Fishman, G. I., Ron, R. The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load

Background: Hypertension is associated with the development of atrial fibrillation, however the electrophysiological consequences of this condition remain poorly understood. K(ATP) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Methods and Results: Elevated blood pressure was induced in mice with a high salt diet (HS) for four weeks. High resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP) and action potential duration (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS compared to control hearts. ERP and APD(90) were significantly shorter in the RAA and LAA of HS compared to control hearts. Perfusion with 1 muM glibenclamide or 300 muM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared to untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS compared to control animals. SUR1 protein expression was increased in the HS LAA (415% of NS) and RAA (372% of NS). Conclusion: K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches

During the life span of The FASEB Journal, the decline in cardiovascular mortality was astonishing as the fundamental bases of the complex syndromes of cardiovascular disease were illuminated. In this Silver Anniversary Review, we highlight a few pivotal advances in the field and relate them to research in Pasteur's quadrant, the region of investigation driven by both a desire for fundamental understanding and the consideration of its use. In the second half of the 20th century, we advanced from little pathophysiologic understanding to a near-complete understanding and effective, evidence-based therapeutics for vascular disorders and a similar development of pharmacotherapy to address heart failure, primarily through agents that antagonize the excessive concentration of circulating neurohumoral agents. In the current era, we have witnessed 'the rise of the machines,' from stents to cardiac resynchronization therapy. The next wave of treatments will build on an increasingly sophisticated understanding of the molecular determinants of cardiovascular disorders. We briefly consider the promise of regenerative medicine and are intrigued by the possibility for the direct reprogramming of resident cardiac fibroblasts into cardiomyocytes. As for the future, genomic profiling should help physicians recommend individualized risk factor modification targeted to prevent specific manifestations of cardiovascular disease. Transcriptional and biomarker analyses will almost surely be used individually to tailor therapy for those at risk of or experiencing cardiovascular disease. Given the ongoing exponential expansion of scientific knowledge, all of human ingenuity will be needed to fully utilize the power of Pasteur's quadrant and to unleash another quarter century in cardiology as scientifically fruitful and effective on human health as the last.-Levin, R. I., Fishman, G. I. The power of Pasteur's quadrant: cardiovascular disease at the turn of the century

BACKGROUND: Sodium channel alpha-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc, or to lateral membranes, where they associate with region-specific molecules. OBJECTIVE: To determine the functional properties of sodium channels as a function of their location in the cell. METHODS: Local sodium currents were recorded from adult rodent VMs and Purkinje cells using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M), or cell end (area originally occupied by the intercalated disc; ID). Channels were identified as TTX-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM TTX. RESULTS: Average peak-current amplitude was larger in ID than M, and largest at site of contact between attached cells. TTX-S channels were found only in M region of VMs, and not in Purkinje myocytes. TTX-R channels were found in M and ID, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by TNF-alpha. The magnitude of current increase was largest in M, but this difference was abolished by 100 nM TTX. CONCLUSIONS: Our data suggest that: a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID; b) cell-cell adhesion increases functional channel density at ID. c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but represent a functional reserve that can be upregulated by exogenous factors

Rationale: Posttranslational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathological gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood. Objective: To establish whether specific Cx43 phosphorylation events influence gap junction expression and pathological 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 nonphosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathological gap junction remodeling 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 electric remodeling, and were highly susceptible to inducible arrhythmias. Conclusions: These data demonstrate a mechanistic link between posttranslational phosphorylation of Cx43 and gap junction formation, remodeling, and arrhythmic susceptibility

Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia

The QRS interval, from the beginning of the Q wave to the end of the S wave on an 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 individuals of European descent from 14 studies, with further genotyping in 7,170 additional Europeans, and we identified 22 loci associated with QRS duration (P < 5 x 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 previously unidentified biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a candidate gene at the most significantly associated locus in this study, 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