Faculty Bibliography

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

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

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

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

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

Rationale: The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. Objective: We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. Methods and Results: We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R4496C/+) mice, the Purkinje cells were preferentially responsive to drug. In contrast, the RyR2 blocker tetracaine was equally efficacious in mutant Purkinje cells and ventricular myocytes. Conclusions: Purkinje cells display a greater propensity to develop abnormalities in intracellular Ca(2+) handling than ventricular myocytes. This proarrhythmic behavior is enhanced by disease-causing mutations in the RyR2 Ca(2+) release channel and greatly exacerbated by catecholaminergic stimulation, with the development of arrhythmogenic triggered beats. These data support the concept that Purkinje cells are critical contributors to arrhythmic triggers in animal models and humans with CPVT and suggest a broader role for the Purkinje fiber network in the genesis of ventricular arrhythmias

BACKGROUND: Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes. METHODS AND RESULTS: Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express beta-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca(2+) transients in Cntn2-EGFP(+) cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations. CONCLUSIONS: Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells