Faculty Bibliography

BACKGROUND: Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. METHODS AND RESULTS: In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor gamma 1 (PPARgamma1) via the cardiac alpha-myosin heavy-chain promoter. The PPARgamma transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPARgamma agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPARgamma transgenic mice. CONCLUSIONS: Our findings support an important link between PPARgamma activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.

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

Aims High recurrence rates after complex radiofrequency ablation procedures, such as for atrial fibrillation, remain a major clinical problem. Local electrophysiological changes that occur following cardiac ablation therapy are incompletely described in the literature. The purpose of this study was to determine whether alterations in conduction velocity, action potential duration (APD), and effective refractory period resolve dynamically following cardiac ablation. Methods and results Lesions were delivered to the right ventricle of mice using a subxiphoid approach. The sham-operated control group (SHAM) received the same procedure without energy delivery. Hearts were isolated at 0, 1, 7, 30, and 60 days following the procedure and electrophysiological parameters were obtained using high-resolution optical mapping with a voltage-sensitive dye. Conduction velocity was significantly decreased at the lesion border in the 0, 7, and 30 day groups compared to SHAM. APD(70) at the lesion border was significantly increased at all time points compared to SHAM. Effective refractory period was significantly increased at the lesion border at 0, 1, 7, and 30 days but not at 60 days post-ablation. This study demonstrated that post-ablation electrophysiological changes take place immediately following energy delivery and resolve within 60 days. Conclusions Cardiac ablation causes significant electrophysiological changes both within the lesion and beyond the border zone. Late recovery of electrical conduction in individual lesions is consistent with clinical data demonstrating that arrhythmia recurrence is associated with failure to maintain bi-directional conduction block

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

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

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

Cardiac fibrosis occurs in a number of cardiovascular diseases associated with a high incidence of arrhythmias. A critical event in the development of fibrosis is the transformation of fibroblasts into an active phenotype or myofibroblast. This transformation results in functional changes including increased proliferation and changes in the release of signaling molecules and extracellular matrix deposition. Traditionally, fibroblasts have been considered to affect cardiac electrophysiology indirectly by physically isolating myocytes and creating conduction barriers. There is now increasing evidence that cardiac fibroblasts may play a direct role in modulating the electrophysiological substrate in diseased hearts. The purpose of this review is to summarize the functional changes associated with fibroblast activation, the membrane currents that have been identified in adult cardiac fibroblasts, and describe recent studies of fibroblast-myocyte electrical interactions with emphasis on the changes that occur with cardiac injury. Further analysis of fibroblast membrane electrophysiology and their interactions with myocytes will lead to a more complete understanding of the arrhythmic substrate. These studies have the potential to generate new therapeutic approaches for the prevention of arrhythmias associated with cardiac fibrosis

Ventricular preexcitation, which characterizes Wolff-Parkinson-White syndrome, is caused by the presence of accessory pathways that can rapidly conduct electrical impulses from atria to ventricles, without the intrinsic delay characteristic of the atrioventricular (AV) node. Preexcitation is associated with an increased risk of tachyarrhythmia, palpitations, syncope, and sudden death. Although the pathology and electrophysiology of preexcitation syndromes are well characterized, the developmental mechanisms are poorly understood, and few animal models that faithfully recapitulate the human disorder have been described. Here we show that activation of Notch signaling in the developing myocardium of mice can produce fully penetrant accessory pathways and ventricular preexcitation. Conversely, inhibition of Notch signaling in the developing myocardium resulted in a hypoplastic AV node, with specific loss of slow-conducting cells expressing connexin-30.2 (Cx30.2) and a resulting loss of physiologic AV conduction delay. Taken together, our results suggest that Notch regulates the functional maturation of AV canal embryonic myocardium during the development of the specialized conduction system. Our results also show that ventricular preexcitation can arise from inappropriate patterning of the AV canal-derived myocardium.

Introduction: Spinal cord stimulation (SCS) has been shown to modulate atrial electrophysiology and confer protection against ischemia and ventricular arrhythmias. We hypothesized that SCS may reduce susceptibility to tachypacing (TP) induced atrial fibrillation (AF). Methods: Spinal cord leads (Octrode, St. Jude Medical) were implanted in the upper thoracic spine (T1-T5) of canines and connected to pulse generators (EonC, St. Jude Medical). The AV node was ablated and atrial effective refractory period (AERP) was measured at baseline and with SCS (n=10). In separate animals the AV node was ablated and endocardial RA and RV pacing leads were connected to dual chamber pacemakers for ambulatory AF induction. Custom firmware provided continuous 30s periods of atrial TP followed by 6s sense windows. TP was interrupted by detection of AF (atrial rate >250 bpm) and resumed upon return to sinus rhythm. AF Index was defined as the fraction of time the animal did not receive TP relative to the total allowable TP time. The effect of SCS delivered intermittently for 6 hr/day (SCS ON; n=3) on AF index was followed for 8 weeks and compared to control (SCS OFF; n=3). Results: Right (p=0.002) and left (p=0.009) AERP were significantly longer during SCS (168+/-15.1, 168+/-14.8 ms) compared to baseline (130+/-8.7, 152+/-10.3 ms). AF Index was significantly decreased in the SCS ON compared to SCS OFF (p<0.0001). AF Index was >70% in the SCS OFF group and <5% in the SCS ON animals starting at week 3 (Figure). Conclusions: These data demonstrate that SCS prolongs AERP and prevents TP-induced AE (Graph presented)

Formins dynamically regulate actin microfilament assembly, influencing processes such as cell division, signal transduction, migration, and cell-cell contact formation. The actin cytoskeleton affects ion channel activity and formation of mechanical cell-cell junctions. As in vitro differentiation of embryonic stem cells (ESC) results in phenotypical immature cardiomyocytes, differences in formin expression might reflect alterations to the actin cytoskeleton of adult cardiomyocytes. Objective: To compare formin expression during heart development to the profile of in vitro differentiated cardiomyocytes. Methods: Samples: ESC and cardiac tissue from embryonic day 14.5, neonatal day 2 or 2 month old mice of the 129P2 strain (heart development); spontaneously contracting cardiomyocytes differentiated from 129P2 ESC (in vitro differentiation). Transcript analysis performed by qRT-PCR analysis (15 mouse formin genes, 2 stemness genes, 4 cardiomyocyte differentiation genes). Expression of candidate formin proteins was analyzed using commercially available Diap1 and Fhod1 antibodies. Results: qRT-PCR analysis revealed significant changes in formin expression for 9 of 15 formins during heart development. Four patterns of transcript changes were observed: formins expressed in ESC and decreasing during development (Diap1, Diap3); transcripts peeking during fetal heart development (Fhod1, Fmnl1, Grid2ip); transcripts altered during heart development compared to ESC and adult heart (Daam1, Daam2, Fmnl2, Fmnl3); transcript increasing during development (Fhod3). In vitro differentiated cardiomyocytes revealed significant increases in expression of embryonic formins (Grid2ip, Fhod1, Fmnl2) and formins otherwise not differentially expressed during heart development (Diap2, Fmn1, Fmn2, Inf2). Expression of Fhod1 and Diap1 protein was studied in ESC, neonatal cardiomyocytes and in vitro differentiated cardiomyocytes, verifying the observed changes in transcript expression. Conclusion: Formins are dynamically regulated during cardiac development. Modifying formin expression of in vitro differentiated cardiomyocytes could improve functional maturity and therefore their potential use in cell replacement therapy for cardiac repair