Faculty Bibliography

RATIONALE: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. The electrophysiological consequences of this phenotypic switch remain largely unknown. OBJECTIVE: Determine whether fibroblast activation following cardiac injury results in a distinct electrophysiological phenotype that enhances fibroblast-myocyte interactions. METHODS AND RESULTS: Neonatal rat myocyte monolayers were treated with media (CM) conditioned by fibroblasts isolated from normal (Fb) and infarcted (MI-Fb) hearts. Fb and MI-Fb were also plated on top of myocyte monolayers at 3 densities. Cultures were optically mapped after CM treatment or fibroblast plating to obtain conduction velocity and action potential duration (APD(70)). Intercellular communication and connexin43 expression levels were assessed. Membrane properties of Fb and MI-Fb were evaluated using patch clamp techniques. MI-Fb CM treatment decreased conduction velocity (11.1%) compared to untreated myocyte cultures. APD(70) was reduced by MI-Fb CM treatment compared to homocellular myocyte culture (9.4%) and Fb CM treatment (6.4%). In heterocellular cultures, MI-Fb conduction velocities were different from Fb at all densities (+29.8%, -23.0%, and -16.7% at 200, 400, and 600 cells/mm(2), respectively). APD(70) was reduced (9.6%) in MI-Fb compared to Fb cultures at 200 cells/mm(2). MI-Fb had more hyperpolarized resting membrane potentials and increased outward current densities. Connexin43 was elevated (134%) in MI-Fb compared to Fb. Intercellular coupling evaluated with gap fluorescence recovery after photobleaching was higher between myocytes and MI-Fb compared to Fb. CONCLUSIONS: These data demonstrate cardiac injury results in significant electrophysiological changes that enhance fibroblast-myocyte interactions and could contribute to the greater incidence of arrhythmias observed in fibrotic hearts

Introduction: Hypertension (HTN) is associated with the depletion of myocardial energy stores and the development of atrial fibrillation (AF). The electrophysiological alterations in the setting of HTN are poorly understood. We hypothesized that chronic HTN leads to the activation of atrial KATP channels and the creation of an arrhythmogenic substrate. Methods: HTN was induced in mice by administration of a high salt diet (HS) for 2-4 weeks. Two control groups were studied: one was given a normal salt diet (NS) and the second was given the high salt diet with the diuretic hydrochlorothiazide (HCTZ). The development of HTN and changes in cardiac function were assessed with tail-cuff plethysmography and echocardiography. Optical mapping was used to evaluate electrophysiological parameters and AF inducibility. Results: Systolic blood pressure was significantly increased in HS (121.2+/-1.3mmHg) compared to NS (103.4+/-1.0mmHg) animals. Average fractional shortening was not significantly different. ERP was unchanged in the RAA and significantly decreased in the LAA of HS (11.8+/-1.6ms) compared to NS animals (26.5+/-2.5ms). Average LAA (10.8+/-0.7ms) and RAA (15.5+/-1.6ms) APD90 was significantly shorter in HS compared to NS animals (14.4+/-1.0 and 19.9+/-0.9ms, respectively). Importantly, AF inducibility was higher in the HS (N=6/16) compared to the NS group (N=0/12). Perfusion with the KATP antagonist glybenclamide (GLY; 10muM) restored LAA ERP (31.0+/-2.7ms) and APD90 (20.7+/-1.7ms) in the HS group. AF inducibility following GLY perfusion decreased in the HS (N=1/11) and was unchanged in the NS (N=0/12) group. No differences were observed between NS and HCTZ groups, suggesting that the elevated salt intake did not play a significant role in the observed differences. In addition, no differences in CV or interstitial fibrosis were observed between HS and NS animals, suggesting changes in refractoriness are responsible for the increased vulnerability to AF. Conclusions: These data indicate HTN leads to regionally specific activation of atrial KATP channels and an increase in the vulnerability to induction of AF, making IK(ATP) an attractive therapeutic target

Introduction: Hyperlipidemia has long been associated with arrhythmias and sudden death, generally due to ischemic infarction. However, recent evidence suggests cholesterol levels may also directly modify membrane ion channel function. We hypothesized that feeding with a Western-type diet (WD) will directly alter epicardial conduction velocity (CV) and action potential durations (APD). Methods: Control C57BL/6 and ApoE knockout hearts were extracted, Langerdorff perfused and optically mapped. APD50, APD70 and CV were determined for the right atrium (RA), left atrium (LA), right ventricle (RV) and left ventricle (LV) while pacing at 10 Hz. The ApoE knockout mice were either fed a normal diet (ND) or WD for 1, 2, or 4 weeks. Control mice received ND. Results: There were no significant differences in APD50, APD70, or CV between control and ND ApoE knockout mice for any of the chambers (p>0.05). APD70 (ms) was significantly (p<0.05) increased at 1 week (13.8+/- 0.8 RA; 14+/-1 LA), 2 weeks (14.1+/-0.96 RA; 20.1+/-2.7 LA), and 4 weeks (15.2+/-1.8 RA; 15.2+/-0.9 LA) of WD in both atria compared to controls (10.4+/-0.38 RA; 9.5+/-0.37 LA). APD50 values showed similar changes. RV CV (mm/s) was significantly slower in the ApoE mice fed WD at 1 week (0.51+/-0.02 RV), 2 weeks (0.45+/-0.05 RV), and 4 weeks (0.47+/-0.06 RV) compared to controls (0.62+/-0.03). LV CV was significantly slower in ApoE mice on WD for 2 weeks (0.355+/-0.02) compared to control (0.53+/-0.02). Conclusions: WD resulted in chamber specific changes in CV and APD. These results suggest that elevated cholesterol levels may be arrhythmogenic in the absence of myocardial ischemia (Figure presented)

Introduction: A critical event in the development of cardiac fibrosis is the transformation of fibroblasts into myofibroblasts. Fibroblasts isolated from healthy hearts and grown under standard tissue culture conditions express alpha-SMA and have been referred to as myofibroblasts. However, recent data suggest the in vitro transformation does not fully replicate the in vivo activation process. The purpose of this study was to investigate the potential of activated fibroblasts to contribute to an arrhythmogenic substrate through paracrine and direct coupling effects. Methods: Confluent neonatal rat myocyte monolayers were treated with media (CM) conditioned by cardiac fibroblasts isolated from ventricles of healthy (Fb) and infarcted (MI-Fb) hearts and optically mapped 16-20 hours later. To study the combined paracrine and direct coupling effect, Fb and MI-Fb were plated on top of myocyte monolayers. Results: Treatment with both Fb CM (16.6+/-0.4 cm/s) and MIFb CM (15.8+/-0.4 cm/s) significantly decreased conduction velocity (CV) compared to homocellular myocyte monolayers (Myo; 19.7+/-0.7 cm/s). Action potential duration (APD70) was significantly reduced by MI-Fb CM (143.6+/-1.7 ms) treatment compared to Myo (159.4+/-4.0 ms) and Fb CM (153.4+/-2.7 ms). In heterocellular cultures, Fb significantly decreased (17.0+/-0.5 cm/s) and MI-Fb increased (22.0+/-0.6 cm/s) average CV compared to Myo. In addition, CV was significantly faster with MI-Fb compared to Fb (p=1.95E-8). Fb (145.0+/-3.9 ms) and MIFb (131.1+/-3.7 ms) significantly reduced APD70 compared to Myo (159.4+/-4.0 ms), and APD70 was significantly shorter with MI-Fb compared to Fb (p=0.01). Analysis of Cx43 levels showed a significant upregulation of Cx43 in MI-Fb compared to Fb. Conclusions: These data demonstrate Fb exert predominantly paracrine effects while MI-Fb affect myocyte electrophysiology through a combination of paracrine and direct coupling mechanisms. Moreover, APD shortening and increased Cx43 levels in MI-Fb could contribute to the greater incidence of arrhythmias observed in fibrotic hearts. These findings may lead to the development of new anti-arrhythmic therapeutic approaches targeting the fibroblast activation process

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose towards arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization, but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiologic effects of GJR. We paced wildtype (normal Cx43 abundance) and heterozygous Cx43 knockout mice (Cx43(+/-), 66% mean reduction in Cx43) for six hours at 10-15% above their average sinus rate. We investigated the electrophysiologic effects of pacing on the whole animal using programmed electrical stimulation, and in isolated ventricular myocytes with patch clamp studies. Cx43(+/-) myocytes had significantly shorter action potential durations (APD) and increased steady state and inward rectifier potassium currents (Iss and IK1, respectively) compared to wildtype littermate cells. In Cx43(+/-) hearts, pacing resulted in significant prolongation of ventricular effective refractory period and action potential duration, and significant diminution of Iss compared to unpaced Cx43(+/-) hearts. However, these changes were not seen in paced wildtype mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiologic changes which may contribute to the worsened prognosis often associated with pacing in the failing heart. Key words: Connexin43, ventricular myocytes, mouse, gap junction

Impulse propagation in cardiac tissue is a complex process in which intercellular coupling through gap junction channels is a critical component. Connexin40 (Cx40) is an abundant gap junction protein that is expressed in atrial myocytes. Alterations in the expression of Cx40 have been implicated in atrial arrhythmogenesis. The purpose of the current study was to assess the role of Cx40 in atrial impulse propagation. High-resolution optical mapping was used to study conduction in the right and left atrial appendages of isolated Langendorff-perfused murine hearts. Wild-type (Cx40(+/+)), heterozygous (Cx40(+/-)), and knockout (Cx40(-/-)) mice, both adult and embryonic, were studied to assess the effects of reduced Cx40 expression on sinus node function and conduction velocity at different pacing cycle lengths (100 and 60 ms). In both adult and late-stage embryonic Cx40(+/+) mice, heterogeneity in CV was found between the right and left atrial appendages. Either partial (Cx40(+/-)) or complete (Cx40(-/-)) deletion of Cx40 was associated with the loss of conduction heterogeneity in both adult and embryonic mice. Additionally, sinus node impulse initiation was found to be ectopic in Cx40(-/-) mice at 15.5 days postcoitus, whereas Cx40(+/+) mice showed normal activation occurring near the crista terminalis. Our findings suggest that Cx40 plays an essential role in establishing interatrial conduction velocity heterogeneity in the murine model. Additionally, we describe for the first time a developmental requirement for Cx40 in normal sinus node impulse initiation at 15.5 days postcoitus

The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death.

Loss of connexin43 (Cx43) gap junction channels in the heart results in a marked increase in the incidence of spontaneous and inducible polymorphic ventricular tachyarrhythmias (PVTs). The mechanisms resulting in this phenotype remain unclear. We hypothesized that uncoupling promotes regional ion channel remodeling, thereby increasing electrical heterogeneity and facilitating the development of PVT. In isolated-perfused control hearts, programmed electrical stimulation elicited infrequent monomorphic ventricular tachyarrhythmias (MVT), and dominant frequencies (DFs) during MVT were similar in the right ventricle (RV) and left ventricle (LV). Moreover, conduction properties, action potential durations (APDs), and repolarizing current densities were similar in RV and LV myocytes. In contrast, PVT was common in Cx43 conditional knockout (OCKO) hearts, and arrhythmias were characterized by significantly higher DFs in the RV compared to the LV. APDs in OCKO myocytes were significantly shorter than those from chamber-matched controls, with RV OCKO myocytes being most affected. APD shortening was associated with higher levels of sustained current in myocytes from both chambers as well as higher levels of the inward rectifier current only in RV myocytes. Thus, alterations in cell-cell coupling lead to regional changes in potassium current expression, which in this case facilitates the development of reentrant arrhythmias. We propose a new mechanistic link between electrical uncoupling and ion channel remodeling. These findings may be relevant not only in cardiac tissue but also to other organ systems where gap junction remodeling is known to occur.-Danik, S. B., Rosner, G., Lader, J., Gutstein, D. E., Fishman, G. I., Morley, G. E. Electrical remodeling contributes to complex tachyarrhythmias in connexin43-deficient mouse hearts