Discrepancy Between Acute and Long-Term Effects of the Calmodulin-Camkii-Calcineurin Pathway on Arrhythmogenesis in the CAVB Dog

Abstracts myocardium with fibrous and fatty tissue, cardiac arrhythmias, and sudden death. ARVC has been associated with mutations in desmosomal proteins, most prominently in the gene coding for plakophilin-2 (PKP2). How disruption of desmosomal proteins in cardiac myocytes leads to the disproportionate extent of fibrosis, and fat accumulation, observed in ARVCafflicted hearts is unclear. Here, we propose that fibrofatty infiltration has its origins (at least in part) in nonmyocyte cardiac cells. Our attention centers on the epithelial cells that conform the cardiac epicardium and act as progenitors of the cardiac fibroblast lineage. We speculate that desmosomal integrity is necessary for proper control of migration, proliferation, and transdifferentiation of epicardium-derived cells (EPDCs). Through our studies, we explore a possible link between EPDC biology and the fibrofatty infiltration characteristic of ARVC. Methods and Results: EPDC cultures were subjected to a PKP2 siRNA oligo, an oligo containing the PKP2 silencing sequence but randomized to not silence the protein, or an untreated control group. Immunolocalization studies using antibodies against E-cadherin and alpha-smooth muscle actin (*sma) served as markers for epithelial or myofibroblast cells, respectively. Quantification of these experiments revealed an increase in the population of *sma-positive cells after loss of PKP2 expression (79% ⫾ 4% of total labeled cells [mean ⫾ SEM], n ⫽ 10) compared to cells exposed to the nonsilencing oligo (4%5 ⫾ 3%, n ⫽ 6) or not treated and kept in control conditions (61% ⫾ 6%, n ⫽ 9). Wound healing assays using time-lapse videomicroscopy revealed that cells lacking PKP2 moved at a faster speed but in a more tortuous path compared to cells treated with the nonsilencing construct or kept in control conditions. Conclusions: Our data indicate that loss of PKP2 expression alters the migration of EPDCs and their transformation into the fibroblast lineage. We speculate that these cells may play a role in the genesis of the fibrofatty infiltration observed in patients with ARVC.

OVEREXPRESSION OF DIPEPTIDYL-AMINOPEPTIDASE-LIKE PROTEIN 6 (DPP6) ALTERS ACTION POTENTIAL CHARACTERISTICS THROUGH EFFECTS ON Ito1 Tamara T. Koopmann, Arie O. Verkerk, Geert J. Boink, Carol A. Remme, Maaike Buskermolen, Hanno L. Tan, Arthur A. Wilde, Connie R. Bezzina. Heart Failure Research Center, Academic Medical Centre, Amsterdam, The Netherlands Background: Through genetic studies in multiple linked families, we previously identified dipeptidyl-aminopeptidase-like protein 6 (DPP6) as a gene for idiopathic ventricular fibrillation and proposed increased expression of this gene as a likely pathogenic mechanism in this disorder. Co-expression studies in heterologous expression systems point to DPP6 as a putative ␤-subunit of human cardiac Kv4.3 transient outward current, but the function of DPP6 in native cardiac myocytes is unknown. Here we studied the effects of DPP6 overexpression on action potential and transient outward current characteristics in adult ventricular myocytes. Methods and Results: Freshly isolated adult rabbit myocytes were infected with adenovirus for overexpression of human DPP6 (TripleFLAG-DPP6-IRES-GFP). Myocytes were also infected with adenovirus encoding GFP for use as control. Action potential and transient outward K⫹ current (Ito1) characteristics were studied using patch-clamp methodology after 2 days of culture. Immunocytochemistry demonstrated localization of human DPP6 and FLAG on the sarcolemma of DPP6infected myocytes but not of GFP-infected control cells. DPP6 overexpression decreased action potential duration at 20% and 50% of repolarization and caused a more negative action potential plateau phase, particularly at long cycle lengths. Resting membrane potential, upstroke velocity, action potential amplitude, and action potential duration at 90% of repolarization were not affected. Ito1 density and voltage dependency of activation and inactivation were not affected by DPP6 overexpression. However, the fast time constant of current inactivation was slower, and the recovery from inactivation was faster in DPP6-overexpressing myocytes than in GFP-overexpressing myocytes. Conclusion: Overexpression of DPP6 affects the early phase of cardiac action potentials due to enhanced function of Ito1. Gain of function of Ito1

1693 is expected to be proarrhythmic; therefore, DPP6 overexpression may predispose to sudden cardiac death. COUPLING OF ISOLATED ADULT RABBIT VENTRICULAR MYOCYTES TO FIBROBLASTS UNDER STRESS INDUCES AFTERDEPOLARIZATIONS Thao P. Nguyen, James N. Weiss. Medicine/Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, CA Background: Ventricular fibrillation (VF) is the most lethal cardiac arrhythmia, yet the underlying mechanisms are poorly understood. VF occurs most commonly in the presence of markedly increased tissue fibrosis, as seen in aging or heart disease. We hypothesize that electrotonic coupling of ventricular myocytes to fibroblasts under stress can augment stress effects to impair myocyte repolarization reserve and promote the emergence of known VF triggers such as early afterdepolarizations (EADs) and triggered activity. Methods & Results: Patch-clamped isolated rabbit ventricular myocytes were exposed to an oxidative or metabolic stressor (1 mM H2O2 or 2.7mM Ko), which typically induced EADs at pacing cycle length (PCL) 6 seconds but not at 1 second. Single myocytes were coupled by dynamic clamp to virtual fibroblasts with programmable properties. The virtual gap junction current generated by this coupling was summated in real time with native myocyte ionic currents, and their combined effects on myocyte action potentials were observed. Electrotonic coupling to a virtual fibroblast induced EADs and triggered activity at PCL 1s and increased EADs and repolarization failure at PCL 6s during exposure to stress, but not under basal conditions. The extent of EAD induction by fibroblasts correlated with the coupling parameters over a physiologically realistic range. The virtual gap junction current had 2 components: a rapid early transient outward phase, followed by a late sustained phase that reversed polarity from outward to inward. When calcein-loaded rabbit ventricular myocytes were co-cultured with monolayers of rabbit fibroblasts, calcein dye was detected in neighboring fibroblasts after 24 hours, most likely by transport across gap junctions. Conclusions: Our findings indicate that functional heterocellular gap junction coupling between myocytes and fibroblasts spontaneously forms in co-cultures. Moreover, electrotonic coupling of myocytes to virtual fibroblasts can further reduce myocyte repolarization reserve already impaired by stress to promote EADs, triggered activity, and repolarization failure that would not have emerged under stress alone. Our observation supports the link between fibrosis, stress, and ventricular arrhythmias.

DISCREPANCY BETWEEN ACUTE AND LONG-TERM EFFECTS OF THE CALMODULIN-CAMKII-CALCINEURIN PATHWAY ON ARRHYTHMOGENESIS IN THE CAVB DOG Vincent J.A. Bourgonje, Marieke Schoenmakers, Jet D.M. Beekman, Roel van der Nagel, Leon J. de Windt, Toon A.B. van Veen, Marc A. Vos. Medical Physiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands Background: Activation of calcium/calmodulin-dependent protein kinase II (CaMKII) has been linked to arrhythmias and heart failure. Through calcineurin, CaMKII has also been implicated in inducing electrical remodeling. Enhanced susceptibility to arrhythmias based on electrical remodeling is also present in the chronic AV block (CAVB) dog, although in the setting of physiologic hypertrophy. Because the stimuli involved (increased angiotensin II, norepinephrine, and intracellular calcium) are known CaMKII activators, this study addressed the involvement of the CaMKII-calcineurin pathway at different time points (0, 2, and 9 weeks) in the remodeling process of the CAVB dog. Methods and Results: To prevent activation of calcineurin and electrical remodeling, cyclosporin A (CSA) was given to 5 dogs prior to AV block. Seven CAVB dogs not treated with cyclosporin served as control. Dofeti-

1694 lide was given to induce torsades de pointes (TdP). In 5 different inducible CAVB dogs, the calmodulin inhibitor W-7 was administered as an acute arrhythmia suppressant to determine if CaMKII was involved in arrhythmogeneity. Left ventricular biopsy samples were taken at 0 (sinus rhythm), 2, and 9 weeks. AV block and expression of phosphorylated (P) CaMKII and Rcan1-4 (downstream target of calcineurin) were determined. Progressive electrical remodeling in the CAVB dog during the first 2 weeks was not prevented by CSA treatment (QTc time increase: ⫹21% control vs ⫹17% CSA), and CSA did not prevent TdP arrhythmias (4/7 control vs 4/5 CSA). Contractile remodeling also was not blocked (⫹LV dP/dtmax in-

Heart Rhythm, Vol 6, No 11, November 2009 crease ⫹36% vs ⫹48%). At 2 and 9 weeks of CAVB, Rcan1-4 expression was unaltered, thus supporting the CSA data, and the total amount of CaMKII protein and CaMKII-P was not different. However, W-7 was highly antiarrhythmic (32 ⫾ 18 before treatment vs 4 ⫾ 7 TdP per 10 minutes after W-7 treatment, P ⫽ .002). Conclusions: Despite an abundance of stimuli, the CAMKII-calcineurin pathway is not involved in the ventricular remodeling processes of the CAVB dog. However, calmodulin inhibition by W-7 appears to be a valid antiarrhythmic strategy. This finding suggests that dofetilide-induced arrhythmogenesis in CAVB is due to a calmodulin-dependent pathway.