Abstract 2814: Loss of Plakophilin-2 Expression Leads to Decreased Sodium Current and Slower Conduction Velocity in Cultured Cardiac Myocytes
Plakophilin-2 (PKP2) is a protein associated with the cardiac desmosome. Mutations in PKP2 have been linked to up to 70% of cases of familial Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) with an identifiable genotype. Here, we show a cross-talk of PKP2 with the voltage-gated sodium channel complex. A recombinant protein formed by GST concatenated to the head domain of PKP2 (amino acids 1 to 355) pulled-down Nav1.5 from adult heart lysate, and Nav1.5 antibodies co-precipitated native PKP2 from the same preparation. In separate experiments, PKP2 was knocked down (KD) from adult rat ventricular myocytes and patch clamp used to record sodium currents. Data were compared to either cells untreated (UNT), or treated with a construct that failed to silence PKP2 (ΦKD). PKP2 silencing led to an average 50% decrease in peak current density measured at −40 mV (n=6). Steady-state inactivation curves showed that V1/2 of inactivation shifted from −81.51±2.62mV (UNT) and −85.01±3.54mV (ΦKD) to −98.81±2.07mV in KD cells (p<0.02.; n=6 in all groups). Time course of recovery from inactivation was best described by a two-exponential function. Both time constants were prolonged (77% and 128%) in KD cells when compared to either UNT or ΦKD cells (n=6 for all treatments). Additional experiments tested the relevance of PKP2 expression to action potential conduction velocity (CV). Rat neonatal ventricular myocytes were cultured in monolayers, and propagation mapped by optical methods. At a pacing frequency of 1Hz, average CV for UNT monolayers was 24.55±0.99cm/s (n=9), and for ΦKD cells was 28.23±1.29cm/s (n=10). Silencing of PKP2 caused a drastic decrease in CV (17.76±0.84cm/s; n=7; p<0.001). Similar results were found in the range 1– 8 Hz. Pacing at 9 –10 Hz was not possible in KD monolayers, reflecting frequency-dependent activation failure. Our data show that expression of PKP2 affects the function of the sodium channel complex, and the ability of cells to propagate an action potential. The molecular mechanisms by which mutations in mechanical junction proteins affect electrical cardiac stability remain to be defined. Whether these results, in an in vitro system, bear relevance to the pathogenesis of ARVC, is a hypothesis that awaits future investigation.