Molecular electrophysiology underlying repolarization in canine cardiac purkinje cells : characterization and significance

Cardiac Purkinje fibers (PFs) play a very important role in cardiac electrophysiology. They are crucial for assuring appropriate timing and sequence of ventricle contraction and play an important role in cardiac arrhythmogenesis, largely via abnormalities in repolarization. Little work has been done to define the molecular electrophysiology of cardiac Purkinje cells (PCs). The primary hypothesis of this thesis was that PCs have unique molecular determinants of repolarization. The specific objectives were to characterize the repolarizing currents in isolated canine PCs, to clarify the molecular basis for these currents and to study role of PC current remodeling in a cardiac disease state. To achieve these goals, we used approaches at three different levels: the cellular level (with microelectrode techniques), the ionic level (with whole-cell patch clamp techniques) and molecular level (with competitive RT-PCR, Western blot analysis and immunocytochemistry).We first optimized PC isolation techniques, which allowed us to characterize repolarizing currents and to visualize channel protein distribution by immunolocalization in cardiac PCs. We then characterized an important repolarizing current, the transient outward current (Ito) in canine PCs. We found that Purkinje Ito has some unique properties compared to those of atrial and ventricular Ito, suggesting a different molecular basis. We therefore characterized the expression of alpha-subunits encoding Ito-like currents and the K+-channel interacting protein 2 (KChIP2) beta-subunit. We demonstrated important differences in the expression of Kv3.4; encoding a TEA-sensitive Ito channel, and of KChIP2, that might play an important role in the specific molecular composition of Purkinje Ito. We also characterized another important repolarizing current, the delayed rectifier (IK) that had been reported to be absent or small in PCs. We found that I K· in PCs has properties typical of those observed in other regions of the heart; and IK channel subunits ERG, KvLQT1 and minK were more sparsely expressed in PCs than in ventricular muscle (VM), potentially explaining the tendency of PCs to generate arrhythmias due to abnormal repolarization. We also noted important differences in the expression of the Ca2+-channel subunits (Cav1.2, Cav3.1, 3.2 and 3.3), the Na+/Ca2+-exchanger subunit NCX1 and the hyperpolarization-activated channel subunits HCN1, 2 and 4. Studies in human PCs confirmed that some of the unique PC ionic properties observed in dogs are also present in man. Finally, we showed that an experimental cardiac disease paradigm (congestive heart failure) causes characteristic ionic remodeling in PCs that may explain their role in potentially lethal arrhythmias associated with heart failure.Our findings support the hypothesis of a unique and important molecular basis for the control of repolarization in cardiac PCs