Ca2+-dependent mechanisms cause arrhythmia in inherited arrhythmia such as catecholaminergic polymorphic tachycardia (CPVT) as well as in cardiomyopathy. The inward rectifier K current (IKI) is responsible for resting potential and terminal repolarization in heart and is carried through the alpha subunit protein KIR2.1 encoded by KCNJ2. >Addifional alpha subunits in this KIR2.X family (KIR2.2, KIR2.3, KIR2.4) may play a role in IKL Relative to voltage-dependent Na and K channels, the subunits underlying IKI and arrhythmia mechanisms involving IKI are less well studied. We report preliminary data for a novel mutafion KIR2.1-V227F from a pafient with CPVT, a syndrome not previously associated with mutations in KCNJ2. In voltage clamp experiments KIR2.1-V227F co-expressed with KIR2.1-WT in heterologous cells showed loss of function only when sfimulated by PKA. KIR2.1-V227F transfected into cultured myocytes caused triggered activity only after isoproterenol in support of a mechanism for CPVT. We hypothesize that some KIR2.1 mutations respond to beta adrenergic activity with loss of function and subsequent altered cellular electrophysiology and Ca2+ handling, including direct effects of Ca2+ on IKI, leading to triggered arrhythmia. We further hypothesize that heteromers with other KIR2.X subunits in the heart are important for mechanism, and that novel mutations in KIR2.X cause inherited arrhythmia. This project will study three arrhythmia mutations in KIR2.1, one hypothesized to require adrenergic activity (V227F), one hypothesized to have less or no adrenergic dependence (R82W) and one unknown (R67Q). These channels will be expressed in heterologous systems and transgenic mice and studied by an integrated approach including voltage clamp studies of ion currents, Ca2+ imaging, acfion potenfials, and ECG to determine the biophysical, cellular, and whole animal arrhythmia phenotypes that account for arrhythmogenic mechanisms.
Aim 1 will characterize the biophysical phenotype of IKI including adrenergic and Ca2+ dependence of KIR2.1-V227F, R82W, and R67Q mutafions in heterotetramers with KIR2.2, KIR2.3, and KIR2.4 in heterologous cells. As part of this aim we will determine the presence and relative quantities of KIR2.X in human and mouse myocardium, and search for and characterize novel arrhythmia mutations in KIR2.X.
Aim 2 will characterize the cellular phenotype including action potentials, Ca2+ transients, and ion currents in native myocytes from transgenic mice with the KIR2.1 mutafions, and Aim 3 will determine the arrhythmia phenotype by analyzing arrhythmia in these mice. Achievement of these aims will elucidate arrhythmia mechanism for known and recently discovered mutations in genes affecfing IKI, and may lead to the discovery of mutations in novel arrhythmia genes. These discoveries will impact diagnosis and treatment of Ca2+-dependent arrhythmia disorders. Because loss of IKI function also plays a role in acquired arrhythmia in heart failure, these findings will have application beyond these relatively uncommon arrhythmia syndromes, and will also contribute insights into the IKI macromolecular complex.
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