While prolongation of the action potential (AP) can be anti-arrhythmic, it is also the cause of many arrhythmias due to drugs, congenital disease, and heart failure. Much evidence implicates early after depolarizations (EADs) due to L-type Ca2+ current (ICa-L) as a trigger for arrhythmias associated with prolonged action potentials. We and others nave shown that multifunctional Ca2+/calmodulin - dependent protein kinase II (CaM kinase) augments ICa-L in response to increased intracellular Ca2+ ([Ca2+]i ), and increased [Ca 2+]i occurs during EADs and AP prolongation. Importantly, after initial activation, CaM kinase activity (unlike that of other kinases) becomes independent of [Ca2+]i. Therefore, the central hypothesis to be tested in this research is that CaM kinase acts as a [Ca2+]i dependent and a [Ca2+]i - independent positive feedback effector for ICa-L and thus serves as a key proarrhythmic signaling molecule for EADs. Moreover, our preliminary data also indicate CaM kinase is localized in myocytes to the Z-lines, raising the possibility that the action of CaM kinase on ICa-L and EADs is specified by an interaction among the kinase, specific anchoring proteins, and the cytoskeleton.
Four specific aims i n this research will determine (1) the role of [Ca2+]i and ICa-L in EADs in cardiac cells with prolonged action potentials; (2) the effect of CaM kinase inhibition on ICa-L, [Ca2+]i and EADs; (3) the effect of constitutively active CaM kinase on ICa-L, [Ca 2+]i, and EADs; and (4) the role the cytoskeleton and anchoring proteins in modulation of ICa-L, [Ca 2+]i, and EADs by CaM kinase. EADs will be studied in isolated rabbit ventricular myocytes with prolonged action potentials, using current clamp and whole cell mode voltage clamp with action potential wave forms, under conditions where [Ca2+]i is controlled and directly measured, ICa-L is directly measured, and CaM kinase activity is controlled with specific inhibitory peptides or the use of a constitutively active (Ca2+-independent) form of the enzyme. By furthering our understanding of the mechanisms underlying arrhythmias related to prolonged repolarization, this research should result in identification and initial validation of novel targets for suppression or these arrhythmias.
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