Pacemaker gene mutations, bradycardia, arrhythmias and the coupled clock theory

To the Editor: In a recent paper, Nawathe et al.1 showed that M54T LQTS6 mutation in neonatal rat ventricular myoctes that were co-transfected with human HCN4, the dominate component of f-channels, is associated with bradycardia. The bradycardiac effects that were associated with these mutations, as claimed by the authors, are attributable soley to direct If inhibition. It is well established that normal automaticity of sinoatrial node cell (SANC) is regulated by integrated functions within a system of coupled-clocks. The sarcoplasmic reticulum (SR), traditionally defined as “Ca2+ clock”, spontaneously generates diastolic Ca2+ releases that activate an inward current (specifically Na+-Ca2+ exchanger current (INCX)), that accelerates the rate of diastolic membrane depolarization. The f-channel current (If) another member of the “Membrane clock” (i.e., the ensemble of sarcolemmal electrogenic molecules), concurrently drives the diastolic membrane depolarization with INCX.2 The two clocks are tightly coupled by a common chemical axes: Ca2+-calmodulin activated adenylyl cyclases that generates the second messenger cAMP, which activates protein kinase-A (PKA), and Ca2+ activated calmodulin-dependent kinase II (CaMKII). Both kinases phosphorylate different proteins of the M clock (e.g., L-type and K+ currents) and of the Ca2+ clock (phospholamban and ryanodine receptors). Additionally, cAMP positively shifts the f-channel activation curve. According to the coupled-clock theory, the membrane and Ca2+ clocks should crosstalk. Thus, a change in action potential (AP) firing rate in response to any disturbance signal that directly perturbs either clock entrains the function of the other clock. Consequently, the resultant steady-state AP cycle length change embodies contributions of both clocks. Therefore, the interpretation that bradycardia associated with mutation in HCN4 or with pharmacological blockade of If, occurs solely on the basis of direct inhibition of If function may not be correct. In this regard, a recent study demonstrated that ivabradine, at a concentration that specifically inhibits If, but does not directly suppress L-type current, SR Ca2+ cycling and other surface membrane ion channels, indirectly suppresses intracellular Ca2+ cycling.3 Similar clock coupling effects should occur in the presence of HCN4 mutations. A plausible, indirect effect on SR Ca2+ in response to HCN4 mutations or pharmacological blockade of If can be explained on the basis of this net reduction in Ca2+ influx associated with the bradycardic effect (fewer APs per unit time). A reduction in If initiates a small reduction in number of APs per unit time, which reduces the Ca2+ influx per unit time. This reduction in net Ca2+ influx results in a reduction in intracellular Ca2+, and Ca2+ available for pumping, reducing the SR Ca2+ load and prolonging the restitution time required for spontaneous diastolic Ca2+ releases to occur, i.e., the period of local Ca2+ release becomes prolonged. Consequently, the Ca2+ activation of INCX to a later time during diastolic depolarization, which further reduces the heart rate, leading to further reduction in Ca2+ influx per unit time. Moreover, as a result of reduced intracellular Ca2+, Ca2+ activated CaMKII and adenylyl cyclases-cAMP/PKA signaling axes become damped. The resultant reduction in phosphorylation of Ca2+ cycling proteins that further reduces the net Ca2+ influx, further reduces SR Ca2+ loading that further reduces AP rate and, in parallel, the resultant reduction in cAMP further shifts the If activation curve. Thus, the steady-state bradycardia measured experimentally in response to HCN4 mutations or pharmacological blockade of If is achieved when the induced reduced Ca2+ influx is balanced by a reduction in Ca2+ efflux via INCX. Similar to mutations in the f-channel within the membrane clock, mutations in the intracellular Ca2+ clock proteins are associated with bradicardia. A mutation in ryanodine receptor, axon-3, carried by patients with catecholaminergic polymorphic ventricular tachycardia, is associated with arrhythmias.4 Isolated SANC from mice that express this mutation have a slower spontaneous AP firing rate.5 Patients with a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Cav1.3 (Cav1.3 is largely absent from the ventricles, but is highly expressed in atria, atrioventricular node and sinoatrial node6) experience pronounced bradycardia in 12–24-h ECG recordings, and their heart rate variability is increased.7 Similar to HCN4 mutations, the steady-state bradycardia associated with mutations in Ca2+ clock proteins may be mediated only in part by a reduction in available Ca2+ that inhibits Ca2+ cycling, and in part, by the resultant changes in cross-talk induced change in the membrane clock. In summary, crosstalk occurs between the Membrane and Ca2+ clocks whenever either the membrane or Ca2+ clock is directly perturbed the other clock is indirectly perturbed. Therefore, the steady-state bradycardia associated with different HCN4 mutations, ivabradine or Ca2+ regulatory protein is likely mediated, only in part by If inhibition, and in part, by change in Ca2+ clock due to clock cross-talk. Only an understanding of the full profile underling mechanisms of bradycardia that is associated with HCN4 mutations will promote the discovery of optionally effective treatments for bradycardia.