Rebuttal: “The funny current in the context of the coupled clock pacemaker cell system” Dario DiFrancesco, PhD*, Denis Noble, CBE, FRS, FmedSci, FRCP (Hon) † From the *Department of Biomolecular Sciences and Biotechnology, The PaceLab, University of Milano, Italy, and the † Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom. It is easily agreed that pacemaking is a complex cellular phenomenon involving several processes, which does not exclude that specific functional roles can be assigned to individual mechanisms. Maltsev and Lakatta 1 view pacemaking as consequent to the periodicity of local Ca releases (LCRs). Contrary to this view, experimental and computational data 2 indicate a spe- cific role of I f in driving pacemaker activity by the gener- ation of diastolic depolarization and control of its early slope, which allows fine control of the pacing rate. Recent evidence for such a pacemaking function in- volves data from a cardiac-specific, inducible ciHCN4 knockout mice showing that the sino-atrial node (SAN) rate decreases (Figure 1A) in proportion to the reduction in I f conductance (Figure 1B) in knockout mice, supporting a direct HCN4 role in rate determination. On the other hand, the view that LCRs contribute essen- tially to rate control was challenged recently by evidence that spontaneous action potentials (APs) persist, with little change in frequency, even in the absence of Ca 2+ -depen- dent contractions (Figure 1C). LCR-induced pacemaking cannot be reconciled with the persistence of regular voltage rhythm in the absence of contraction-associated Ca 2+ tran- sients. Finally, new evidence from Cav1.3 knockout mice sug- gests that LCRs may not be spontaneous after all. In Cav1.3 -/- SAN cells, diastolic LCRs are reduced by 71% relative to wild-type cells. 5 If LCRs are associated with the opening of L-type Cav1.3 channels, they must be re- garded as membrane voltage– operated mechanisms, ac- tivated by diastolic depolarization. This evidence op- poses the view that LCRs are caused by spontaneous Ca 2+ -cycling processes. We suggest that the primary role of Ca 2+ cycling is the repriming of Ca 2+ stores for maximal contraction effi- ciency; to achieve this, rates of release and storing of Address reprint requests and correspondence: Dr Dario DiFrancesco, PhD, Department of Biomolecular Sciences and Biotechnology, The PaceLab, University of Milano, via Celoria 26, 20133 Milano, Italy. E-mail address: dario.difrancesco@unimi.it. Figure 1 A: Time course of telemetric rate from control and ciHCN4 knockout (KO) mice (top: representative records; middle: mean traces) upon treatment with tamoxifen (arrows). Bottom: survival curves. B: Percent decrease in the in vivo rate of KO mice is paralleled by the decrease in single-cell rate (squares, sample traces on top) and in I f conductance. Modified from Baruscotti et al. 3 C: From top: APs, contraction, cycle time, and APD50 in a SAN cell during transition from perforated to ruptured patch clamp with 1,2-bis(o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid (BAPTA)-containing pipette. From Himeno et al. Am J Physiol 2011 Am Physiol Soc, used with permission. 4 1547-5271/$ -see front matter © 2012 Heart Rhythm Society. All rights reserved. doi:10.1016/j.hrthm.2011.09.023