Sino-atrial node (SAN) pacemaker activity is generated by ion channels of the plasma membrane, such as hyperpolarization-activated “funny” f-(HCN), Ca²⁺ channels and ryanodine receptor (RyR) – dependent Ca²⁺ release from the sarcoplasmic reticulum (SR). It is currently disputed whether Ca²⁺ release from RyRs could sustain viable pacemaker activity provided preserved SR Ca²⁺ content. While working myocytes express L-type Ca_v1.2 channels to maintain SR Ca²⁺ content, SAN cells express also L-type Ca_v1.3 and T-type Ca_v3.1 channels to generate pacemaking.

We used mutant mice carrying concomitant ablation of Ca_v1.3 and Ca_v3.1 (Ca_v1.3^−/−/Ca_v3.1^−/−) to study the importance of these channels in automaticity. We also investigated the role of f-HCN channels and RyR-dependent Ca²⁺ release in residual pacemaker activity of mutant mice.

We employed in vivo telemetric recordings of heart rate (HR) in Ca_v1.3^−/−, Ca_v3.1^−/− and Ca_v1.3^−/−/Ca_v3.1^−/− mice. We studied the consequences of pharmacologic inhibition of f-HCN and TTX-sensitive Na⁺ channels in mutant mice using Langendorff perfused hearts or optical mapping (OM) of the pacemaker impulse in intact SAN preparations (SANs).

Ca_v ablation reduced HR in mice: Ca_v3.1^−/− (−7.6%, n=11), Ca_v1.3^−/− (−24.4%, n=8), Ca_v1.3^−/−/Ca_v3.1^−/− (−35%, n=11). In OM experiments on SANs, concomitant inhibition of f-HCN and Na_v1.1 channels slowed pacemaking in wild-type (−48%, n=7) and Ca_v3.1^−/− (−37%, n=7), while arresting automaticity in 4/6 of Ca_v1.3^−/−, 3/6 of Ca_v1.3^−/−/Ca_v3.1^−/−. When present, residual pacemaking was reduced by 82%. Similar results were obtained using isolated Ca_v1.3^−/−/Ca_v3.1^−/− pacemaker cells were automaticity arrested in 5/9 cells tested, or was reduced by 80% in 4/9 cells.

Heart automaticity is primarily generated by Ca_v1.3 and f-HCN channels. RyR-dependent Ca²⁺ release cannot sustain automaticity following concomitant targeting of Ca_v1.3 and f-HCN channels.