Functional microdomain in the cardiac pacemaking in mouse

Background Heart rhythm imitates from the leading pacemaker called sinoatrial node (SAN). SAN cell contains a number of unique ion channels and transporters responsible for cardiac pacemaking which were prevailingly believed freely mobile in the cell membrane. Recently, it has been reported that Caveolin‐3 (Cav3), a myocyte‐specific scaffolding protein, compartmentalizes these ion channels and transporters within caveolar membranes. These proteins are attributed to a surface “membrane clock” which is spatiotemporally coupled with subcellular calcium handling machinery (i.e. “Ca 2+ ‐clock”) forming a coupled‐clock pacemaker cell system. Such coupling is achieved via extremely complex crosstalk between the two clocks and is hypothesized to rely on functionality of a macro‐molecular pacemaker signaling complex targeted to caveolae. Methods and Results In vivo ECG telemetry, ex vivo high‐resolution calcium/voltage optical mapping, and in vitro confocal imaging of [Ca 2+ ] i were performed in wild type (WT, n=17) and conditional cardiac‐specific Cav3 knockout (Cav3 −/− , n=14) mice. Cav3 −/− mice exhibited SAN pacemaking abnormalities characterized by alternating periods of tachycardia‐bradycardia rhythm originated from distinct anatomical locations, including SAN (during tachycardia) and ectopic foci located within the intercaval region outside of the SAN (during bradycardia). The ectopic foci areas consisted of non‐tubulated myocytes and had prolonged Ca release time and Ca transient duration due to desynchronized sarcoplasmic reticulum Ca release. This was reproduced in non‐tubulated (but not in tubulated) WT atrial myocytes by disruption of caveolae by methyl‐β‐cyclodextrin. To investigate unstable SAN pacemaking, we characterized subsarcolemmal local Ca 2+ releases (LCRs) from ryanodine receptors (RyR), a critical component of Na + /Ca 2+ exchanger (NCX)‐mediated late diastolic depolarization of SAN action potential (AP). In Cav3 −/− SAN myocytes, regular LCRs were recorded; however, a half of them failed to initiate APs resulting in irregular SAN rate. We found significantly prolonged LCR‐to‐AP period (73.9±6.7 ms in WT vs. 130.6±15.0 ms in Cav3 −/− , p <0.01) and increased LCR amplitude (normalized value: 0.54±0.046 %/μm in WT vs. 0.90±0.13 %/μm in Cav3 −/− , p <0.01). A longer time and a higher driving force needed to activate NCX and boost AP may indicate redistribution of NCX from their canonical location in caveolae to non‐native membrane regions where their functional coupling with RyRs is lost. Conclusion Our findings demonstrate that Cav3 plays a crucial role in supporting functional integrity of the SAN by synchronizing calcium‐voltage coupling and regulating rhythmic LCRs. Loss of Cav3 disrupts caveolae‐associated macro‐molecular pacemaker signaling complex which results in functional uncoupling of NCX from RyRs and affects calcium‐voltage synchronization leading to SAN dysfunction.