Ca2+ dye loading and live-cell imaging

To simultaneously record cytosolic and SR Ca²⁺ signals³⁵, freshly isolated CMs from AAV9-Tnnt2-srCES2 injected mice were incubated with low-affinity Ca²⁺ indicator Fluo5N-AM (5 μmol/L, Invitrogen, #{"type":"entrez-nucleotide","attrs":{"text":"F14204","term_id":"860757","term_text":"F14204"}}F14204) at 37 °C for 10 min, washed once, and incubated with Rhod2-AM (5 μmol/L, Invitrogen, #R1244) at 37 °C for 8 min, and then gently washed two times. Dye loading, washing, and Ca²⁺ imaging were conducted in Tyrode solution containing 1 mM Ca²⁺.

Confocal imaging was performed with an Olympus FV3000RS microscope with a ×60 1.4 NA oil immersion objective and a line scan speed of 3.78 ms/line. The pinhole was set for a nominal 1 μm optical section. For single-channel measurement of ASPH-G6f, excitation was at 488 nm, and fluorescence emissions were collected at between 490 and 540 nm. For simultaneous measurement of Fluo5N and Rhod2, excitation was at 488 and 543 nm, and fluorescence emissions were collected at between 490 and 520 nm and >560 nm, respectively. For Ca²⁺ transients recording, CMs were perfused with Tyrode solution containing 10 mM butanedione monoxime to avoid motion artifacts, and field stimulation was applied at 1 Hz.