Extended Data Fig. 2. Ca²⁺ handling and cell-shortening analysis in 8-week-old and 20-week-old adult mouse ventricular myocytes loaded with FuraRed.

Analysis of (a) SERCA and (b) NCX function, and the fluxes needed to balance their activity (SR backflux and sarcolemmal leak, respectively), from electrically-evoked Ca²⁺ transients (CaT) and caffeine-evoked Ca²⁺ release (CaffT) in myocytes isolated from mice at 8 weeks of age. SERCA pump activity was described in terms of a rate constant (unit: s⁻¹) which was calculated from the slope of the relationship between flux and cytoplasmic [Ca²⁺]. To produce this relationship, flux was calculated from the recovery phase of the CaT, and multiplied by buffering capacity to give d[Ca²⁺]/dt. Backflux was calculated as the rate required to balance SERCA at diastolic [Ca²⁺]. The analysis for NCX used the recovery from caffeine-evoked Ca²⁺ release. Sarcolemmal leak was calculated as the rate required to balance NCX at resting [Ca²⁺]. Time-course shown as mean only. Hierarchical analysis of 226–240 myocytes from N = 7, 7, 8, 9 isolations. Mean ± SEM. (c) Hierarchical analysis of cell-shortening of myocytes from 8 week mice, obtained from the cell area during electrically-evoked CaT. Mean ± SEM. (d) Analysis of electrically evoked Ca²⁺ transients and caffeine evoked Ca²⁺ release in myocytes isolated from mice at 20 weeks of age. See Fig. 3 for details of methods. Hierarchical analysis of results from 4-5 isolations per sex and genotype, yielding 25–37 myocytes per group. * P < 0.05. Mean ± SEM. (e) Recordings of electrically-evoked Ca²⁺ transients and caffeine-evoked Ca²⁺ release made in adult rat (ar) ventricular myocytes exposed for 10–30 mins to control (CON) or 6 mM propionate (PRO). Time-course shown as mean ± SEM. Hierarchical analysis of 54-67 myocytes from N = 5 isolations (N.S; not significant).

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