Figure 5. Casq2^–/– myocytes have largely preserved SR Ca²⁺ release and SR Ca²⁺ content under basal conditions, but isoproterenol application causes increased SR Ca²⁺ leak.

(A) Representative examples of rapid application of caffeine (10 mmol/l) to a Casq2^+/+ (top) and a Casq2^–/– myocyte (bottom). Myocytes were field stimulated at 1 Hz to maintain consistent SR Ca²⁺ load. Note the increased twitch transient and caffeine-induced transients in the presence of ISO (1 μmol/l; right). The height of the caffeine-induced Ca²⁺ transient was used as a measure of total SR Ca²⁺ content (30). Fractional SR Ca²⁺ release was calculated by dividing the height of the last twitch transient by the height of the caffeine transient. (B) Comparison of average SR Ca²⁺ content (left) and fractional SR Ca²⁺ release (right). *P < 0.05, **P < 0.01. Casq2^+/+ myocytes: n = 41 (baseline) and 27 (ISO); Casq2^–/–myocytes: n = 70 (baseline) and 37 (ISO). (C) Protocol used to measure SR Ca²⁺ leak as described in ref. 32. Plasma membrane Ca²⁺ flux is eliminated by removal of extracellular Na⁺ and Ca²⁺. The drop in steady-state [Ca²⁺]_i (double arrow) represents a shift of Ca²⁺ from the cytosol to the SR when RyR2 channels are inhibited by tetracaine (1 mmol/l) and was used as a measure of SR Ca²⁺ leak. (D) Comparison of average SR Ca²⁺ leak (left) and SR Ca²⁺ content in the presence of tetracaine (right). Note that when SR Ca²⁺ leak was blocked by tetracaine, SR Ca²⁺ content was not significantly different between the 2 groups. **P < 0.01. Casq2^+/+ myocytes: n = 32 (baseline) and 45 (ISO); Casq2^–/–myocytes: n = 29 (baseline) and 42 (ISO). (E) SR Ca²⁺ leak in the presence of ISO plotted as a function of SR Ca²⁺ content. Note that the SR Ca²⁺ leak of Casq2^–/– myocytes remained SR load dependent but was shifted to the left compared with that of Casq2^+/+ myocytes.