IP₃Rs regulate the frequency of the spontaneous SR calcium releases. (A) RyR (top) and IP₃R (bottom) immunolabeling in isolated E10 embryonic cardiomyocytes. Red arrowheads indicate specific staining pattern around nuclei (red n). (B) When IP₃Rs are inhibited with 2-APB, rapid application of 10 mM caffeine activates RyRs to release calcium (n = 10), and slower application of caffeine (n = 5) induces frequent oscillatory calcium releases (insert). In an identical experimental arrangement, when RyRs are inhibited with 50 μM ryanodine and IP₃Rs are stimulated with IP₃AM, a sustained calcium leak is triggered that is proportional to the applied [IP_3-AM] (bottom). (C) Application of membrane-permeable IP₃ (IP_3-AM) increases the frequency of spontaneous calcium oscillations in isolated E10 cardiac myocytes.