Figure 4.

PKA-independent increase in intracellular Ca²⁺ is essential for the β₁AR apoptotic effect. After β₁β₂ DKO myocytes were infected by Adv-β₁AR, cells were incubated with designated reagents for 1 hour, then ISO (1 μM) was added and cells were incubated for another 3–6 hours (a, b, d, and e) or 24 hours (c). (a) Prolonged β₁AR stimulation elevated basal intracellular free Ca²⁺ in unpaced cardiac myocytes. This effect was blocked by the L-type Ca²⁺ channel antagonist nifedipine (1 μM), but not the PKA inhibitor PKI (5 μM). *P < 0.01 vs. ISO-untreated groups and those pretreated by nifedipine (n = 20–35 cells from six hearts). (b) Intracellular Ca²⁺ transients were measured in a subset of cells electrically paced at 0.5 Hz for at least 10 minutes in the absence (n = 29 cells from four hearts) and presence (n = 22 cells from four hearts) of sustained β₁AR stimulation by ISO. *P < 0.05 vs. ISO-untreated myocytes. (c) Effects of nifedipine, EGTA-AM (1 μM), or the SR ATPase inhibitor thapsigargin (1 μM) on β₁AR-induced increase in TUNEL-positive cells. *P < 0.01 vs. ISO-untreated myocytes and those pretreated with EGTA-AM, nifedipine, or thapsigargin (n = 4–8). (d) Representative confocal linescan images of caffeine-elicited SR Ca²⁺ release in ISO-treated (1 μM, 3 hours, bottom) and untreated cells (top). The x axis shows the time courses for caffeine treatment, and the y axis shows the spatial profiles of Ca²⁺ transients along a scan line inside the cell. (e) Average amplitude of caffeine-elicited Ca²⁺ transients in ISO-treated or untreated group. *P < 0.01 vs. ISO-untreated myocytes. n = 25–30 cells from six hearts in each group. Nif, nifedipine; TG, thapsigargin.