Modulation of focal and global Ca2+ release in calsequestrin‐overexpressing mouse cardiomyocytes

In cardiac muscle the influx of Ca2+ through the Ca2+ channel is critical in triggering Ca2+ release from the sarcoplasmic reticulum (SR). The released Ca2+ in turn inactivates the L-type Ca2+ channel by interacting with the ‘Ca2+ sensing’ domains of the C-terminal tail (Soldatov et al. 1998) expressing calmodulin-binding properties (Zuhlke et al. 1999; Peterson et al. 1999; Qin et al. 1999). Ca2+ mediated cross-signalling between the Ca2+ channel and ryanodine receptor (RyR) allows for tight functional coupling between these proteins, such that the influx of Ca2+ through the Ca2+ channel activates Ca2+ release from the RyR and the released Ca2+ in turn inactivates the Ca2+ channel, thereby inhibiting the release process (Sham et al. 1995). Quantitative analysis of the contribution of the released Ca2+ to the inactivation of the Ca2+ channel suggests that about 70 % of Ca2+ channel inactivation is determined by the Ca2+ released via the ryanodine receptor (Adachi-Akahane et al. 1996). In the cardiac Ca2+ signalling cascade, the Ca2+ storage capacity of SR and the extent to which Ca2+ can be released from the low affinity (Kd≈ 1 mM) Ca2+ binding protein calsequestrin (CSQ) (MacLennan & Wong, 1971; Ikemoto et al. 1989) may also play a critical role in the release process (Kawasaki & Kasai, 1994). The structural properties of CSQ and its localization within the lumen of the SR suggest that this protein may interact with the RyR via the junctional proteins junctin and triadin (Jones, et al. 1995; Guo et al. 1996; Zhang et al. 1997). Recently we reported that mouse hearts overexpressing canine CSQ hypertrophied by 50–100 % and developed heart failure (Jones et al. 1998). Isolated myocytes from such hearts were 50–100 % larger and had significant electrophysiological and electromechanical alterations (Knollmann et al. 1998b). The most prominent features of electrical remodelling of these CSQ-overexpressing hearts were prolongation of the action potential and Q-T interval, downregulation of the transient outward K+ current (Ito), and greatly slowed kinetics of inactivation of ICa (Jones et al. 1998; Knollmann et al. 1998b). Since ICa-gated Ca2+ release was impaired in CSQ-overexpressing myocytes despite the presence of 10- to 15-fold larger caffeine-releasable Ca2+ pools (Jones et al. 1998), we suggested that impaired Ca2+ release was mostly responsible for the marked slowing of the inactivation kinetics of ICa. In a subsequent report on another strain of transgenic mice overexpressing murine calsequestrin, similar hypertrophy and slowing of the kinetics of ICa were described, but the extent of impairment of ICa-gated Ca2+ release was not quantified (Sato et al. 1998). In the present report we have quantified the properties of focal and global Ca2+ release in this model and have studied the nature of impaired Ca2+ signalling in CSQ-overexpressing myocytes by interventions that modify the Ca2+ channel or the Ca2+ sensitivity of the RyR. We find that agents that enhance Ca2+ current are less effective in re-establishing normal Ca2+ signalling than those that sensitize the Ca2+ release channel. A preliminary report of this work has already appeared (Wang & Morad, 1999).