Calsequestrin (CSQ) is a SR resident Ca2+-binding protein that may also modulate other proteins involved in excitation- contraction (E-C) coupling. To evaluate the effect of CSQ over-expression on cardiac excitation-contraction (E-C) coupling, a recombinant adenovirus coding for human calsequestrin (Ad-CSQ) was used to transfect rabbit ventricular myocytes.
Rabbits were humanely killed with an overdose of Euthatal (100 mg kg-1) and their hearts removed. The ventricular myocytes were isolated and placed in culture and infected with Ad-CSQ or an adenovirus coding for β-galactosidase (Ad-LacZ) as a control. Data are expressed as means ± S.E.M. Comparisons were performed using Students’ unpaired t test and differences were considered significant when P < 0.05.
Twenty-four hours after infection with Ad-CSQ (100MOI), CSQ expression was increased 1.58 ± 0.18 times (mean ± S.E.M., n = 10, P < 0.05) as assessed by Western blots. Fura-2 loaded myocytes were voltage clamped from a holding potential of -80 mV to -40 mV (50 ms) and then to 0 mV (150 ms) every 2 s at room temperature in the presence of 5 µM TTX to inhibit inward Na+ current. An increased peak-systolic [Ca2+] was observed after Ad-CSQ transfection (329 ± 32 nM, n = 23 vs. 192 ± 25 nM, n = 16, P < 0.05). L-type Ca2+ current amplitude was also increased (3.22 ± 0.33 pA pF-1, n = 23 vs. 1.85 ± 0.27 pA pF-1, n = 17, P < 0.05). Measurements of intracellular Ca2+ and associated Na+-Ca2+ exchanger current (INCX) upon rapid application of 10 mM caffeine were used to assess SR Ca2+ content. The integral of the INCX revealed a significantly higher SR content in the Ad-CSQ group (4.13 ± 0.42 C/F, n = 8) compared to the Ad-LacZ group (2.57 ± 0.39 C/F, n = 6). When caffeine was applied in the presence of Ni2+ the rate of extrusion of Ca2+ from the cell was significantly reduced in the Ad-CSQ transfected cells. This latter result suggests that Ca2+ extrusion via the sarcolemmal Ca2+ ATPase is reduced in the Ad-CSQ group.
In summary, CSQ over-expression has complex effects on E-C coupling including modulation of Ca2+ influx and efflux processes.