Ca2+ influx through Ca2+ channels in rabbit ventricular myocytes during action potential clamp - Influence of temperature

Ca2+ influx via Ca2+ current (I-Ca) during the action potential (AP) was de termined at 25 degrees C and 35 degrees C in isolated rabbit ventricular my ocytes using AP clamp. Contaminating currents through Na+ and K+ channels w ere eliminated by using Na+- and K+-free solutions, respectively. DIDS (0.2 mmol/L) was used to block Ca2+-activated chloride current (I-Cl(Ca)). When the sarcoplasmic reticulum (SR) was depleted of Ca2+ by preexposure to 10 mmol/L caffeine, total Ca2+ entry via I-Ca during the AP was approximate to 12 mu mol/L cytosol (at both 25 degrees C and 35 degrees C). Similar Ca2influx at 35 degrees C and 25 degrees C resulted from a combination of high er and faster peak I-Ca offset by more rapid I-Ca inactivation at 35 degree s C, During repeated AP clamps, the SR gradually fills with Ca2+, and conse quent SR Ca2+ release accelerates I-Ca inactivation during the AP. During A Ps and contractions in steady state, total Ca2+ influx via I-Ca was reduced by approximate to 50% but was again unaltered by temperature (5.6+/-0.2 mu mol/L cytosol at 25 degrees C, 6.0+/-0.2 mu mol/L cytosol at 35 degrees C) . Thus, SR Ca2+ release is responsible for sufficient I, inactivation to cu t total Ca2+ influx in half, However, because of the kinetic differences in I-Ca, the amount of Ca2+ influx during the first 10 ms, which presumably t riggers SR Ca2+ release, is much greater at 35 degrees C. Ic, during a firs t pulse, given just after the SR was emptied with caffeine, was subtracted from Ic, during each of 9 subsequent pulses, which loaded the SR. These dif ference currents reflect lc, inactivation due to SR Ca2+ release and thus i ndicate the time course of local [Ca2+] in the subsarcolemmal space near Ca 2+ channels produced by SR Ca2+ release (eg, maximal at 20 ms after the AP activation at 35 degrees C). Furthermore, the rate of change of this differ ence current may reflect the rate of SR Ca2+ release as sensed by L-type Ca 2+ channels. These results suggest that peak SR Ca2+ release occurs within 2.5 or 5 ms of AP upstroke at 35 degrees C and 25 degrees C, respectively. I-Cl(Ca) might also indicate local [Ca2+], and at 35 degrees C in the absen ce of DIDS (when I-Cl(Ca) is prominent), peak I-Cl(Ca) also occurred at a t ime comparable to the peak I-Ca difference current. We conclude that SR Ca2 + release decreases the Ca2+ influx during the AP by approximate to 50% (at both 25 degrees C and 35 degrees C) and that changes in I-Ca (and I-Cl(Ca) ), which depend on SR Ca2+ release, provide information about local subsarc olemmal [Ca2+]. The full text of this article is available at http://www.ci rcresaha.org.