Study Of Mechanism Of Calcium Transport In Excitation Contraction Coupling Of Guinea-pig Ventricular Myocytes

Cardiac excitation-contraction ( E-C ) coupling is the process, which include membrane depolarization, myocyte excitation, calcium ( Ca²⁺) transport and Ca²⁺ as the stimulus for the contraction. The second messenger Ca²⁺ is essential in cardiac electrical activity and is the direct activator of the myofilaments. Myocyte mishandling of Ca²⁺ is a central of both contractile dysfunction and arrhythmias in pathophysiological conditions.OBJECTIVE:(1) To study the physiological role of the L-type calcium channel and sodium/calcium exchange ( NCX ) as a trigger of calcium-induced calcium release ( CICR ) in mammalian cardiac myocytes.(2) To compare the characteristics of Ca²⁺ release from the sarcoplasmic reticulum (SR) triggered by L-type calcium current (I_Ca(L)) and by reverse-mode NCX during depolarizing steps in single guinea-pig ventricular myocytes.(3) To study the electrophysiological characteristics of select NCX inhibitor KB-R7943. The select NCX inhibitor is expected to be useful tools to study the physiological roles ofNCX.(4) To discuss the select NCX inhibitor may have therapeutic potential as a new remedy for heart failure, arrhythmias, hypertension and ischemic disease.METHODS:(1) Single guinea-pig ventricular and rat ventricular myocytes were enzymatically isolated from the hearts. We did the experiments by the whole cell patch clamp, perforated patch clamp, current clamp and single cell shortening recording technique. Experiments with the protocol were done in which membrane current, action potential and contraction were recorded simultaneously.(2) K+-based pipette solution with 10 mmol/L [Na+]p, six 200 ms conditioning pulses from - 80 mV to 30 mV were applied at 0.33 1.0 Hz to maintain SR content constant. During the 10 s pause, membrane voltage was held at - 40 mV, a single pulse to + 10 mV, + 50 mV and +100 mV for 50, 200 and 500 ms elicited a contraction. Membrane current and contraction were measured at same times. Ica(L) is taken as Cd2+ -sensitive current.(3) Cells were excited by external stimuli, 10 control stimuli at 0.5 Hz, a 10 s rest interval and 10 test stimuli also at 0.5 Hz. We also tested KB-R7943 on contractions evoked by stimulus trains with 30 stimuli at frequency ranging from 0.5 to 3.0 Hz. Each stimulus trains was separated by a 30 s rest.(4) Experiments with the protocol were done in which action potentials and contractions were recorded simultaneously. The protocol included two trains of action potentials at 0.5 Hz separated by a 10 s rest interval.(5) Cs+-based pipette solution, membrane voltage was stepped from - 80 mV to - 40 mV for 200 ms. A second voltage jump to + 10 mV for another 200 ms elicited Ica(L> The clamp protocol was repeated at 0.1 Hz. We observed concentration-dependent block of Ica(L) and membrane current by KB-R7943 in steady state.(6) Calcium channel inhibitor and NCX blocker were applied to myocytes by rapid superfusion. All the experiments were done at 35 ± 0.5 °C. The external solution was superfused by gravity.RESULTS:(1) We observed the bell-shaped relationship between L-type calcium current and voltage. The voltage dependent of cell shortening was not bell-shaped but sigmoid. If we plot contractions at 80 ms after the onset of the depolarizing step as a function of the test membrane potential. The bell-shaped voltage dependence of contraction suggests a close relation to Ica(L>(2) The results showed there was a linear relationship between block Ica(L) and contractions (r = - 0.99., p < 0.01). The results indicated that the more block of Ica(L), the more likely contraction would be abolished at + 10 mV test potential with different concentration Nifedipne ( Nif) or Nif 30 umol/L plus Cd2+ 30 umol/L. We got same results between internal and external stimulation.(3) The results indicated that the longer the test pulse duration, the less likely contraction would be decreased at + 50 mV test potential by Nif 30 umol/L plus Cd2+ 30 umol/L. It showed duration-dependent block of contractions by Nif/ Cd2+, which decreased contractions by 58 90 %. The residual contractions onset was delayed. At + 100 mV, contractions were greater than + 50 mV and cell shortening varied little as a function of test pulse duration, addition of Nif 30 umol/L plus Cd2+ 30 umol/L had no effect on cell shortening and the latency to contraction onset at any test pulse duration. 5 mmol/L Ni+ eliminated the residual contractions.(4) With action potential stimulation, 30 umol/L Nif did not block contraction completely on the first test pulse. Nif 30 umol/L plus Cd2+ 30 /umol/L or Nif 100 umol/L completely blocked the test contraction on the first test pulse, and APD50 was decreased by them maximally.(5) Guinea pig myocytes exhibited a positive force-frequency relationship. Rat myocardium exhibited a negative force-frequency relationship. Guinea pig also demonstrated post-rest decay of twich force. In contrast, rat myocardium exhibited post-rest potentiation of contractile force.(6) With Cs+-rich pipette solution, when the holding potential was - 80 mV between testpulse, half-maximal inhibition dose (IC50) of ICa(L) by KB-R7943 was 8.7 /imol/L. 0.3 to 1.0 /imol/L KB-R7943 should have little effect on triggering of contractions by ICa(L)-KB-R7943 shifted the membrane current inward at - 40 mV and - 80 mV.(7) The resting potential was not changed by KB-R7943. The APD50 was not significantly changed by 0.3 /imol/L KB-R7943, but APD90 was increased by 0.3 pimol/L KB-R7943 (7.3%).(8) With external stimulation, 0.3 /miol/L KB-R7943 suppressed the positive staircase. The time to recover 50% of initial amplitude of the tenth contraction after washout of 0.3 /anol/L KB-R7943 averaged 32 ± 11.5 s. Similar experiments were done with 1 and 10 /rniol/L KB-R7943.At 1 and 10 /imol/L, KB-R7943 suppressed the first contraction and the positive staircase in a frequency-independent manner.(9) Experiments at + 10 mV, 0.3 /imol/L KB-R7943 eliminated the positive staircase without appreciably changing Ica(L)-(10)Membrane voltage was clamped at + 100 mV. Block of ICa(L) with Nif 30 /miol/Lplus Cd2+ 30 /rniol/L had negligible effect on the cell shortening. Addition of 1 and 10 /imol/L KB-R7943 decreased contractions by 17.5% and 37.5%. Contraction onset at + 100 mV was delayed by 1 /imol/L KB-R7943. 5 mmol/L Ni+ eliminated the residual contractions.(11) Experiments at + 100 mV, addition of 10 /imol/L ryanodine did not appreciably change end- of-pulse current. Ryanodine completely blocked contractions in 2 of 5 cells tested. In the remaining cells, 10 /imol/L ryanodine decreased the contractions and increased the latency to contraction onset. Addition of 1 and 10 /imol/L KB-R7943, in the continued presence of Nif 30 /imol/L plus Cd2+ 30 /imol/L and ryanodine, reduced remaining contractions, respectively. The latency to contraction onset was delay. 5 mmol/L Ni+ eliminated the residual currents and contractions.CONCLUSIONS:(1) Under normal physiological conditions, ICa(L) will be the major trigger for Ca2+ release from the sarcoplasmic reticulum ( SR ). At more positive potential, Ca2+ entry via the reverse mode NCX could also contribute.(2) Ca2+ entry through the NCX is capable of triggering Ca2+ release from SR but differs substantially from Ica(D- Lower efficiency of reverse mode NCX as a trigger for Ca2+ release is most likely related to lower molecular Ca2+ entry rates and spatial organization. For strong depolarization at > + 60 mV, NCX will trigger release. A striking characteristic of Ca2+ release triggered by NCX is the delay between the onset of depolarization and the actual release.(3) The close linear relationship between Ica(L) block and contraction block is consistent with triggering of contraction by current entering through L-type Ca channels. Ca2+ enter via reverse mode NCX is a much less efficient trigger than entry via L-type calcium channels. NCX will modulate Ca2+ release from the SR as both reverse and forward mode.(4) KB-R7943 is an agent that inhibits selectively the reverse mode NCX. KB-R7943 at low concentrations (0.3 and 1.0 /imol/L) scarcely affect Ica(L), but reduce reverse mode NCX by 50%.(5) Removal of reverse mode NCX by KB-R7943 should prevent SR Ca2+ content from increase to a new steady-state during stimulus trains and interfere with the positive staircase.(6) Reduced contractions in 0.3 /imol/L KB-R7943 more likely reflect suppression of(7) A large contraction component at + 100 mV results from NCX-triggered SR Ca + release. Ryanodine-resistant contractions reflect myofilament activation by Ca entering on reverse mode NCXSUMMARIZE:Under normal physiological conditions, ICa(L) will be the major trigger for Ca2+ release from the SR. Ca2+ enter via reverse mode NCX was a much less efficient trigger than entry via L-type calcium channels. NCX will modulate Ca2+ release from the SR as both reverse and forward mode. In contrast, under special conditions, a large contraction component results from NCX-triggered SR Ca2+ release. Ryanodine-resistant contractions reflect myofilament activation by Ca2+ entering on reverse mode NCX. Reverse mode NCX provides a means to regulate SR Ca2+ content so as to modulate contraction force. KB-R7943 can block reverse mode NCX that regulate excitation-contraction coupling. We conclude that selective block of Lsvca interferes with Ca2+ loading of the SR and suppresses the positive staircase of contractions in frequency-independent manner. KB-R7943 is expected to be useful tools to study the physiological roles of NCX. Such inhibitors may have therapeutic potential as a new calcium regulator for heart failure, arrhythmias, hypertension and ischemic disease.