Inhibitory Protection Of NO Derived From NNOS On L-type Calcium Channel In Rat Cardiomyocytes

BackgroundWith a rapid development of manned aerospace in China in the last few years,astronauts gradually prolong their work in space. However, long-term exposure tomicrogravity will afflict astronauts with post-flight orthostatic intolerance primarilycaused by hypovolemia, depressed cardiac contractility and vascular remodeling. Reducedpre-load as a consequence of hypovolemia is a primary reason to depressed cardiaccontractility, but the underlying mechanism of impaired cardiac function in microgravityhas not been elucidated until now. Our previous work had found a decrease in theexpression and activity of nNOS, which locates at sarcoplasmic reticulum and sarcolemma and targets L-type calcium channel （LTCC）, phospholamban, and ryanodine receptor.Since LTCC is the key point of cardiac excitation-contraction coupling, we hypothesizedthat the decrease in nNOS expression would influence cardiac function by modulating thegating property of LTCC.Aims（1） Acutely isolating high-quality and high-yield cardiomyocytes and preservingcardiomyocytes for long-time.（2） Analyzing changes of nNOS and LTCC （α1C subunit） expression in the myocardiumof4-week tail-suspended rats （4w SUS） and synchronous control rats.（3） Observing the effects of NO on LTCC gating property and LTCC responsiveness toisoproterenol （ISO） in cardiomyocytes from SUS and CON groups.MethodsIn this research, tail-suspended rat model was used to simulate microgravity on theground. Expression of nNOS and LTCC（α1C） was analyzed by Western blots, and nNOSactivity was detected by nitric oxide synthase kit. Whole cellular patch-clamp techniquewas used to record current of LTCC (I_Ca.L) and to analyze gating property includingsteady-state activation, steady-state inactivation and recovery of LTCC.Results（1） Acutely isolated cardiomyocytes could be preserved for10hours.Because the accumulation of acidic metabolics from high-yield cardiomyocytesdecreased pH value in20ml of preserved-buffer solution, the cardiomyocytes wereexposed to a slight acidosis. The slight acidosis increased ratio of abnormal rod-shapedmyocytes, reduced membrane potential of mitochondria and blue autofluorescence ofmitochondrial NADH, and decreased I_Ca.L. The100ml of preserved-buffer solution couldkeep a stable pH value in the media. Therefore, the high-qulity cardiomyocytes could bepreserved for at least10hours.（2） There was a decrease in both nNOS expression and activity, but no alteration in LTCC（α1C） expression in4-week tail-suspended group.The nNOS expression of cardiomyocytes decreased significantly during4-weeksimulated microgravity and there was no change in eNOS expression and α1C expression.Fluorescence probe of DAF-FM DA targeting NO revealed both total NOS activity andspecific nNOS activity decreased in4-week SUS group.（3） NO protected LTCC gating property through its inhibitory effect during ISOstimulation in cardiomyocytes.LTCC gating property at basal condition exhibited a leftward shift in I-V（Current-Voltage） curve and increased I_Ca.Lin the SUS group as compared with the CONgroup. The steady-state activation of LTCC showed a leftward shift of the half maximalactivation voltage （Va0.5） and no change in the slope factor （ka）. These indicated theincreased LTCC open probability in the SUS group. The steady-state inactivation of LTCCwas not changed in the half maximal inactivation voltage （Vi0.5） and the slope factor （ki） inthe SUS group. The time constant （τ） of recovery shortened in the SUS group whichindicated an acceleration of LTCC recovery from inactivation. ISO treatment increasedI_Ca.Lpeak in both CON and SUS groups, but the percent increase of I_Ca.Lpeak was more inthe CON group than in the SUS group. ISO treatment also induced a leftward shift of Va0.5and a decrease in ka, no changes in Vi0.5and ki, and a decrease in τ in the CON group.However, ISO treatment induced a rightward shift of Va0.5and an increase of ka, a leftwardshift of Vi0.5and no change of ki, no change in τ but incomplete recovery in SUS group.These results illustrated LTCC exhibited a reduced responsiveness to ISO and impairedLTCC gating property in SUS group.LTCC in CON group showed an increase in I_Ca.L, a leftward shift of Va0.5and nochange in ka, no changes in Vi0.5and ki, a decrease in τ after nNOS inhibitor treatment for5min. Compared to ISO treatment alone, LTCC in CON group showed a decrease in I_Ca.L, arightward shift of Va0.5and an increase in ka, a leftward shift of Vi0.5and no change in ki,no change in τ and the incomplete recovery after treatment of nNOS inhibitor together with ISO for5min. These results suggested LTCC had a reduced responsiveness to ISOand impaired LTCC gating property after nNOS inhibition in the CON group.LTCC in CON group showed no change in I_Ca.L, a leftward shift of Va0.5and adecrease in ka, no changes in Vi0.5and ki, and a decrease in τ after NO donor （SNAP）treatment for5min. Compared to ISO treatment alone, LTCC in CON group showed adecrease in I_Ca.L, a rightward shift of Va0.5and an increase in k_a, no changes in Vi0.5and ki,and no changes in τ and complete recovery after treatment of SNAP together with ISO for5min. These results suggested that excessive NO inhibited LTCC and reduced I_Ca.LafterISO stimulation, but did not impair LTCC gating property.ConclusionThe above results suggest that NO derived from nNOS can slightly inhibitresponsiveness of LTCC to ISO and protect LTCC gating property during ISO stimulationin rat cardiomyocytes.