Overexpression Of Na⁺-HCO₃^-Cotransporter Contributes To The Exacerbation Of Cardiac Remodeling In Mice With Myocardial Infarction By Increasing Intracellular Calcium Overload

BackgroundHeart beat starts with the initial phase with Ca2+ influx into cells and Ca2+release from sarcoplasmic reticulum to trigger contraction of cardiac muscles.Reabsorption of the Ca2+ through Ca2+ pump into the sarcoplasmic reticulum will switch the cardiomyocyte from contraction to diastole.Intracellular Ca2+ homeostasis is important for maintaining cardiac contractile function.The Ca2+ pump is delicately affected by a large number of factors in a coordinated manner including intracellular pH,Na+ concentration and Ca2+ concentration.Under physiological conditions,the Na+/H+ exchanger(NHE)and the Na+/HCO3 cotransporter(NBC1)play important roles in regulating intracellular pH,Na+ concentration,and Ca2+ concentration.Previous studies have shown that inhibition of the sarcolemma Na+/H+ exchanger(NHE)has a beneficial effect on cardiac remodeling and heart failure caused by ischemia-reperfusion injury by reducing the accumulation of Na+ and Ca2+.Another study suggested that NBC1 is upregulated in hypertrophic and failing heart,implying an increased ability of cardiomyocytes to export H+.Along with the large amount of H+ excret from the cells,excessive Na+ excret into the cells and subsequently leads to Ca2+ overload via Na+/Ca2+ exchanger,which then collectively inducing cardiac dysfunction.NBC1 is up-regulated in the heart of hypertrophy and failure,but the mechanism is not clear.We hypothesized that NBC1 overexpression might exacerbate cardiac remodeling through increasing incellular Ca2+ overload,promoting cardiomyocyte apoptosis and inhibition of cell contraction.Contents:1.Transgenic(Tg)mice overexpressing NBC1 were used to make a myocardial infarction model,and the influence of NBC1 on cardiac remodeling were investigated through evaluation of cardiac function,hemodynamics,histology,etc.in the mouse model of myocardial infarction(MI).2.In vitro experiments,we isolated and cultured adult mouse ventricular myocytes(AMVMs),overexpressing NBC1 with adenovirus,and detecting cellular calcium kinetics with or without hypoxic injury,clarifying that NBC1 increases intracellular calcium overload and causes decline of cell systolic function.Methods:Including methods in vivo and in vitro.1.Animal study protocols:NBC1 transgenic(Tg)mice were produced.We developed a myocardial infarction model on Tg and wild-type(WT)mice.Effects of NBC1 overexpression to cardiac function,hemodynamics,and ventricular remodeling were observed in the chronic phase(6 weeks).2.Cardiomyocyte study protocols:Isolated adult mouse ventricular myocytes(AMVMs)were used for in vitro experiments.Construction of NBC 1 overexpressing adenovirus,NBC 1 were overexpressed in AMVMs.The treatment factors were overexpression of NBC1,hypoxia for 6 hours,and use of NBC1 inhibitor S0859.Related indicators including calcium kinetics,myocardial cell contractile function and calcium transients are measured after 6 hours of stimulation.Results:1.The mortality rate after MI was markedly increased in Tg mice than in their WT littermates.Survival analysis results indicated that the mortality of mice in the Tg group was significantly higher than that in the WT mice 6 weeks after MI.The mortality rate of NCB1 transgenic mice was 77.5%(31/40),which was significantly higher than that of wild-type mice(30%,6/20).Most of the death occurred from day 3 to day 10 after MI.Autopsy of dead mice revealed pulmonary edema,pleural effusion,blood clots,or left ventricular free wall perforation.These results suggested that the main cause of death after MI was acute heart failure or heart rupture.2.Overexpression of NBCI worsened cardiac function 6 weeks after myocardial infarction.6 weeks after Ml,LVEDd,LVEDs,the exponential time constant of relaxation(Tau),heart weight/body weight(HWf/BW)ratio,and lung weight/body weight(LW/BW)ratio were significantly increased.Left ventricular diastolic anterior wall thickness(LVAWd),LVFS,LVEF,LVSP,dp/dt max,dp/dt min,and contraction index were significantly decreased relative to sham group.Compared with the WT group 6 weeks after MI,dp/dt max,dp/dt min,and contraction index of the Tg group were significantly lower than those of the WT group while Tau was significantly increased.The results of ultrasonography and left ventricle hemodynamics examination showed that NBC1 overexpression aggravated left ventricular dysfunction after 6 weeks of MI.3.Overexpression of NBC1 inhibited cardiomyocyte contraction and worsened calcium kinetics of AMVMs.In AMVMs,overexpression of NBC1 aggravated the inhibition of the shortening of sarcomere and amplitude of calcium transients by hypoxia;overexpression of NBC1 further prolonged the Tpeak and ?d of calcium transients;and administration of NBC1 inhibitor S0859 can partially reduced the effects on the shortening of sarcomere,the amplitude of calcium transients,Tpeak and ?d by hypoxia and overexpression of NBC14.Overexpression of NBC 1 increased the concentration of sodium and calcium ions in AMVMs under hypoxic conditions.Overexpression of NBC 1 or hypoxia increased the concentration of sodium and calcium ions in AMVMs.Overexpression of NBC 1 further aggravated the increase the sodium and calcium concentrations induced by hypoxia in intracellular.The NBC1-specific inhibitor S0859 was used to attenuate the increase of the sodium and calcium ions caused by hypoxia and overexpression of NBC 1 in AMVMs.5.Overexpression of NBCI aggravated hypoxia-induced cardiomyocyte apoptosis.Overexpression of NBC1 significantly increased cardiomyocytes apoptosis at 6 weeks after myocardial infarction and hypoxia-induced apoptosis of AMVMs,and this phenomenon was partially antagonized by NBC1 specific inhibitor S0859 in AMVMs.Conclusion:NBC1 overexpression promotes cardiac remodeling in mice with myocardial infarction by increasing intracellular calcium overload.Therefore,NBC1 may be a potential target for treatment of cardiac remodeling.