Extracellular PH Regulates Autophagy Via AMPK Pathway In Rat Cardiomyocytes

BackgroundMaintaining the intracellular pH and extracellular pH within a physiological range is fundamental for cellular metabolism and function.Despite the efficient pH control mechanisms that have evolved within mammalian cells,local and even systemic pH fluctuations occur frequently due to severe burn and trauma,hypoxia,ischemia,metabolic alkalosis and so on.Changes in pH can affect a variety of signals,thus modulating multiple pathophysiological processes.Cardiac myocytes are terminally differentiated cel ls that are susceptible to changes in pH.Downregulation of cardiac pH is associated with a decreased level of cellular metabolism and myocardial contractile dysfunction,which promotes cardiovascular morbidity and mortality.Upregulation of the cardiac pH enhances myocardial metabolism and protein synthesis,whereas the persistent alkalization may contribute to the development of the myocardial hypertrophic phenotype.These results suggest that pH plays an important role in regulation of myocardial cell function.Nevertheless,the molecular mechanisms underlying pH changes-induced cardiomyocytes dysfunction are poorly understood.Autophagy is a metabolic process that helps to prevent the build-up of protein aggregates and damaged organelles,and basal autophagy plays an essential cytoprotective role.In the cardiovascular system,autophagic activity contributes to a wide range of cellular events under both physiological and disease-related pathophysiological conditions.It is widely accepted that upregulation of autophagy promotes cardiomyocytes survival upon nutrient and energy stress,such as in trauma,starvation,hypoxia and pressure overload,whereas excess autophagy provokes cell death and cardiac failure by destroying too many essential cellular components.Therefore,autophagy may play both beneficial and deleterious roles in cardiomyocyte function.In some mammalian cells,it has been demonstrated that acidic or alkaline stress upregulate autophagy,thus contributing to a protective or cytotoxic effect.Those reports have indicated that pH can modulate autophagic activity and play a critical role in specific pathophysiological processes.However,whether changes in pH affect autophagy in cardiomyocytes remains unclear.AMP-activated protein kinase（AMPK）is a αβγ heterotrimeric serine-threonine kinase that positively regulates autophagy under various stress conditions.In the myocardium,starvation,ischemia,and oxidative stress upregulate the activity of AMPK and autophagy,thus playing a critical role in alleviating myocardial injury and promoting cardiomyocytes survival.Activated AMPK functions as an activator of autophagy through different mechanisms,such as activating ULK1 and inhibiting the activity of mammalian target of rapamycin（mTOR）.To date,there is no information available regarding whether AMPK participates in pH-modulated autophagy in cardiomyocytes.Moreover,the signals downstream of AMPK that mediate pH-modulated autophagy are unknown.Here,we investigated the effect of pH on autophagy in rat cardiomyocytes and to identify the molecular mechanism underlying its effects.Methods1.DMEM/F-12 with different pH values were obtained by adding different concentrations of NaHCO₃.Cell viability was measured using CCK8 assay and the release of LDH.Western blot（WB）and immunofluorescence were used to detect autophagy activity upon different pH.Then the acidic compartments were stained by using Lyso Tracker red and Lyso Sensor Green.Bafilomycin A1（BafA1）was used to inhibit the acidification of organelles,and then detect autophagy activity upon different pH.2.WB were used to detect AMPK activity upon different pH,then recombinant adenovirus vectors for silencing AMPKα2 were constructed and used to infect cardiomyocytes.WB and IF were used to detect autophagy activity upon different pH.Cardiomyocytes were treated with Met or sh AMPKα2 upon different pH and then the autophagy activity were analysed using WB and immunofluorescence.To show the role of AMPK and autophagy in cardiomyocytes in response to changed pHe,cell viability was measured using CCK8 assay,the release of LDH,and SYTOX green nucleic acid stain.3.The effect of pH on mTORC1 activity was evaluated using WB.Cardiomyocytes were treated with Met or sh AMPKα2 upon different pH and then the mTORC1 activity were analysed using WB.The influences of mTORC1 on AMPK and autophagy activity were assessed.WB was used to detect ULK1 activity upon different pH,then the effect of AMPK on ULK1 activity were assessed.Recombinant adenovirus vectors for silencing ULK1 were constructed and used to infect cardiomyocytes.Cardiomyocytes were treated with Met or sh ULK1 upon different pH and then the autophagy activity were analysed using WB and immunofluorescence.To evaluate the role of ULK1 and autophagy in cardiomyocytes in response to changed pHe,cell viability was measured using CCK8 assay,the release of LDH,and SYTOX green dye.Results1.Acidic treatment significantly reduced cell viability;alkaline treatment increased cell viability in the early,and these changes were reversed at 9 h.LC3-II conversion was decreased upon acidic treatment and was increased upon alkaline treatment,both with downregulated p62 expression.Consistently with the WB results,the LC3 puncta was markedly reduced upon acidic treatment and was increased upon alkaline treatment,and most of the LC3 staining was co-localized with lysosomal associated membrane protein 1（LAMP1）staining.Additionally,the staining of acidic compartments in cardiomyocytes showed that lysosomal acidity remained unabated for 6h under both acidic and alkaline conditions,and Baf A1 treatment significantly enhanced the accumulation of LC3-II and p62.2.Acidic treatment significantly downregulated AMPK activity and alkaline treatment upregulated it.Under acidic treatment,Met treatment effectively induced AMPK activity and upregulated autophagy,and that transfection with the sh AMPKα2 reversed this effect.Under alkaline treatment,knockdown of AMPKα2 expression blocked the alkaline medium-induced AMPK activity and downregulated autophagy.Immunofluorescence data is consistent with the WB results.Besides,acidic treatment significantly reduced cell viability and Met treatment attenuated it.Interestingly,in combination with sh AMPKα2,the effect of Met was acounteracted.Alkaline treatment increased cell viability;in contrast,knockdown of AMPKα2 had the opposite effect.3.mTORC1 activity was inhibited upon acidic treatment and was stimulated upon alkaline treatment.The changes in AMPK activity had little effect on the mTORC1 activity upon different pH.Also,the change of mTORC1 activity had no appreciable effect on the AMPK activity or the LC3-II conversion upon different pH.ULK1 activity was decreased upon acidic treatment and was increased upon alkaline treatment.Furthermore,activation of AMPK in acidic pH upregulated ULK1 activity and inhibition of AMPK blocked this effect,transfection with sh AMPKα2 in alkaline pH reduced ULK1 activity.Transfection of sh ULK1 blocked the LC3-II conversion that was induced by activation of AMPK via Met upon acidic treatment,and also blocked the LC3-II conversion regardless of whether AMPK was activated upon alkaline treatment.Immunofluorescence data is consistent with the WB results.Moreover,in combination with sh ULK1,the effect of Met and alkaline treatment on cell viability was attenuated.Conclusion1.Acidic treatment significantly reduced cell viability;alkaline treatment increased cell viability in the early,and the change was reversed at 9 h.Autophagy activity was decreased upon acidic treatment and was increased upon alkaline treatment.2.AMPK play a crucial role in pH-regulated autophagy and cell viability in cardiomyocytes.3.ULK1 activity is indispensable for the regulation of autophagy by AMPK upon changed pHe.mTORC1 pathway was not involved in AMPK or autophagy modulation upon changed pHe in cardiomyocytes.4.Early autophagy may exert a protective role in cardiomyocytes upon different pH.5.In the fluid resuscitation of severe burn,keeping the blood pH does not affect the release of oxygen from hemoglobin andnormalcellactivity is important to alleviate theinjuryinduced by anoxia and ischemic and improve the viability of patients.6.These data may potentially introduce a new insight on understanding the therapeutic value of modulating autophagic activity to prevent pH changes-elicited cardiac injury.