Study On The Effect Of Low-dose Aconitine On The Metabolism Of HiPSCs-CM And Mechanism Involved

Purpose: Aconitine(AC)is the main active component of Aconitum carmichaelii Debx,a plant of the Ranunculaceae family,which has cardiotonic effect,but also has potential cardiotoxicity.At present,the studies on AC-induced cardiotoxicity mostly carried out at high doses,which mechanisms limited to cardiomyocyte damage and perturbations of intracellular ion homeostasis.Our studies had provided the first confirmation that low-dose AC is non-cell death toxic but it has arrhythmia potential.However,whether low-dose AC induce myocardial toxicity by affecting energy metabolism in cell is still unclear.In this study,whether sequential administration of low-dose AC affect,and mechanisms involved may be explored from the perspective of energy metabolism.Methods:(1)hiPSCs were cultured and induced differentiation into hiPSCs-CM.The expression of myocardial marker and stem cell markers were detected by immunofluorescence to identify whether the differentiation of hiPSCs-CM was successful.(2)After low-dose AC continuously administration,the effects of electrophysiological parameters of hiPSCs-CM were measured by microeletrode arrays.(3)Mitochondrial function,glycolysis function,ATP production rate and energy metabolism phenotype of hiPSCs-CM were determined by energy metabolism technology(Seahorse).(4)Western-blot and immunofluorescence were used to detect the expression of hiPSCs pluripotency transcription factors in hiPSCs-CM;(5)Quantitative PCR and western blot were used to detect the expression of glucose and lipid metabolism related m RNA and protein in hiPSCs-CM;(7)After adding AMPK inhibitor CC,western-blot,immunofluorescence,real-time PCR,Seahorse,and MEA were repeated to verify whether low-dose AC affects the energy metabolism of hiPSCs-CM via the AMPK pathway.Results:(1)After the induction of differentiation,the immunofluorescence results showed that only the expression of the cardiomyocyte marker Actinin(α-Actinin)could be detected in hiPSCs-CM,while the expression of Oct4 A was not found;indicating that hiPSCs-CM were differentiated successfully and could be used for experiments of subsequent low-dose aconitine continuous administration.(2)After 0.1 μmol/L AC was administered,a dose-dependent decrease in the expression of α-Actinin in hiPSCs-CM were measured.(3)MEA results found that after the continuous administration of 0.1 μmol/L AC acted on hiPSCs-CM,the beat period,the field potential and the duration of local extracellular action potentials significantly shortened,the conduction velocity increased,and the delayed excitement contraction decreased.(4)Seahorse results showed that maximum oxygen consumption,energy requirement,glycolytic capacity and ATP production rate were increased in hiPSCs-CM,and energy metabolism phenotype changed from mitochondrial oxidation phosphorylation to glycolysis.(5)The expression of pluripotent transcription factors c-Myc,Sox2 and Oct4 A increased,AMPK was activated to induce phosphorylation.The expression of m RNA and protein level in lipid metabolism key enzyme ACC was decreased while the ratio of Glut1/Glut,a key gene of glucose metabolism,was increased.(6)After adding AMPK pathway inhibitor CC,comparing with the AC group,the expression of pluripotency transcription factors Oct4 A and Sox2 in hiPSCs-CM decreased,and the expression of key genes engaged in glucose and lipid metabolism increased;the energy requirement and the glycolysis were reduced.Conclusion: This study provides the first confirmation that continuous administration of low-dose AC could change the metabolic pattern of hiPSCs-CM.Further confirmation that the mechanism is related to increasing the pluripotency of hiPSCs-CM and activating AMPK pathway.Using cell biology technology to investigate several parameters related to hiPSCs-CM.The results showed that low-dose AC decreased myocardial fibers proteins in a dose-dependent manner and resulted in high-frequency contraction of cardiomyocytes to produce cardiotonic effect.The myocardial contractility was reduced although no arrhythmia was induced,which has an impact on the electrophysiology of cardiomyocytes.The mitochondria of cardiomyocytes provide energy quickly,and the energy supply mode shifts from mitochondrial oxidative phosphorylation to glycolysis;the pluripotency of cardiomyocytes is increased,and AMPK pathway is activated.Energy substrate changes from fatty acids to glucose.