Study On The Effects Of Electrical Stimulation On Human Induced Pluripotent Stem Cells Derived Cardiomyocytes And The Underlying Mechanisms

Heart dysfunction and even cardiac death caused by tachycardia and long-term pacing is not rare in clinical practice.But little is known about the changes occurred at the cellular level and the underlying mechanisms.In this study,we mimicked clinical tachycardia and long-term pacing by applying rapid electrical stimulation(RES)or long-term electrical stimulation(LTES)on human induced pluripotent stem cell derived cardiomyocytes(hiPSC-CMs).For RES,stimulation at a frequency of 3 Hz was applied on hiPSC-CMs for 7 days,with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia,respectively.Stimulation at a frequency of 1 Hz and 24h/24h was set as low-frequency electrical stimulation(LES)control.For LETS,stimulation parameters were set at 1.2Hz for 2 weeks and set pacing percentage at 40%and 100%as two subgroups.Age-matched hiPSC-CMs without electrical stimulation were set as no electrical stimulation(NES)control.Following stimulation,immunostaining and TEM were adopted to observe cellular structure.Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide(AV/PI)staining flow cytometry analysis.Patch-clamp was used to evaluate cellular electrophysiology.JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions.Western blot and qPCR were performed to evaluate the expression of involved genes.When compared with control group,hiPSC-CMs following RES presented mitochondrial dysfunction and increased apoptotic percentage.Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES.Molecular analysis demonstrated upregulated expression of Caspase3 and pro-apoptotic BAX,while downregulated expression of anti-apoptotic BCL.Genes related to calcium re-sequence were downregulated,while phosphorylated Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKII)was significantly upregulated following RES.There was no significant difference between the NES control and LES control groups in these aspects.Inhibition of CaMKII with 1μM KN93 partly reversed these adverse effects of RES.When compared with control group,hiPSC-CMs following LTES presented increased cardiac apoptosis and decreased expression of contractile protein cTnT.The action potential duration was shortened and currents density were decreased.Endoplasmic reticulum(ER)stress and Calpain activity were promoted.All of these adverse effects were pacing percentage dependent.Pharmacal inhibition of Calpain with Calpeptin can partly attenuate the adverse effects of long-term pacing.In this study,RES on hiPSC-CMs disturbed calcium homeostasis and activate mitochondrial apoptosis pathway,which led to cell loss and systolic dysfunction.RES can also increase phosphorylation of CaMKII,pharmacological inhibition of CaMKII activity partly reversed the adverse effects of RES.LTES have adverse effects on both structural and electrophysiological properties,LTES can promote ER stress and Calpain activity,pharmacological inhibition of Calpain with Calpeptin can partly attenuate the adverse effects of long-term pacing.CaMKII and Calpain might be chosen as new therapeutic targets of clinical arrythmias.