Abstract 4366694: Branched Chain Amino Acids Promote Proarrhythmic Electrical Remodeling in hiPSC-derived Cardiac Atrial Microtissues

Introduction: Metabolic syndrome has become a global health crisis affecting 25-35% of adults. Conditions of metabolic syndrome increase the risk of cardiovascular disease, stroke, and diabetes. Branched-chain amino acids (BCAAs) are elevated in metabolic syndrome and play important roles in ATP production and modulation of metabolic processes. The dysregulation of BCAA metabolism has been linked to atrial arrhythmias but the mechanisms underlying this link are not clearly understood. Goal: To show that BCAA elevation promotes mitochondrial dysfunction and oxidative stress through mTOR activation causing proarrhythmic electrophysiological remodeling using an in vitro human induced pluripotent stem cell (hiPSC)-derived 3D atrial microtissue platform. Methods: hiPSC-derived 3D atrial microtissues (MTs) were cultured under two conditions for 5 days: 1) control (CTR) media: RPMI 1640 Media+B27+albumin+ antibiotics, 2) BCAA media: control media+7.5 mM Leucine+4.5 mM isoleucine+10 mM valine. Spontaneous or stimulated (1 Hz) atrial microtissue action potentials (APs) were recorded by imaging voltage sensitive dye (3 culture batches, 157~244 MTs/condition). The Seahorse assay was used to assess mitochondrial respiration (2 batches, 21~23 MTs/condition). Mass spectrometry (2 batches, 180~210 MTs/condition) and bioinformatics were used to compare metabolite profiles. Results: Compared to CTR, BCAA treatment significantly increased (mean ± SD, p<0.05, independent t-test) automaticity (cycle length: BCAA=1360±524 vs CTR=1506±220 ms), increased pacemaker potential amplitude (BCAA=46±26 vs CTR=34±28 mV ), and shortened AP duration (APD: BCAA=200±67 vs CTR=236±64 ms) (panel A&B). Preliminary data indicate that adding the mTOR inhibitor rapamycin to the BCAA media (10 nM, 5 days) can restore the APD in BCAA-treated microtissues (panel C), aligning with the hypothesis that BCAA dysregulation operates via the mTOR pathway to promote atrial arrhythmias. BCAA treatment also reduced basal and ATP linked mitochondrial respiration (panel D). Changes in metabolites associated with excitability and ion channel regulation were detected by mass spectrometry: BCAA increases AMP, AMP/ATP ratio, and spermidine (panel E) and decreases NAD levels. Conclusion: The observed effects of treating hiPSC atrial microtissues with BCAA on their action potentials, mitochondrial dysfunction, and changes in metabolite profiles favor ectopic activity and reentry in cardiac atrial tissue.