Suzan J.G. Knottnerus1,2*, Isabella Mengarelli 3*, Rob C.I. Wüst1, Vincent Portero3, Lodewijk IJlst1, Jeannette C. Bleeker 1,2, Sacha Ferdinandusse1, Ronald J.A. Wanders1, Frits A. Wijburg4, Gepke Visser1,2,Kaomei Guan6, Arie O. Verkerk4,5, Riekelt H. Houtkooper1*, Connie R. Bezzina3*
*: equal contribution
Corresponding author: Suzan J.G. Knottnerus Corresponding author’s e-mail: firstname.lastname@example.org
Background: Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that catalyzes the first step in the mitochondrial beta-oxidation, are at risk of developing cardiac symptoms including arrhythmias. Treatment options are scarce, partly because the exact underlying mechanism leading to arrhythmias in these patients is currently unknown. The electrophysiological derangement may be related to either (1) energy shortage because of defective fat utilization, or (2) the accumulation of fatty acid oxidation intermediates i.e. long-chain acyl-CoAs and/or notably long-chain acylcarnitines. To address this question, we have used a pharmacological approach involving either enhanced mitochondrial biogenesis or substrate reduction in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from VLCAD deficient (VLCADD) patients.
Methods: We measured electrophysiological and biochemical parameters in cardiomyocytes from one control and two VLCADD patient-derived hiPSC lines: VLCAD1 (p.Val283Ala/p.Glu381del), and VLCAD2 (homozygous for c.104delC). We used two strategies to reduce the accumulation of acyl-CoAs and/or acylcarnitines by pre- incubating the cells 48h with (1) 50 µM resveratrol to increase mitochondrial biogenesis, or (2) 100 µM etomoxir – a powerful inhibitor of the rate controlling enzyme carnitinepalmitoyl transferase 1 (CPT1) – to reduce the fatty acid oxidation flux.
Results: When cultured under standard conditions, the cardiomyocytes from the two VLCADD hiPSC lines accumulated long-chain acylcarnitines. Action potentials, measured with patch clamp and Kir2.1 injection via dynamic clamp, were shorter and displayed lower amplitudes in VLCADD compared to control. Moreover, the susceptibility -to delayed afterdepolarizations (DADs) was increased in cardiomyocytes from the two VLCADD lines. In VLCAD1, but not in VLCAD2, long-chain acylcarnitine accumulation was decreased by pre-incubation with resveratrol. Accordingly, action potentials were normalized and susceptibility to DADs generation was reduced in VLCAD1. Pre-incubation with etomoxir led to a reduction of acylcarnitine production in both patients, and reduced the amount of DADs in cardiomyocytes from both cell lines.
Conclusion: Cardiomyocytes from hiPSC-lines of VLCADD patients show accumulation of long-chain acyl-CoAs and/or long-chain acylcarnitines, shorter action potentials, and a higher susceptibility to DAD generation, an important cellular mechanism for arrhythmias. The reduction of acylcarnitine accumulation by resveratrol or etomoxir reduced DAD generation in the two patients’ cell lines, indicating that DADs in VLCADD hiPSC-CM are caused by acylcarnitine accumulation rather than energy shortage.