Increased cytosolic calcium buffering contributes to a cellular arrhythmogenic substrate in iPSC-cardiomyocytes from patients with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([Ca 2+ ] i ) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca 2+ ] i -alternans development. AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca 2+ ] i -alternans. Myofilaments are known to play a role as intracellular Ca 2+ buffers and here we show increased Ca 2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca 2+ . Similarly, EMD57033, a myofilament Ca 2+ sensitiser, replicated the abnormal [Ca 2+ ] i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca 2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca 2+ dynamics, normalising Ca 2+ transient profile and minimising the occurrence of Ca 2+ alternans at physiological frequencies. This finding suggests that increased Ca 2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca 2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.