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]) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca]-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]-alternans. Myofilaments are known to play a role as intracellular Ca buffers and here we show increased Ca affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca. Similarly, EMD57033, a myofilament Ca sensitiser, replicated the abnormal [Ca] dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca dynamics, normalising Ca transient profile and minimising the occurrence of Ca alternans at physiological frequencies. This finding suggests that increased Ca 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 sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.