Electrophysiological abnormalities associated with a CACNA1D variant are rescued by AAV6-Cav1.3-C-terminus gene therapy in patient-iPSC-CMs

Abstract

Inherited arrhythmia syndromes are caused by genetic variants that alter cardiac ion channel function. We investigated a complex presentation in a pediatric patient with ventricular tachycardia and conduction abnormalities, harboring a de novo CACNA1D (c.3786G>T) variant, and two inherited variants, the SCN5A (c.2618C>G), and a DSP desmosome (c.1582C>G). The CACNA1D variant, which encodes Cav1.3 L-type calcium channel is the focus of this study, because the C-terminus fragment of Cav1.3 has recently been identified as a transcription auto-enhancer of its own gene and able to prevent arrhythmic events in a mouse model of ischemic heart failure. Leveraging this intrinsic property, we hypothesized that the Cav1.3-C-terminus could reverse the arrhythmic events associated with the CACNA1D variant. Patch-clamp and optical mapping experiments demonstrated a loss of Cav1.3 function, characterized by reduced L-type calcium current densities, and decrease of conduction velocity, leading to inducible re-entrant arrhythmias in human induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs). RNA sequencing confirmed this loss-of-function via the downregulation CACNA1D gene expression. Interestingly, Cav1.3-C-terminus treatment of hiPSC-CMs successfully normalized Cav1.3 gene expression, restored calcium currents, and conduction velocity, and prevented the susceptibility to arrhythmias. These findings highlight the electrophysiological consequences resulting from a de novo Cav1.3 variant and demonstrate an important transcriptional role of Cav1.3-C-terminus as a transcriptional regulator and as a promising therapeutic tool to restore normal electrical properties in patients with calcium channels loss of function.