Modelling the impact of the patient-specific TRPC3 mutation using induced pluripotent stem cell-derived cardiomyocytes

Canonical transient receptor potential channel member 3 (TRPC3) is a non-selective cation channel, which is predominantly permeable for Ca²⁺ ions. TRPC3 has been shown to play a pivotal role for the pathogenesis of arrythmia, cardiac hypertrophy, cardiac fibrosis, and cerebellar disorders respectively. Mutations of TRPC3 appear very rare and only a few studies reported on patients with TRPC3 mutations. Recently, a previously not described de-novo TRPC3-mutation (c.1874G>A) was identified in a juvenile patient with spinocerebellar ataxia.
Based on the importance of TRPC3 for cardiac function and to investigate potential complications caused by the TRPC3-mutation, we aimed to characterise the effect of the nanog, oct3-4, ssea4, tra-1-60, lin 28, sox 2 TRPC3-mutation on the development and function of human induced pluripotent stem -derived cardiomyocytes (iPSC-CM). As a control, we used iPSCs reprogrammed from the mother of the patient, who lacks the TRPC3-mutation and shows to clinical phenotype.
Patient-derived iPSCs were generated from skin fibroblasts by using Sendai virus. Sequencing analysis confirmed the presence of the mutation in iPSCs. Immunostaining of pluripotency markers NANOG, OCTt3-4, SSEA4, TRA-1-60, LIN 28, SOX 2, and detection of germlayer markers AFP, beta-3-tubulin and alpha-SMA. From spontaneous differentiation of iPSC spheroids demonstrated revealed the pluripotency of the iPSCs.
Next, iPSC-CMs were differentiated and maturated them for different time periods. Analysis of iPSC-CMs by detection of cardiac troponin T (cTNT) using flow cytometry revealed a proportion of >90% cTNT-positive cells differentiated from patient-derived iPSCs, demonstrating that the TRPC3-mutation does not affect cardiac differentiation. Strikingly, we observed progressing cell death of patient-derived iPSC-CMs in comparison to the control iPSC-CMs during cultivation up to 70 days. In line with these findings, LDH-activity in the medium supernatant was increased in patient-derived iPSC-CMs compared to the control. Furthermore, patient-derived iPSC-CMs showed differences in their contratile function, such as decreased spontaneous beating frequency as well as shortened beating duration, contraction time and relaxation time. Morphological analyses of the sarcomere networks in iPSC-CMs stained for α-actinin revealed no significant differences in cell size, sarcomere length or sarcomere organisation. In further experiments, we aim to characterize how the TRPC3 -mutation affects the electrophysiological function of iPSC-CMs using automated patch clamp  through detection of sodium and calcium currents. To further investigate whether the mutation in TRPC3 is the cause for the observed functional changes in, we aim to correct the mutation in patient-derived iPSCs-  using CRISPR/Cas9.
Taken together, our data provide first evidence that the patient-mutation TRPC3(c.1874G>A) impairs iPSC-CM viability and function.