Background: Peripartum cardiomyopathy (PPCM) is a heart failure caused by left ventricular (LV) systolic dysfunction in late pregnancy, during delivery or early postpartum in previously heart-healthy women. Although 15-20% of PPCM patients carry variants in cardiomyopathy-related genes, their contribution to disease development remains unclear. Whole exome sequencing of 94 PPCM patients identified two sisters (S1, S2) with a heterozygous Myosin-binding protein C3 variant (MYBPC3, c.3052G>C;p.(Glu1018Gln), ACMG class 3) inherited from their asymptomatic father (F). S1 showed a mild PPCM (LV ejection fraction (LVEF) 45%), whereas S2 developed a severe PPCM (LVEF 17%) and additionally carries a de novo heterozygous Titin variant (TTN, c.44988del;p.(His14997Metfs*24), ACMG class 4). Mother (M) and brother (B) of the sisters carry neither variant and are heart-healthy. Since these patients were asymptomatic before pregnancy, peripartum (patho-)physiology in mutation carriers likely triggers heart failure.
Methods and results: Induced pluripotent stem cells (iPSCs) were generated from S1, F, B and M. An isogenic S1 control (S1-Ctrl) was created via CRISPR/Cas9 correction of the MYBPC3 variant. All iPSC lines showed normal karyotypes, common pluripotency markers and the ability of trilineage differentiation.
Ca2+ transient experiments under basal conditions in S1 and F iPSC-cardiomyocytes (CMs) revealed reduced caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR), and a shorter recovery time than in S1-Ctrl and M. Under 48 h estradiol (E2) treatment as peripartum-associated stressor, S1 and F iPSC-CMs also showed a reduction in the caffeine-induced Ca2+ release parameters compared to S1-Ctrl and M, which was further decreased under combined treatment of cyclic stretch (1 Hz) and E2 for 48 h. Combined stress also resulted in a shorter time to the first Ca2+ transient.
RNA sequencing of iPSC-CMs under E2 treatment and with or without stretch showed an altered expression of contractility-related genes in S1 versus S1-Ctrl iPSC-CMs. Under combined stretch and E2, the expression of ATP2A2 was 2.2-fold and RYR2 3.1-fold higher in S1 than in S1-Ctrl cells, suggesting an insufficient compensation for the MYBPC3 variant. Further, under combined stretch and E2, S1 cells had a 2.4-fold higher expression of ATP2A2 than stretch, which was not seen in S1-Ctrl cells, indicating a stress-dependent transcriptional dysregulation in S1 cells.
Conclusions: We generated patient-specific PPCM-iPSCs carrying a heterozygous MYBPC3 gene variant and corresponding controls. S1 and F iPSC-CMs show reduced SR Ca2+ release in response to caffeine compared with control iPSC-CMs, suggesting impaired Ca2+ handling and contractility of the iPSC-CMs by the MYBPC3 variant, exacerbating under peripartum-associated stress, potentially leading to cardiomyocyte dysfunction, which may contribute to PPCM. Consistent with the functional defects, RNA sequencing demonstrated stress-dependent dysregulation of contractility genes in S1 iPSC-CMs, indicating an insufficient transcriptional compensation for the MYBPC3 variant.
