Background: Peripartum cardiomyopathy (PPCM) is defined as heart failure (HF) secondary to left ventricular (LV) systolic dysfunction, that occurs at the end of pregnancy, during delivery or in the first months after delivery in previously heart-healthy women. 15-20% of PPCM patients carry gene variants known to induce genetic cardiomyopathies. However, it is unclear whether these gene variants, mostly heterozygous missense or frame shift mutations, are disease causing. Since these patients were symptom-free prior pregnancy, pregnancy and/or the postpartum (patho-)physiology seem to trigger HF in mutation-carriers. The aim of this project is to generate induced pluripotent stem cells (iPSCs) from mutation carrying patients and their relatives for differentiation of cardiomyocytes (CM) to evaluate the disease potential of pathogenic gene variants.

Methods and results: Whole exome sequencing in 65 PPCM patients from the German PPCM registry identified several gene variants in cardiomyopathy-associated genes such as Titin (TTN) or Myosin Binding Protein C, cardiac (MYBPC3). Here, we identified two sisters who both developed PPCM. Both are carrying the same MYBPC3 (c.3052G>C:p.(Glu1018Gln)) gene variant inherited from their father who is apparently healthy. One daughter showed a severe form of PPCM with cardiogenic shock (baseline (BL) LV ejection fraction (LVEF): 17%) in contrast to her sister (S1) who developed a mild form of PPCM (BL LVEF: 45%). The more severely affected sister carries a de novo truncating TTN (c.44988del;p.(His14997Metfs*24)) gene variant leading most likely to a hemizygous condition for TTN, which in combination with MYBPC3 mutation may aggravate the PPCM phenotype. The mother and brother of the sisters are not carrying one of these variants and are heart-healthy. Human iPSCs from sister S1, brother, father and from healthy controls were generated, in order to gain iPSC-generated cardiomyocytes (iPSC-CMs). These iPSCs showed a normal karyotype and the ability to differentiate into the three germ layers (confirmed by pos. staining of α-Fetoprotein, Sox-17, cardiac Troponin T, α-Actinin, β3-Tubulin or Desmin after spontaneous in vitro differentiation). In S1 and father iPSCs, the MYBPC3 gene variant was verified. In immunofluorescence staining, all iPSCs are positive for the pluripotency markers Oct4, Sox-2, Nanog, TRA-1-60 and TRA-1-81. Thus far Ca²⁺ transient and contraction experiments on S1 and healthy female control iPSC-CMs were performed. The application of caffeine to S1 iPSC-CMs induced a significantly lower Ca²⁺ release from the sarcoplasmic reticulum (SR) and a shorter recovery time than in healthy female control iPSC-CMs. Further, stimulation with isoprotenerol (Iso) led in S1 iPSC-CMs to a blunted positive inotropic response, and a significantly lower increase in maximal rate of [Ca²⁺]_i rise, maximal contraction rate, maximal rate of [Ca²⁺]_i decay and maximal relaxation rate compared to healthy female control iPSC-CMs.

Conclusions: We were able to generate patient-specific PPCM iPSCs carrying a MYBPC3 gene variant. S1 iPSC-CMs exhibit lower SR Ca²⁺ release in response to caffeine and blunted β-adrenergic inotropic responsiveness compared to healthy female control. Therefore, the MYBPC3 gene variant affects the function and contractility of the iPSC-CMs leading to cardiomyocyte dysfunction, which may underlie PPCM-related HF.