Impact of peripartum stress on Ca2+ handling in iPSC-derived cardiomyocytes from Peripartum Cardiomyopathy patients carrying a MYBC3 gene variant

Background: Peripartum cardiomyopathy (PPCM) is defined as heart failure (HF) secondary to left ventricular (LV) systolic dysfunction, occurring 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 variants in cardiomyopathy-associated genes. However, it is unclear whether these gene variants are disease causing. Since these patients were symptom-free prior last month of pregnancy, it seems that peripartum (patho-)physiology triggers HF in mutation-carriers. Here, we generated induced pluripotent stem cell (iPSCs)-derived cardiomyocytes (iPSC-CMs) from mutation-carrying PPCM patients, isogenic controls and their relatives to evaluate the disease potential of pathogenic gene variants under peripartum stress.

Methods and results: Whole exome sequencing in 94 PPCM patients from the German PPCM registry identified several gene variants in cardiomyopathy-associated genes. Here, we identified a family with two sisters who both developed PPCM. Both are carrying the same heterozygous Myosin Binding Protein C, cardiac (MYPBC3, c.3052G>C;p.(Glu1018Gln), variant of uncertain significance, VUS) gene variant inherited from their asymptomatic father (F). One sister (S1) showed a mild form of PPCM (baseline (BL) LV ejection fraction (LVEF): 45%) in contrast to her sister (S2) who developed a severe form of PPCM with cardiogenic shock (BL LVEF): 17%). The more severely affected sister S2 carries a de novo truncating Titin (TTN) (c.44988del;p.(His14997Metfs*24), frameshift) gene variant leading most likely to a hemizygous condition for TTN. The mother (M) and brother (B) of the sisters are not carrying one of these variants and are heart-healthy. CMs were differentiated from iPSCs generated from sister S1, F, B and M. Additionally, an isogenic iPSC control of S1 (S1-Ctrl) was created via CRISPR/Cas9 correction of the MYBPC3 gene variant. In addition, the de novo TTN mutation of S2 was introduced into the S1 iPSC line. All generated iPSCs lines showed a normal karyotype, common pluripotency markers and the ability to differentiate into the three germ layers. The MYBPC3 gene variant was confirmed in S1 and F iPSCs.
Thus far, Ca²⁺ transient experiments with caffeine under basal conditions in S1 and F iPSC-CMs showed a significantly lower caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR), and a shorter recovery time than in S1-Ctrl and M iPSC-CMs. Following treatment with peripartum-associated stress conditions with estradiol for 48 h, S1 and F iPSC-CMs showed a significant decrease in the caffeine-induced Ca²⁺ release parameters compared to S1-Ctrl and M iPSC-CMs, but no difference in the recovery time. Combined treatment of cyclic stretch (1 Hz) and estradiol for 48 h resulted in an even more impaired Ca²⁺ release signal compared to the other conditions, with a shorter time to the first Ca²⁺ transient, compared to S1-Ctrl and M iPSC-CMs.

Conclusions: We were able to generate patient-specific PPCM iPSCs carrying a heterozygous MYPBC3 gene variant and the corresponding controls. S1 and F iPSC-CMs exhibit lower SR Ca²⁺ release in response to caffeine compared to S1-Ctrl and healthy M iPSC-CMs. Therefore, the MYBPC3 gene variant seems to affect the Ca²⁺ handling machinery and contractility of the iPSC-CMs, which is exacerbated under peripartum-associated stress, potentially leading to cardiomyocyte dysfunction, which may underlie PPCM-related HF.