Introduction: Hypertrophic cardiomyopathy (HCM) is a disease that affects 3 in 20 cats and 1 in 500 humans [1]. The MYBPC3 R820W mutation causes HCM in ragdoll cats and humans [2], and is characterised by cardiomyocyte (CM) remodelling, myofiber disarray, interstitial fibrosis, left ventricular hypertrophy and hypercontractility. Despite some breakthrough drugs such as Mavacamten, there are limited treatments available to reverse all aspects of HCM, such as interstitial fibrosis [3]. The exact pathological mechanisms leading to HCM caused by the MYBPC3 R820W mutation have not been studied in detail. However, they likely involve interactions between dysfunctional CMs and other cardiac cells, including cardiac fibroblasts (CFs) [4, 5]. Our lab has generated a novel induced pluripotent stem cell (iPSC) line homozygous mutant for the MYBPC3 R820W mutation (R820W+/+), and an isogenic control line.
Our overall aim for this project was to generate an engineered heart tissue (EHT) model of HCM and use it to understand the aberrant interactions between R820W+/+ iPSC-derived CMs (iPSC-CMs) and CFs. Our objectives were:
- To use a novel ImageJ and R script to measure differences in contractility parameters between EHT groups.
- To use immunohistochemistry to measure differences in iPSC-CM area (hypertrophy), iPSC-CM alignment and fibrosis.
Methods: We generated EHTs containing R820W+/+ or isogenic control iPSC-CMs, either alone or with CFs (n=5-10 per group). EHTs were cultured for 2 weeks, then electrically stimulated at 1Hz and 10 second videos were taken to measure contractility. Then EHTs were either fixed for staining or processed for RNA and protein extraction. Two-way ANOVA was used for all statistical analyses.
Results: EHTs with CFs showed a significantly shorter time to peak of contraction and contraction duration compared to EHTs with iPSC-CMs only, regardless of iPSC-CM mutation (p<0.0001 isogenic control, p=0.0093 R820W+/+). R820W+/+ EHTs with iPSC-CMs only had a significantly shorter time to peak of contraction compared to isogenic control EHTs with iPSC-CMs only (p=0.0363). Also, R820W+/+ EHTs with iPSC-CMs and CFs had a significantly shorter relaxation time, compared to the R820W+/+ EHTs with iPSC-CM only. For iPSC-CM area, results revealed a larger iPSC-CM area in R820W+/+ EHTs with CFs, compared to isogenic control EHTs with CFs. For iPSC-CM alignment, we found that adding CFs to isogenic control EHTs caused significantly more iPSC-CM alignment, compared to isogenic control EHTs with iPSC-CMs only (p<0.0001). Also, R820W+/+ EHTs with CFs showed significantly less iPSC-CM alignment, compared to the isogenic control EHTs with CFs (P=0.0186). Analysis of Masson’s trichrome staining showed no significant difference in fibrosis between groups (P>0.05).
Conclusions: our model of HCM shows a mild hypercontractile phenotype, which is ameliorated by the addition of CFs. Our results also show the expected interactions between isogenic control iPSC-CMs and CFs, indicating that the model is working. We have revealed a possible interaction between R820W+/+ iPSC-CMs and CFs which leads to hypertrophy and myocardial disarray. Future work will focus on investigating differences at the protein and gene level, and maturing the EHT model further with electrical stimulation in culture.