Background:
Investigations of Bcl2-associated athanagene 3 (BAG3) related myopathies focus on the severe phenotypes caused by a handful of localized mutations; however translational research has been difficult and heavily reliant on the investigative muscle tissue model. In this study we demonstrated generation of purely induced pluripotent stem cell (iPSC) derived components with a novel BAG3 variant (p.Asp466Glu) for the engineered human myocardium (EHM). We hypothesized that dysfunctional mitochondria contribute to the development and progression of the disease in a 3D muscle environment.
Methods and Results:
We generated iPSC-derived cardiomyocytes and cardiac fibroblasts from three BAG3 genotypes (wild type, heterozygous, homozygous) which were later utilized to construct EHM for long term functional assessment. Differences in metabolic and transcriptome patterns of the 2D cells were investigated, indicating similar amounts of baseline ATP generation (WT: 0.8±0.06, HET: 0.5±0.05, HOM: 0.72±0.05 pmol O2/min [n=6-8]) and stress tolerance (WT: 449.6±22.27%, HET: 397.2±29.78%, HOM: 452.4±27.96% of basal oxygen consumption rate [n=6-8]) in the cardiomyocytes, while the cardiac fibroblasts showed minimal differences in glycolytic function (WT: 142.7±5.4%, HET: 142.8±5.94%, HOM: 131.4±8.0% of basal extracellular acidification [n=6-8]) and no perturbations in the transcriptome of differentiating cardiac fibroblasts. Subjecting both cell types to a 3D tissue environment gave rise to functional differences between the three genotypes which were observed via longitudinal live video-optical analysis (WT: 6.03±0.53%, HET: 4.4±0.57%, HOM: 3.1±0.24% shortening [n=11]) and isometric force analysis (WT: 1.0±0.14, HET: 0.76±0.17, HOM: 0.41±0.04 mN [n=5-6]). Analysis of sarcomere size indicated no differences (WT: 1.80±0.026, HET: 1.78±0.037, HOM: 1.78±0.023 μm [n=8]), but with an impact on sarcomere organization and mitochondrial alignment (WT: 2441±194, HET: 2476±252, HOM: 1802±166 RFU [n=8]).
Conclusion:
The EHM model is well suited for contractile and metabolic investigations of BAG3-associated cardiomyopathies and may serve as a platform for therapeutic screening.
