Disrupted Sarcomere Reorganization of Cardiomyopathy-Prone Human iPSC-Derived Cardiomyocytes on a Dynamic Mechanical Substrate.

Introduction: Approximately 15% of dilated cardiomyopathy (DCM) cases are associated with Bcl2-associated athanogene 3 (BAG3) gene mutations, which play a crucial role in myofilament organization and contractile behavior. Previous studies have highlighted the role of dynamic mechanical stress in myofibril alignment in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). In this study, we employed thermo-responsive shape memory polymers (SMPs) to mimic the dynamic mechanical environment of the extracellular matrix (ECM) and investigated their impact on myofibril assembly in healthy wild-type (WT) and BAG3 knockout (BAG3-/-) hiPSC-CMs. Methods: We synthesized Tert-Butyl Acrylate (TBA) and Butyl acrylate (BA)-based SMP substrate. hiPSC-CMs were cultured on 30oC on 40% strained (dynamic) and un-strain (static) SMPs for two days before proceeding with polymer recovery at 37oC. Myofibril components of BAG3 knock-out (KO) and WT CMs were evaluated by immunocytochemistry fluorescent images at 5 hours and 24 hours after triggering the shape changes of the SMP substrate. We quantified Z-lines and M-lines of hiPSC-CMs to evaluate sarcomere remodeling on static and dynamic substrates. Results: Our findings revealed that BAG3-/- hiPSC-CMs exhibited persistent Z-line disruption in sarcomeres compared with WT hiPSC-CMs, but M-line structures were less sensitive to mechanical stress at 5 hours, highlighted a temporal distinction in the assembly and regulation of Z-lines over M-lines. While no significant changes were detected at 5 hours, BAG3-/- CMs exhibited similar impairments in M-line organization as seen with Z-lines. These findings suggest that M-lines in BAG3-/- CMs display heightened sensitivity to dynamic mechanical actuation, but this phenotype emerges only after prolonged culture. Conclusions: This study highlights the interplay between genetic deficiency and mechanical stress to facilitate disease progression in BAG3-associated DCM. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-025-00880-z.