Stage-associated differences in injury and stress responses of hiPSC-derived cardiomyocytes under macrophage-derived inflammatory stimulation

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Background: Macrophage-driven inflammation contributes to myocardial injury in pediatric myocarditis, but the extent to which cardiomyocyte maturation status influences responses to inflammatory stimulation remains unclear. This study aimed to investigate stage-associated differences in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to THP-1 macrophage-derived conditioned media and to explore the involvement of stress-response pathways. Methods: A macrophage-derived inflammatory microenvironment was modeled using THP-1-derived macrophage conditioned media (M0-CM and M1-CM). hiPSC-CMs at day 7 (D7) and day 21 (D21) were used as early-stage and relatively more mature in vitro stages, respectively. Cell viability was assessed using Cell Counting Kit-8 (CCK-8), and lactate dehydrogenase (LDH) release and cardiac troponin I (cTnI) levels were measured to evaluate injury. C/EBP homologous protein (CHOP) expression was analyzed by Western blot, and mitochondrial membrane potential (ΔΨm) was determined using tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining following small interfering RNA (siRNA)-mediated CHOP knockdown. Results: M1-CM significantly increased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels compared with M0 and control groups (P<0.001), along with upregulation of inducible nitric oxide synthase (iNOS) (P<0.001). Under inflammatory stimulation, D7 hiPSC-CMs showed greater decreases in viability and greater LDH and cTnI release than D21 hiPSC-CMs in the stage-stratified analyses. CHOP expression was induced by M1-CM (P<0.001), and CHOP knockdown decreased LDH release (P<0.001) and partially restored ΔΨm (P<0.05). Conclusions: model. CHOP-associated endoplasmic reticulum (ER) stress contributed partly to mitochondrial dysfunction, but this system does not fully recapitulate pediatric myocarditis and requires validation in more physiologically complex models.

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