Modeling restrictive cardiomyopathy caused by tnni3 mutations using patient-derived ipsc-cardiomyocytes, cardiac spheroids, and engineered heart tissues

Abstract Background Restrictive cardiomyopathy (RCM) is the rarest cardiomyopathy, representing only 2–5% of pediatric cases. It is defined by impaired diastolic relaxation with preserved systolic function and has a poor prognosis, with almost 50% mortality within two years of diagnosis. Mutations in TNNI3, encoding the adult cardiac troponin I (cTnI), are major contributors to severe inherited RCM. During development, cardiomyocytes express TNNI1, the fetal troponin I isoform, which is gradually replaced by TNNI3 as cells mature. Because RCM-causing mutations occur in TNNI3, in vitro models must correctly induce the TNNI1-TNNI3 switch to reproduce disease mechanisms. This study aimed to use patient-specific hiPSCs carrying pathogenic TNNI3 mutations and to determine how well different cardiac models, 2D monolayers, 3D spheroids, and micro-engineered heart tissues (µEHTs), recapitulate the molecular and functional features of RCM. Methods We used hiPSCs derived from two pediatric RCM patients carrying TNNI3 Arg192Cys and Arg170Gln mutations, alongside their isogenic controls and two non-carrier relatives. Cardiomyocytes differentiated from these lines were used to generate (i) 2D monolayers, (ii) 3D self-assembled cardiac spheroids, and (iii) 3D µEHTs. Expression of TNNI1 and TNNI3 was assessed by immunofluorescence across all platforms. Functional characterization included calcium-handling measurements in spheroids, and force analysis in µEHTs to identify mutation-specific contractile abnormalities. Results All hiPSC lines were efficiently differentiated into cardiomyocytes. In 2D cultures, TNNI3 expression increased overtime, while TNNI1 levels remained stable, indicating partial maturation. In cardiac spheroids, TNNI1 was enriched at the periphery, whereas TNNI3 localized predominantly in the inner regions, reflecting the strongest TNNI1-TNNI3 transition, with TNNI3 clearly dominating while only minimal TNNI1 persisted. Functionally, calcium-handling analyses in spheroids revealed a tendency toward prolonged contraction and relaxation times in patient-derived lines compared with non-carrier controls. The µEHTs exhibited both reduced force generation and prolonged contraction and relaxation times in the mutant tissues, consistent with early diastolic dysfunction characteristic of RCM. Conclusion 2D cultures showed partial maturation, with increasing TNNI3 and stable TNNI1 levels. Spheroids exhibited a more advanced pattern, with inner TNNI3 enrichment and a tendency toward prolonged contraction and relaxation times in patient-derived lines. µEHTs showed the most mature profile, with dominant TNNI3 expression and reduced force and slower kinetics, highlighting them as the most relevant model for TNNI3-related RCM.