Purpose: The ability to generate patient-specific human induced pluripotent stem cells (ps-iPSCs) provides a unique opportunity to gain new insights into the underlying mechanism of inherited heart diseases, such as dilated cardiomyopathy (DCM). Mutations in many genes have been implicated in the pathogenesis of DCM, including those occurring in the giant elastic protein titin (TTN). We aimed to apply iPSCs from a DCM patient with compound heterozygous TTN mutations to analyze the functionality and cell biology of cardiomyocytes derived from ps-iPSCs with regard to the cardiac DCM phenotype.

Methods and Results: Skin fibroblasts from the DCM patient with compound heterozygous mutations in A-band and M-band TTN were reprogrammed to ps-iPSCs using the Sendai virus reprogramming system. The generated ps-iPSCs showed pluripotent characteristics evident by expressing pluripotency markers on mRNA and protein level similar to human embryonic stem cells. In addition, ps-iPSCs were able to differentiate into derivatives of all three germ layers including cardiomyocytes (ps-iPSC-CMs). Despite its heterozygous phenotype, ps-iPSC-CMs exhibited loss of M-band TTN and an increased N2BA:N2B TTN ratio, for which the observed reduction in expression of RBM20, the TTN mRNA splicing factor, may account. A significant higher percentage of ps-iPSC-CMs showed disruption of the striated patterns of Z-disc-based α-actinin with no alteration in its protein expression, and reduction in A-band-based β-MHC, accompanied by disorganized sarcomere structures compared to control iPSC-CMs. Functional assessment studies under basal conditions revealed faster and irregular beating and higher maximal contraction and relaxation velocities, suggesting abnormal β-adrenergic signaling in ps-iPSC-CMs.

Conclusion: We demonstrate that in vitro disease modeling using ps-iPSC-CMs from a DCM patient harboring compound heterozygous TTN mutations recapitulate abnormalities that are commonly found in individuals with DCM caused by mutations in the giant sarcomere protein titin, including sarcomere disassembly, alterations of functional parameters and activation of the β-adrenergic signaling cascade, all of which may be attributed to increased TTN protein instability and its degradation as part of disease progression.