[II-AEPCYIA-3] Impaired relaxation in induced pluripotent stem cell-derived cardiomyocytes with pathogenic TNNI3 mutation of pediatric restrictive cardiomyopathy
Keywords:Restrictive cardiomyopathy, iPSC, isogenic
BACKGROUND AND AIM:Restrictive cardiomyopathy (RCM) is characterized by impaired diastolic function with preserved ventricular contraction. Several pathogenic variants in sarcomere genes, including TNNI3 are reported to cause Ca2+ hypersensitivity in cardiomyocytes (CMs) in overexpression models; however, the pathophysiology of induced pluripotent stem cell (iPSC)-derived CMs specific to a patient with RCM remains unknown.
METHOD:We established an iPSC line from a pediatric patient with RCM and a heterozygous TNNI3 missense variant R170W. We conducted genome editing via CRISPR/Cas9 technology to establish an isogenic correction line harboring wild-type TNNI3 as well as a homozygous TNNI3-R170W. iPSCs were then differentiated to CMs to compare their cellular physiological, structural, and transcriptomic features.
RESULTS:CMs differentiated from heterozygous and homozygous TNNI3-R170W iPSC lines demonstrated impaired diastolic function in cell motion analyses as compared with that in CMs derived from isogenic-corrected iPSCs and three independent healthy iPSC lines. The intracellular Ca2+ oscillation and immunocytochemistry of troponin I were not significantly affected in RCM CMs with either heterozygous or homozygous TNNI3-R170W. Electron microscopy showed that the myofibril and mitochondrial structures appeared to be unaffected. RNA sequencing revealed that pathways associated with muscle development and structures were altered in RCM-iPSC-derived CMs.
CONCLUSIONS:Patient-specific iPSC-derived CMs could effectively represent the diastolic dysfunction of RCM. Myofibril structures including troponin I remained unaffected in the monolayer culture system, although gene expression profiles associated with muscle development were altered.
METHOD:We established an iPSC line from a pediatric patient with RCM and a heterozygous TNNI3 missense variant R170W. We conducted genome editing via CRISPR/Cas9 technology to establish an isogenic correction line harboring wild-type TNNI3 as well as a homozygous TNNI3-R170W. iPSCs were then differentiated to CMs to compare their cellular physiological, structural, and transcriptomic features.
RESULTS:CMs differentiated from heterozygous and homozygous TNNI3-R170W iPSC lines demonstrated impaired diastolic function in cell motion analyses as compared with that in CMs derived from isogenic-corrected iPSCs and three independent healthy iPSC lines. The intracellular Ca2+ oscillation and immunocytochemistry of troponin I were not significantly affected in RCM CMs with either heterozygous or homozygous TNNI3-R170W. Electron microscopy showed that the myofibril and mitochondrial structures appeared to be unaffected. RNA sequencing revealed that pathways associated with muscle development and structures were altered in RCM-iPSC-derived CMs.
CONCLUSIONS:Patient-specific iPSC-derived CMs could effectively represent the diastolic dysfunction of RCM. Myofibril structures including troponin I remained unaffected in the monolayer culture system, although gene expression profiles associated with muscle development were altered.