Graduate school of medicine, Kyoto University, Department of pediatrics, United States
Abstract: Fukuyama congenital muscular dystrophy (FCMD) is a progressive neuromuscular disease caused by mutations in the FKTN gene. The phenotypes of FCMD include developmental retardation, muscle weakness and dilated cardiomyopathy. Particularly, the prognosis is determined by the severity of respiratory failure and cardiomyopathy. Fukutin protein coded by the FKTN gene elongates sugar chains linked with α-dystroglycan, a membrane protein that connects to the extracellular matrix. However, it is unclear how sugar chains on membrane proteins are involved in the onset of cardiomyopathy. This study aims to clarify the mechanism by human iPS cell (hiPSC)-derived cardiomyocytes. HiPSC lines were established from two FCMD patients and two healthy controls, F-hiPSC and C-hiPSC, respectively. Furthermore, two control hiPSC lines were generated from F-hiPSCs by the CRISPR-Cas9 system. First, the elongation of sugar chains linked to α-dystroglycan was evaluated by comparing the fluorescence intensity of sugar chains antibodies in differentiated cardiomyocytes. Furthermore, the size and beat rate were measured at the single-cell level, and the field potential duration (FPD), which is the same meaning as clinical QT duration, was calculated by a multi-electrode array system. For analysis, a two-sample t-test with unequal variance was used, and a p-value of less than 0.05 was used as the significant value. The fluorescence intensity of sugar chains in F-hiPSC-derived cardiomyocytes (F-CM) was reduced compared to that in C-hiPSC-derived cardiomyocytes (C-CM) and gene-corrected F-hiPSC-derived cardiomyocytes (C-F-CM) (C-CM vs. F-CM: p=0.0032, F-CM vs. C-F-CM: p=0.0013). Although the cell size varied, there was no significant difference between C-CM and F-CM, and C-F-CM was significantly larger than F-CM (p=0.030). The beat rate was 62.4±22.4 bpm for C-CM, 50.1±23.3 bpm for F-CM, and 60.7±30.7 bpm for C-F-CM, with no significant difference. The FPDs corrected for beat rate by Fridericia formula was 241.7±29.3 msec for C-CM and 236.3±41.6 msec for C-F-CM, while it was 283.0±46.3 msec for F-CM, which was longer, but no significant difference was detected (C-CM vs. F-CM: p=0.13, F-CM vs. C-F-CM: p=0.11). These results indicate that the F-CM might have cardiac vulnerability.
