Friedreich's ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency

Abstract

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Keywords: Friedreich’s ataxia; cardiomyopathy; induced pluripotent stem cells; modelling.

Figure 1. Generation of iPSC lines from FRDA-patients

Immunostaining of FA6 CL1 (A, D), CL2 (B, E), CL3 (C, F) for OCT4 (A-C) and TRA-1-60 (D-F); FA8 CL1 (G, J), CL2 (H, K), CL3 (I, L) for OCT4 (G-I) and TRA-1-60 (J-L); FA9 CL1 (M, P), CL2 (N, Q), CL3 (O, R) for OCT4 (M-O) and TRA-1-60 (P-R). (S) Negative isotype. Cells were counterstained with DAPI (blue). Scale bars: 50 μm.

Figure 2. FRDA-iPSCs and - cardiomyocytes retain low levels of FXN and are mainly of ventricular phenotype

(A, B) qPCR and (C) dipstick analysis showing low levels of FXN mRNA (A, B) and protein (C) in undifferentiated cells (A) and their cardiac derivatives (B, C). Significance was assessed by comparing FRDA-iPSCs to undifferentiated H9 controls (A) or FRDA-iPSC derived cardiomyocytes to H9 derived cardiomyocyte controls (B, C). One-way ANOVA followed by Bonferroni's multiple comparison test, ** p<0.01, **** p< 0.0001. (D) qPCR analysis of cardiomyocytes showing significantly higher expression of MYL2 than MYL7 across all cell lines (p<0.05, paired t-test). (A-D) Data are mean ± SEM of combined clones or 3 individual experiments, normalized to ACTB and relative to undifferentiated cells (A, B, D) or normalized to the control line cardiomyocyte (C). (E-G) Representative images of FA6- cardiomyocytes (E), FA8- cardiomyocytes (F) and FA9- cardiomyocytes (G) for MYL2/MLC2v (green), MYL7/MLC2a (red, weak or absent) and counterstained with DAPI (blue).

Figure 3. Assessment of phenotypes in FRDA iPSC-derived cardiomyocytes

(A) Beating rates (beats per minute, BPM), (B) corrected extracellular field potential durations (cFPD) and (C, D) the root of the mean of the sum of the square of the difference in the RR interval (RMSSD) (ms). Data shows RMSSD at basal (C) and 10⁻⁶M (D) isoprenaline ± nifedipine (NIF). (E) Representative Ca²⁺ transients. (F) Diastolic, (G) systolic and (H) amplitude Ca²⁺ in Control and FRDA- cardiomyocytes. (A-D) Data are mean ± SEM of combined values of 3 clones of each line (n=3 per line) and as a pool of all 3 clones of all FRDA lines (n=9 FRDA independent samples) and control cardiomyocytes (n=6). (F-H) Data are mean ± SEM of combined values of control cardiomyocytes (n=5), all 3 clones of each FRDA line (n=8-14 per line) and as a pool of all 3 clones of all FRDA lines (n=35 FRDA independent samples). Statistics: (A-D, F-H) One-way ANOVA followed by Bonferroni's multiple comparison test, * p<0.05, **, p<0.01, ***, P<0.001.