Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome

Abstract

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 μm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 μm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent's iPSCs recapitulated the phenotype of the proband's iPSC-CMs, and conversely, correction of the R443P variant in the proband's iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.

Keywords: CRISPR/Cas9; HLHS; MYH6; contractility; hypoplastic left heart syndrome; iPSC-cardiomyocytes; sarcomere.

FIGURE 1

MYH6-R443P VAR-inserted iPSC-CMs recapitulate phenotypes of patient-specific iPSC-CMs. (A) Sequences at the MYH6-R443 locus in VAR-inserted lines. * is R443 locus and * is locus for silencing mutation. (B) Scheme for differentiating CMs using small molecule Gsk3 inhibitor (CHIR99021) with Activin-A and Wnt inhibitor (IWP). (C) Representative immunostaining of VAR-inserted iPSC-CMs with MF20 at D12. (D) Representative flow cytometry of cardiac troponin T (cTnT) positive cells in CRISPRed iPSC-CMs at D12. (E) Flow cytometry of cells cultured in parallel with those in (D), showing % cTnT-positive cells in VAR-inserted iPSC-CMs at D10-15. Data were compiled from 16 replicates in each cell type and 5 separate experiments. (F) Sarcomeres are dysmorphic in VAR-introduced iPSC-CMs as compared to WT_CC. α-actinin immunostaining (red) is seen in high density cultures at D50. (G) Comparative sarcomere organization in single cell iPSC-CMs isolated after sub-culturing at a low density at D69-D73. (H) % of cells with normal organized sarcomeres in both VAR-inserted iPSC-CMs, +/VAR and VAR/VAR in parallel to those in (G). WTcc, CRISPRed control wild-type; +/VAR, MYH6-R443P heterozygous inserted; VAR/VAR, MYH6-R443P homozygous inserted. Values are means ± SE. Student’s t-test (two-tailed, equal variance), ***P < 0.0005.

FIGURE 2

MYH6-R443P VAR-corrected iPSC-CMs rescues the phenotype of proband iPSC-CMs. (A) Sequences at the MYH6-R443P locus in corrected line. * is the R443 locus and * is the locus for a silencing mutation. (B) Representative immunostaining of VAR-corrected iPSC-CMs with MF20 (red) at D10. (C) Representative flow cytometry of cardiac troponin T (cTnT) positive cells in corrected iPSC-CMs at D12. (D) Flow cytometry of cells cultured in parallel with those in (C), showing % cTnT-positive cells at D10. Each dot is a technical replicate (Probandcc n = 2 and corrected WT n = 4). (E) Sarcomeres appear more “normal” in corrected WT iPSC-CMs compared to Probandcc iPSC-CMs. α-actinin immunostaining is seen in red and GATA4 in green in high density cultures at D63. Probandcc, CRISPRed control proband; corrected WT, CRISPRed and corrected MYH6-R443P variant.

FIGURE 3

Representative traces of contractile properties of single iPSC-CMs at D20. (A) Spontaneously contracting WT and VAR single iPSC-CMs. Best-fit lines are shown to demonstrate the measurement of maximal cell shortening and relaxation rates (IonOptix). (B) Representative calcium transients of WT and VAR single iPSC-CMs (C) Representative action potentials of WT and VAR single iPSC-CMs, followed by a 5 s recording from the same cell. CPM, contraction per minute; WT, heart-healthy parent wild-type; VAR, proband with MYH6-R443P.

FIGURE 4

Contractile differences between Probandcc and corrected WT iPSC-CMs at D20-D22. (A) Contraction rate and (B) Velocity of iPSC-CMs were recorded at D20-D22 using an automated contractility software with four technical replicates for each genotype (Probandcc n = 9 and corrected WT n = 10). Probandcc, CRISPRed control proband; corrected WT, CRISPRed and corrected MYH6-R443P variant. The lines in the box plots indicate median, with maximum and minimum calculated values shown as whiskers. Each dot is a technical replicate. Wilcoxon signed-rank test, *P < 0.05 and ***P < 0.001.

FIGURE 5

Upregulation of β-MHC in VAR at D15-D34. (A) Silver stained 5% SDS PAGE gel with each lane corresponding to the differentiation days and % of β-MHC expression listed beneath, as determined by densitometric analysis. (B) Protein expression in WT and VAR at D15, D20 and D27, as determined by densitometric analysis. Left: WT and VAR expression of MHC (left) and MLC2v (right), normalized to GAPDH. Values are means ± SEM of three technical replicates for each day and cell type. Right: Representative Western blot showing MHC, MLC2v, and GAPDH expression in WT and VAR at D20. L, ladder; I, type I skeletal muscle fiber; IIA, type IIA skeletal muscle fiber; WT, heart-healthy parent wild-type; VAR, proband with MYH6-R443P.

FIGURE 6

Dysmorphic sarcomeres were observed in atrial tissues from HLHS patients with MYH6 variants, but not in ventricles. Sarcomeric α-actinin is in red, MF20 in green and dapi in blue. (A–D) Atrial tissues from HLHS free patients without MYH6 variants. (E–H) Atrial tissues from HLHS patients without MYH6 variants. (I–L) Atrial tissues from HLHS patients with MYH6 variants. (M–P) Ventricular tissues from HLHS patients without MYH6 variants. (Q,R) Ventricular tissues from HLHS patients with MYH6 variants.