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.
Highlights
Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease (CHD) characterized by atresia/stenosis of the aortic and mitral valves, and severe hypoplasia of the left ventricle and aorta (Noonan and Nadas, 1958)
The CRISPR target site has one basepair mismatch, and the sequences of both MYH6 (Figure 1A) and its close homologue MYH7 (Supplementary Figure S1) were confirmed in CRISPRed induced pluripotent stem cells (iPSCs) with Sanger sequencing for any off-target effects
The efficiency of cardiomyogenic differentiation during D10–D15 was reduced in both the inserted +/VAR and inserted VAR/VAR iPSC-CMs determined by MF20 immunostaining (Figure 1C) and flow cytometry assessment of % cardiac troponin T (cTnT)-positive cells (Figures 1D,E)
Summary
Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease (CHD) characterized by atresia/stenosis of the aortic and mitral valves, and severe hypoplasia of the left ventricle and aorta (Noonan and Nadas, 1958). HLHS exhibits complex genetic inheritance along with an increased frequency of bicuspid aortic valve (BAV) and coarctation of the aorta (CoA) in relatives of HLHS patients (Hinton et al, 2007, 2009) Variants in genes such as GJA1 (Dasgupta et al, 2001), NKX2.5 (Elliott et al, 2003), NOTCH1 (Garg et al, 2005; McBride et al, 2008; Hrstka et al, 2017; Yang et al, 2017), and MYH6 (Theis et al, 2015; TomitaMitchell et al, 2016), as well as observations of syndromic or rare copy number variants (CNVs) in cardiomyogenic genes (Grossfeld, 2007; Grossfeld et al, 2009; Tomita-Mitchell et al, 2012; Warburton et al, 2014; Glidewell et al, 2015) have been associated with HLHS. Using cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) obtained from an HLHS patient carrying an MYH6R443P variant, we discovered that sarcomere structure was dysmorphic (Tomita-Mitchell et al, 2016)
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