Successful knock-in of Hypertrophic Cardiomyopathy-mutation R723G into the MYH7 gene mimics HCM pathology in pigs

Abstract

Familial Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiac disease. About 30% of the patients are heterozygous for mutations in the MYH7 gene encoding the ß-myosin heavy chain (MyHC). Hallmarks of HCM are cardiomyocyte disarray and hypertrophy of the left ventricle, the symptoms range from slight arrhythmias to sudden cardiac death or heart failure. To gain insight into the underlying mechanisms of the diseases' etiology we aimed to generate genome edited pigs with an HCM-mutation. We used TALEN-mediated genome editing and successfully introduced the HCM-point mutation R723G into the MYH7 gene of porcine fibroblasts and subsequently cloned pigs that were heterozygous for the HCM-mutation R723G. No off-target effects were determined in the R723G-pigs. Surprisingly, the animals died within 24 h post partem, probably due to heart failure as indicated by a shift in the a/ß-MyHC ratio in the left ventricle. Most interestingly, the neonatal pigs displayed features of HCM, including mild myocyte disarray, malformed nuclei, and MYH7-overexpression. The finding of HCM-specific pathology in neonatal R723G-piglets suggests a very early onset of the disease and highlights the importance of novel large animal models for studying causative mechanisms and long-term progression of human cardiac diseases.

Figure 1

Generation of transgenic fibroblasts and pigs. (A) Schematic drawing of the genome editing approach. A donor DNA encoding for the point mutation c.2223 C > G was generated. The point mutation results in an additional BslI restriction site. A double strand break was introduced in the porcine genomic DNA by TALENs and the point mutation was integrated by homology directed repair. Screening was accomplished by PCR and subsequent BslI restriction. The wildtype (WT) DNA generates a 205 bp and the genome edited (R723G) DNA a 138 bp fragment. (B–D) Agarose gel electrophoresis of BslI restriction fragments from PCR on DNA from transfected cell cultures (B), piglets cloned from cell culture H4 (C), and piglets from a subclone of cell culture H4 and the wildtype porcine fetal fibroblasts (PFF) as control (D).

Figure 2

Gross morphology of hearts from genome edited animals. Representative hearts of genome edited (A–C) and control (D) animals. The animals were male, less than 24 h of age and of comparable birth weight. Coronal sections of the ventricles of genome edited (E–G) and control (H) animals show that right and left ventricles are comparably sized in each animal as expected for neonates. Scale bar represents 1 cm. RV: right ventricle; LV: left ventricle; Sep: septum.

Figure 3

Histopathological analysis of cardiac tissue of the transgenic animals. The myofibrils are mostly regular aligned in the transgenic animals (A) and in the wildtype control (B). However, signs of myocyte disarray were detected in the transgenic animals (C) but not in the controls (D) and also mechanically ineffective endings of sarcomeres (E,F). Intriguingly, the nuclei of the cardiomyocytes in the transgenic pigs were often malformed (G), such nuclei can also be found in human HCM-patients with the same mutation (H). The scale bar represents 2 µm unless indicated otherwise.

Figure 4

MyHC expression. (A) The relative expression of the MYH7 c.2193T-allele, that harbors the R723G mutation in the genome edited animals (p1–p7) and the wildtype sequence in the wildtype (WT) animals (p8-p9). Quantifications were performed in two independent experiments each in two replicates. (B) The relative expression of MYH7 mRNA per GAPDH was examined by realtime PCR for the genome edited animals and two wildtype controls. Data are presented as the mean and SEM of the 2^−ΔCt of the transgenic animals vs. the controls of three independent experiments in duplicates. (C) Methylation status of nine CpG sites in the promoter CpG island of the MYH7 gene in the transgenic animals p1–p7 and two wildtype controls p8-p9. The methylation was assessed by bisulfite specific PCR and analyzed semi-quantitatively from the sequence chromatogram. Filled parts of the circles indicate methylated cytosines and open parts of the circles indicate non-methylated cytosines. (D) Western Blot of the α/ß-MyHC isoforms in genome edited piglets (p1, p4, p7) and age- and sex-matched control (p8). Crude protein extracts of porcine left ventricular tissue (LV) were separated by PAGE and transferred to a nitrocellulose membrane. MyHC-isoforms were detected using an α/ß-MyHC-specific antibody. Human and porcine atrium samples (AD) were used as references for α and ß-MyHC isoforms. The α-MyHC shows a higher molecular weight as compared to the ß-MyHC. The full-length blot is presented in Fig. S7.