Modeling Reduced Contractility and Stiffness Using iPSC-Derived Cardiomyocytes Generated From Female Becker Muscular Dystrophy Carrier

Visual Abstract

The life expectancy for patients with DMD is reported to be 22 to 28 years, 4 whereas patients with BMD may live until the sixth decade of life. 5 Cardiac involvement in male patients with BMD is common, 6 with a frequency of 60% to 75% and an average age of cardiac involvement onset of 28.7 years. 5 On the contrary, the prevalence of dilated cardiomyopathy in female BMD/DMD carriers is lower compared with that in male patients and ranges from 7% to 18%. 7 Meanwhile, a cross-sectional study of 130 female BMD/DMD carriers found that the mean left ventricular (LV) ejection fraction was 63%, and only 4 carriers developed contractile dysfunction with a reduced ejection fraction of <40% at ages 30 to 60 years. 10 Furthermore, a study of a cohort of 397 female BMD/DMD carriers revealed that the clinical feature of cardiomyopathy is not associated with a reduced life expectancy or an increased risk of cardiac death. 11 These clinical studies suggest that early onset DCM with advanced HF in female BMD carriers is extremely rare.
During the past decade, numerous studies using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) for modeling BMD/DMD pathogenesis have been reported. 12 More recently, iPSCs have been generated from male patients with BMD 13,14 ; however, there have been no reports of experimental studies using iPSC-CMs generated from female BMD carriers. Functional analysis of female carriers of BMD using iPSC-CMs is challenging because iPSCs generated from girls and women carry 2 X chromosomes, 1 of which undergoes X chromosome inactivation (XCI). 15 Furthermore, large exon deletions in DMD cannot be corrected by conventional genome editing, thus making it difficult to generate isogenic iPSCs.
In the current study, we encountered a young female BMD carrier with an in-frame deletion in DMD of exon 45-48 (D45-48) who developed early onset DCM with severe advanced HF that required implantation of an LV assist device (LVAD). 16 We determined the proportion of the XCI state in the proband's LV myocardium and identified a potential second-hit variant in the proband that was not present in her nonmanifesting sister. To determine the pathologic effect of the genetic variants, we also performed functional analysis using self-organized tissue rings (SOTRs) generated from isogenic iPSC-CMs prepared using the XCI state and genome editing. Our analysis using isogenic iPSC-CMs revealed the pathogenesis underlying advanced HF in a female BMD carrier. Plasmid constructs for genome editing were transfected into iPSCs as described previously. 17,18 GENERATION OF SELF-ORGANIZED TISSUE RINGS.

METHODS
The 3-dimensional SOTRs were generated as described previously. 19  XCI is responsible for sex chromosome dosage compensation in girls and women. 15 Analysis of genomic DNA extracted from peripheral blood demonstrated that a skewed XCI is associated with cardiac dysfunction in DMD carriers 21 and BMD 22 carriers. However, the proportion of XCI in heart tissue of BMD carriers has not been determined yet.

RESULTS
Polymerase chain reaction (PCR) and Sanger sequence analysis using complementary DNA (cDNA) prepared using RNA isolated from the LV heart tissue of the proband confirmed the D45-48 variant in DMD ( Figures 1C and 1D). Specific probes were designed to evaluate the differential expression of WT and D45-48 transcripts from DMD ( Figure 1E). Droplet digital PCR (ddPCR) analysis using cDNA obtained from the proband's LV tissues (n ¼ 6) revealed that the average ratios of WT and D45-48 transcripts were was not observed in her older sister (III-2), whose cardiac function was within normal limits (Supplemental Figure 1B). To compare the XCI states between the sisters, we performed an androgen receptor (AR) methylation-based assay using peripheral blood, 21,22 given that a myocardium sample of the III-2 carrier was unavailable. Analysis of the XCI state in girls and women by using blood samples is considered comparable with that of other tissues that may be inaccessible. 26 XCI detection using methylation-based assays to distinguish XCI states is dependent on the CAG repeat polymorphism in each X chromosome; therefore, 2 different peaks must be detected during the electrophoretic analysis of PCR products spanning the CAG repeats of the AR gene. As expected, a single peak was detected in the proband's older brother (III-1) who was carrying only 1 X chromosome ( Figure 2A). Only 1 peak was also detected in the proband (III-4), a finding suggesting that the number of CAG repeats in the 2 X chromosomes was comparable. However, 2 peaks were detected in the III-2 carrier, with the lower-molecular-weight peak estimated to be derived from the WT-DMD allele, because III-2 and III-4 (sisters) inherited a common X chromosome carrying the D45-48-DMD allele. Digestion with endonuclease HpaII and quantitative analysis revealed that the proportion of WT-DMD expression to D45-48-DMD expression was approximately 7:3 ( Figures 2B and 2C). This suggested that substantial D45-48-DMD expression in heart tissue would also be predicted in the nonmanifesting carrier III-2. These findings prompted us to explore another contributing factor for advanced HF in the proband.
We screened the expression of a total of 404 different genes related to inherited cardiovascular disease in the proband (III-4) and her sister (III-2), 27 and we identified a heterozygous stop-gain variant (c.T1890G p.Y630X) in procollagen-lysine, 2oxoglutarate 5-dioxygenase 3 (PLOD3), which encodes LH3 and was detected only in the proband ( Figures 2D and 2E). LH3 is ubiquitously expressed in various organs, including the heart, and it catalyzes the hydroxylation of lysyl residues of collagens and plays an important role in collagen cross-linking and deposition. [28][29][30] Compound heterozygous variants or homozygous variants in PLOD3 of humans results in a connective tissue disorder. 29,31,32 Both the mRNA expression levels of PLOD3 and the protein expression levels of LH3 were decreased in the proband's LV tissue compared with that of the control DCM patient (Figures 2F and 2G).

GENERATION OF ISOGENIC iPSCs USING THE XCI
STATE AND GENOME EDITING. To determine the functional consequence of variants in DMD and PLOD3, we sought to establish isogenic iPSC-CMs in The polymerase chain reaction product generated from amplification of genomic DNA for each case was analyzed using a microchip electrophoresis system (MultiNA, Shimadzu). The polymerase chain reaction products from the proband (III-4) and her older brother (III-1) showed single peaks (bold arrows, 310 bp), whereas the proband's older sister (III-2) showed bimodal peaks (narrow arrows, 299 bp and 310 bp). The higher-molecular-weight signals (arrowheads) are believed to be oligomers of polymerase chain reaction products. (B) Representative result of the X chromosome inactivation assay before (control, upper) and after (HpaII digestion, lower) enzyme digestion. The lower-molecular-weight band derived from the wild-type (WT) allele was more prone to digestion than that of the higher-molecular-weight band, which was derived from the deletion of exons 45 to 48 (D45-48) allele.  Figure 2C).
On the basis of these results, we performed additional experiments using iPSCs that were cultured for fewer than 20 passages to avoid any effect from Xi erosion.
We also sought to repair the heterozygous Y630X    subjects. 15 The nonrandom inactivation of the second X chromosome in somatic cells of girls and women is called skewed XCI and is considered a potential mechanism underlying the clinical symptoms observed in BMD/DMD carriers. 21 44 The ratio of type I to type III collagen increases in the hearts of patients with DCM, and this increased ratio contributes to reduce elasticity of the LV wall and LV dilation. 52,53 Type I collagen retains substantial tensile strength, whereas type III collagen has the elasticity to maintain the structural integrity of the collagen network. 54 In particular, increased type III collagen expression helps maintain cardiac function in injured myocardium. 53 Although several environmental factors may potentially affect disease progression in BMD, including mechanical stress caused by strenuous muscle exercise, 55