Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1

Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3–9 (Δ3–9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3–9, frameshifting Δ3–7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3–9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3–9 and wild-type hiPSC-CMs; Δ3–7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3–9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8–9 to restore functional dystrophin and electrophysiological parameters in Δ3–7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3–9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3–9 may become a promising therapy for DMD cardiomyopathy.


INTRODUCTION
Duchenne muscular dystrophy (DMD) is a type of X-linked lethal muscular dystrophy that affects 1 in 3,600-6,000 live male births. 1,2his disorder is caused by loss-of-function mutations in the DMD gene, which encodes a 427-kDa cytoskeletal protein, dystrophin (Dp427m).DMD has 79 exons, consisting of 4 major domains: an N-terminal F-actin binding domain (ABD1) (encoded by exons 1-8), a central rod domain containing a second actin-binding domain (ABD2) (encoded by exons 8-64), a cysteine-rich domain (encoded by exons 64-70), and a C-terminal domain (encoded by exons 71-79). 3,4Dystrophin is a major component of the dystrophin-associated protein complex (DAPC), which links the actin cytoskeleton to the extracellular matrix (ECM) 5,6 and plays an essential role in stabilizing the sarcolemma, calcium homeostasis, and maintaining muscle cell integrity in skeletal and cardiac muscles.Dystrophin is also reported to play an important role in the regulation of cell division and propagation in satellite cells. 7Patients with DMD exhibit muscle degeneration and atrophy primarily in skeletal muscle, leading to loss of ambulation at around 10-12 years of age, and gradually develop dilated cardiomyopathy (DCM) and progressive respiratory failure in later stages, usually in the second decade. 4With improved management for cardiopulmonary dysfunction, especially due to advancements in respiratory support technology, patients with DMD can survive into their forties, although heart failure (HF) remains the leading cause of morbidity and mortality. 4,8,9[16][17][18][19][20][21] Exon skipping therapy to restore the reading frame and produce functional truncated dystrophin using antisense oligonucleotides (AOs), chemically synthesized nucleic acid analogs that specifically bind to a target exon during pre-mRNA, is promising and has already been approved for patients with DMD who have mutations in the most common hotspot in the rod domain in exons 45-55 of DMD. 13,22n exon skipping therapy, the phenotype of the in-frame deletion in DMD must be as mild as possible, both in skeletal and cardiac muscles.Therefore, elucidation of the pathogenesis and molecular mechanism underlying the difference in the phenotypes of BMD and DMD is important.One potential candidate region for exon skipping therapy is the minor deletion hotspot, accounting for approximately 7% of patients with DMD, which encompasses exons 3-9 of the DMD gene encoding actin-binding domain 1 (ABD1) in the N-terminus of Dp427m. 22,23This region contains three actin-binding sites (ABSs): ABS1 (encoded by exon 2), ABS2 (encoded by exon 5), and ABS3 (encoded by exon 6).5][26][27][28][29][30] In contrast, patients with the DMD exons 3-9 deletion (D3-9), in which ABS1 is spared but ABS2 and ABS3 are absent, were asymptomatic or had a very mild BMD phenotype. 23,31As such, D3-9 has drawn attention as an ideal in-frame deletion goal for exon skipping or gene editing therapy. 15,23,32AOs targeting exon 8 efficiently induce exon 8 and 9 skipping in myoblasts from patients with DMD with D3-7, possibly because of the frequently occurring endogenous skipping of exons 8 and 9. 32  Kyrychenko et al. (2017)  demonstrated that the aberrant finding in calcium transient and contraction force was the least in D3-9 hiPSC-CMs among isogenic hiPSC-CMs with other in-frame deletions, D6-9 and D7-11, and frameshifting D8-9, and among another isogenic pair of D3-7 hiPSC-CMs and D3-9 hiPSC-CMs.These findings suggest that the D3-9 through genomic editing is an applicable treatment strategy targeting ABD1.
The primary objective of this study was to assess the efficacy of AOmediated exon 3-9 skipping therapy for ABD1 mutation-induced DMD cardiomyopathy, utilizing a newly developed phonotypic analysis platform for hiPSC-CMs, all the while considering potential future clinical applications.Initially, we were focused on dissecting the pathophysiology of hiPSC-CMs expressing frameshifting D3-7 and in-frame D3-9, aiming to precisely characterize each phenotype.
Notably, this study is the first attempt to conduct a comparative analysis of an isogenic hiPSC-CM set, encompassing a DMD model, a mild BMD model with extended adjacent exonic deletions as a reading frame for exon skipping, and the wild-type (WT).Our findings offer novel insights into the pathology of dystrophin-associated DCM and corroborate the similarities between D3-9 hiPSC-CMs and WT hiPSC-CMs, contrasting with those of D3-7 hiPSC-CMs.
Subsequently, we investigated the potential for converting the DCM pathology observed in D3-7 hiPSC-CMs closer to that of D3-9 hiPSC-CMs through the partial restoration of D3-9 dystrophin via AO administration.

RESULTS
Dp427m was absent and Dp71 was increased in D3-7 hiPSC-CMs, whereas internally truncated dystrophin was more abundant in D3-9 hiPSC-CMs than Dp427m in WT hiPSC-CMs To track the action potential and calcium transient in hiPSC-CMs simultaneously, we combined the green fluorescent voltage indicator gene (ASAP2s) 33 with the red fluorescent intracellular calcium ion indicator gene (R-CaMP1.07) 34in a plasmid and transduced it into intron 1 of the adeno-associated virus integration site 1 gene (AAVS1), a safe harbor in the human genome of healthy male hiPSCs (WT hiPSCs), using the CRISPR-Cas9 gene editing method (Figure 1A).Despite the slower kinetics than those of chemical dye, both indicators worked successfully on differentiated CMs (Figures 1B, 1C, and S1A-S1E; Videos S1A and S1B).Subsequently, we generated two hiPSC lines, the DMD D3-7 hiPSC line as a model for DMD and the DMD D3-9 hiPSC line as a model for the mild form of BMD, 23,31 from the WT hiPSCs with dual indicators through electroporation of Cas9 and two single guide RNAs (sgRNAs) targeting sequences in introns 2 and 7 for D3-7 and introns 2 and 9 for D3-9 (Figure 1D; Table S2). 15The sequences, including both the ends of deleted lesions on undifferentiated cell-extracted genomic DNA and mRNA extracted from differentiated CMs of each cell line, were validated (Figures S3A-S3D; Table S1).When compared with WT hiPSC, these cell lines underwent one additional genome editing procedure, which could potentially introduce experimental variability.However, we confirmed the normal sequences of the top candidate exonic regions for off-target effects for the gRNA (Tables S1 and S3).Based on this, we conducted the experiments considering WT, D3-7, and D3-9 hiPSCs as genetically isogenic cell lines.7][38][39] Although previous studies failed to detect a-DG protein in hiPSC-CMs, 20,40 hiPSC-CMs differentiated using this method showed a sufficiently mature phenotype, with abundant expression of dystrophin, a-dystroglycan (a-DG), and b-dystroglycan (b-DG) on day 48 in WT hiPSC-CMs (Figures S2B-S2F).Furthermore, a major sarcomeric maturation marker of hiPSC-CMs, including the switching in mRNA expression of TNNI1 coding slow skeletal troponin I (ssTnI) to TNNI3 coding cardiac TnI (cTnI), MYH6 coding myosin heavy chain 6 to MYH7 coding myosin heavy chain 7, and MYL7 coding myosin light chain 2a (MLC2a) to MYL2 coding MLC2v, was also promoted following T3/Dex treatment (Figures S2G-S2J) as well as electrophysiological maturation. 37,41 verified the loss of Dp427m protein in D3-7 hiPSC-CMs on day 48 after differentiation through immunostaining using an antibody against dystrophin exons 31-32 [mandys8] and western blotting using an antibody against the C-terminus of dystrophin (Figures 1E-1G).Notably, the protein level of $392-kDa internally truncated dystrophin was significantly higher in D3-9 hiPSC-CMs than the level of Dp427m in WT hiPSC-CMs (Figures 1F and 1G).The same results were confirmed when compared with other healthy male-derived hiPSC-CMs (WT#) (Figures S5B and S5C).Almost the same molecular weight of dystrophin was faintly detected in D3-7 hiPSC-CMs (Figure S5A), implying endogenous exon skipping associated with a milder phenotype. 32,42Furthermore, two shorter isoforms of dystrophin with internal promoters, Dp71 and Dp116, were detected in all samples.The protein level of Dp71 in D3-7 hiPSC-CMs was more than double that in D3-9 and WT hiPSC-CMs, while the level of Dp116 was low in all samples.
D3-7 hiPSC-CMs are small, with some aspects of immaturity, representing low adhesion and viability Next, we analyzed the cell size of hiPSC-CMs through forward scatter in flow cytometry using an anti-cardiac troponin T (cTnT) antibody.D3-7 hiPSC-CMs were revealed to be significantly smaller than the WT and D3-9 hiPSC-CMs, consistent with previous studies (Figures 1H, 1I, and S4A). 43,44Regarding maturation, sarcomere length showed no significant difference among the three groups (Figure 1J); however, cTnI protein expression was the lowest in D3-7 hiPSC-CMs and the highest in WT hiPSC-CMs (Figures 2A and  S6A), which indicated some aspects of maturation defect in the hiPSC-CMs with DMD mutations.Furthermore, we found that the number of live cells after replating the cryopreserved hiPSC-CMs on the Matrigel (MG)-coated dish significantly decreased in D3-7 hiPSC-CMs compared with that in D3-9 and WT hiPSC-CMs (Figures S6B and S6C), indicating impaired cell adhesion and viability in D3-7 hiPSC-CMs, which may also reflect proliferative deficiencies.

Desmin intermediate filaments are significantly decreased in D3-7 hiPSC-CMs
To evaluate the expression of cytoskeletal actin, DAPC, and major sarcomeric proteins, we performed a western blotting analysis (Figures 2A and 2B).The protein levels of a-actin were comparable among the three groups.In addition, a-DG and b-DG, which play a structural role in tethering dystrophin to the ECM, were decreased in D3-7 hiPSC-CMs (Figures 2A and 2B), and the molecular weight of a-DG was lower in D3-7 hiPSC-CMs (Figure 2A), which indicates a deficit in glycosylation and is consistent with findings in patients with DMD. 45,46Notably, desmin was increased 2-fold in D3-9 hiPSC-CMs, but decreased 17-fold in D3-7 hiPSC-CMs compared with that in WT hiPSC-CMs.cTnT levels were significantly lower in D3-7 hiPSC-CMs than D3-9 and WT hiPSC-CMs.cTnI levels were lower in both D3-9 and D3-7 hiPSC-CMs than in WT hiPSC-CMs.As for dystrophin-associated cardioprotective proteins, including AHNAK1, Cypher, and crystallin alpha B (CRYAB), which possibly play roles in maintaining normal cardiac contraction and ion channel functions by interacting with Dp427m in CMs, 20,[47][48][49][50] there were no significant differences in protein expression among the three groups (Figures 2C and 2D).
D3-9 dystrophin represents comparable binding ability to desmin, cTnT, and AHNAK, with about one-half the ability to bind a-actin compared with healthy Dp427m As actin binding is the principal function of dystrophin in maintaining the stability of cell constriction and the homeostatic balance mediated by several signaling pathways, we investigated the binding ability of internally truncated dystrophin to a-actin in D3-9 hiPSC-CMs through co-immunoprecipitation (coIP) using an anti-dystrophin antibody.The signal intensity ratio of a-actin to dystrophin on dystrophin IP samples in western blotting analysis reflects the binding ability of dystrophin to a-actin, revealing that D3-9 dystrophin with an incomplete ABD1 (lacking ABS2 and ABS3) could bind to a-actin at the level of 48% of WT Dp427m in hiPSC-CMs.Furthermore, we examined the interaction of D3-9 truncated dystrophin with desmin and cTnT and found that D3-9 dystrophin could comparably bind them to WT Dp427m (Figures 2E and 2F), implying stabilization by dystrophin binding.Regarding dystrophin-associated cardioprotective proteins, the signal ratio of AHNAK to dystrophin in the dystrophin IP sample in D3-9 hiPSC-CMs was comparable with that in WT hiPSC-CMs.However, we could not detect signals against Cypher and CRYAB in the dystrophin IP samples in WT and D3-9 hiPSC-CMs, which might be due to their much weaker binding than AHNAK, as previously demonstrated in mouse hearts. 50ntracellular calcium overload is a promising hypothesis for the pathomechanism of dystrophic features in DMD skeletal and cardiac muscles, and excessive intracellular calcium concentrations have been demonstrated in hiPSC-CMs derived from patients with DMD through live cell imaging using Indo-1. 16We examined whether Ca 2+ /calmodulin-dependent protein kinase (CaMKII) is activated in the current hiPSC-CM model for DMD and BMD using Western blot analysis.We found that T287 phosphorylated and M281/M282 oxidized CaMKII were significantly increased, accompanied by increased protein levels of T17 phosphorylated phospholamban (PLB), which is downstream of activated CaMKII in D3-7 hiPSC-CMs, compared with D3-9 and WT hiPSC-CMs (Figures 3A and 3B).The results indicated that the elevation of intracellular calcium might lead to the activation of other Ca 2+ -dependent proteases, including protein kinase A, and mitochondrial dysfunction, causing apoptosis or necrosis in D3-7 hiPSC-CMs.
Alterations in action potential and calcium transients were detected in D3-7 hiPSC-CMs but not in D3-9 hiPSC-CMs We performed electrophysiological analyses on day-50 hiPSC-CMs.
2][53] First, we performed single-cell analyses to assess the spontaneous beating rhythm.Spontaneous calcium transient recordings revealed a higher beat rate in D3-7 hiPSC-CMs than in the others (Figures 4A-4C), which might correspond with the susceptibility to ventricular tachycardia and sinus tachycardia in patients with DMD. 54,55No significant arrhythmia, including early after depolarization or delayed after depolarization, was detected in any cell line, which is inconsistent with previous studies, 15,17 while the variability of the inter-beat interval indicating accelerated beat rate variability 56 was augmented in D3-7 and D3-9 hiPSC-CMs (Figures 4A-4C).Next, we examined monolayered hiPSC-CMs seeded at a higher concentration synchronizing with gap junction formation (Figure S6D).Action potential and calcium imaging in 1/3 Hz-paced hiPSC-CMs showed that the duration of the action potential and both the time to peak and full-width at half maximum (FWHM) in calcium transient were prolonged in D3-7 hiPSC-CMs compared with those in D3-9 and WT hiPSC-CMs (Figures 4D-4K).
RNA sequencing identified the transcriptomic characteristics of hiPSC-CMs with D3-7 and D3-9 To identify the transcriptomic characteristics of the hiPSC-CMs differentiated from isogenic DMD-mutated hiPSC lines, we performed bulk RNA sequencing (RNA-seq) analysis on D3-7, D3-9, and WT hiPSC-CMs at 48 d after differentiation.The purity of CMs was 95% or greater in each group, and there was no variation between the groups (Figures S4A and S4B).This provided the expression data for 17,354 genes.Hierarchical clustering of normalized expression levels suggested some similarity between D3-7 and D3-9 (Figure S7A).However, principal component analysis (PCA) of the whole dataset showed a strong similarity between WT and D3-9 hiPSC-CMs, while D3-7 hiPSC-CMs seemed to be distinct and separate from them (Figure 5A).The variation observed in PC1 among the samples was inferred to be attributed to the variability in gene expression related to other mesodermal lineages rather than cardiac-related genes according to the Gene Ontology (GO) enrichment analysis of genes that significantly contribute to PC1 and PC2.Volcano plots showed that NPPA, NPPB, CASQ2, ANKRD1, ACTA1, and FSTL3 were markedly downregulated in D3-7 and D3-9 hiPSC-CMs compared with those in WT hiPSC-CMs (Figures 5B, 5C, 5M-a, 5M-b, S8B, and S8C).Among them, CASQ2 and ANKRD1 were downregulated in D3-7 hiPSC-CMs compared with those in D3-9 hiPSC-CMs (Figures 5D, 5M-a, S8B, and S8C).
Notably, these genes are associated with cardiac hypertrophy and remodeling. 57,58In contrast, the expression levels of DES, SPARC, and ALPK3 were downregulated in D3-7 hiPSC-CMs (Figures 5B and  5D).DES encodes desmin intermediate filaments, SPARC encodes secreted protein acidic and rich in cysteine (a matricellular protein that functions as a positive inotrope, possibly by interaction with integrin b1 and integrin-linked kinase [ILK] in CMs), 59 and ALPK3 encodes a transcription factor important in cardiac differentiation and maturation, 60 all of which are causative for cardiomyopathy.
Venn analysis of differentially expressed genes (DEGs) showed that 1,843 genes were significantly downregulated (Figure 5E) and 1,580 genes were upregulated (Figure 5F) in D3-7 hiPSC-CMs compared with those in D3-9 and WT hiPSC-CMs.To examine the genes involved in the pathology exclusively in D3-7 hiPSC-CMs, we performed enrichment analysis on these gene sets.According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, terms highly enriched in the downregulated gene set included many associated with mechano-transduction (Figure 5G), including the AI3K-Akt signaling pathway (Figure S7B Molecular Therapy: Nucleic Acids hiPSC-CMs (Figure S7E).In contrast, no KEGG pathway terms were significantly enriched in the upregulated gene set, indicating that pathways involving downregulated genes have a stronger impact on the pathology of DCM in D3-7 hiPSC-CMs than upregulated genes.
The GO enrichment analysis of the upregulated gene set showed that several mitochondria-associated terms were significantly enriched (Figure S7F), which is speculated to be a compensatory upregulation, as mitochondrial dysfunction is one of the major pathologies of DCM in DMD.
Next, to determine the pathway involved in cardiac pathology, not only in D3-7 hiPSC-CMs, but also in D3-9 hiPSC-CMs, we examined the KEGG pathway enrichment of the 1,680 downregulated (Figures 5H and 5I) and 1,646 upregulated (Figures 5J and 5K Hierarchical clustering of DEGs involved in the sarcomere included numerous genes prominent on the volcano plots (Figures 5B-5D) and overlappingly categorized in DCM, hypertrophic cardiomyopathy, and arrhythmogenic cardiomyopathy (Figures S7C and S7E).
Regarding cardiac maturation markers, 41,61,62 the ratios of TNNI3/ TNNI1, MYH7/MYH6, and MYL2/MYL7 were the lowest in D3-7 hiPSC-CMs and TNNI3/TNNI1 and MYH7/MYH6 were the second highest in D3-9 hiPSC-CMs among the three groups (Figure 5L).These data indicated a maturation defect in dystrophin-deficient hiPSC-CMs.Regarding the genes involved in calcium handling and  and S8B).As for the genes encoding dystrophin-associated cardioprotective proteins, AHNAK and CRYAB were significantly upregulated in both D3-7 and D3-9 hiPSC-CMs compared with those in WT hiPSC-CMs, and LDB3 encoding Cypher was upregulated in D3-7 hiPSC-CMs compared with that in D3-9 hiPSC-CMs, implying a compensatory increase in the loss or aberrant interaction with dystrophin (Figures 5M-5D and S8D).These molecules may be involved in the pathogenesis of DMD cardiomyopathy; however, there has not been a definitive consensus on their expression and function in DMD CMs because there have been limited studies conducted so far. 20Thus, further study is needed to gain a better understanding.
AOs targeting exon 8 converted the reading frame to D3-9, restoring functional dystrophin with reduced Dp71 isoform and improving electrophysiology To explore whether exon skipping induced by AOs changes the phenotype of D3-7 hiPSC-CMs closer to D3-9 hiPSC-CMs, we performed exon skipping using Vivo-Morpholinos targeting exon 8 in D3-7 hiPSC-CMs, with the concentration range of 0.5-4 mM in a growth medium, which was confirmed to be nontoxic to hiPSC-CMs through a lactate dehydrogenase (LDH) cytotoxic assay (Figure S9).We performed RT-PCR and western blotting on cDNA and whole-protein samples extracted from hiPSC-CMs 14 d after treatment.RT-PCR analysis revealed three skipping patterns by the combination of exon 8 skipping induced by AOs and endogenous exon 9 skipping.The frequency of exon 8+9 skipping increased in a dose-dependent manner (Figure 6A), whereas the frequency of single exon 8 skipping was very low.This is consistent with an experiment on myoblasts derived from patients with DMD using 2 0 -O-methylmodified bases on a phosphorothioate backbone. 32Western blotting analysis revealed that internally truncated dystrophin was detected in AO-treated D3-7 hiPSC-CMs and increased in a dose-response manner, reaching 43% of dystrophin protein levels in D3-9 hiPSC-CMs at 4 mM AOs. Dp71 was partially decreased by AO treatment in a dose-dependent manner, with a 33% decrease at 4 mM (Figures 6B and 6C).Immunostaining also showed dystrophin restoration, albeit faintly, in D3-7 hiPSC-CMs (Figure 6D).
Next, we performed an electrophysiological analysis on hiPSC-CMs 20 d after AO administration.The single-cell analysis showed no significant arrhythmic cells in any of the samples, and the variability of the inter-beat interval tended to be reduced in D3-7 hiPSC-CMs, although the difference was not significant (Figure 7A).The monolayered analysis of the action potential with field pacing showed that the APD60 was shortened by the restoration of truncated dystrophin at $43% level of D3-9 hiPSC-CMs at 4 mM of AOs (p < 0.005) (Figure 7B).As for the calcium transient, the time to peak was shortened at 1 mM (p < 0.05), with the restoration of trace amounts of dystrophin and 4 mM with a greater effect (p < 0.001) (Figure 7D), and the FWHM was shortened at 2 mM and a higher dose (p < 0.005) in D3-7 hiPSC-CMs (Figure 7E).In summary, in the calcium transient, the rising phase was accelerated by a small restoration of D3-9 truncated dystrophin, and the duration of calcium transient and action potential was shortened by more restoration of dystrophin in D3-7 hiPSC-CMs.
To further investigate the early effect of exon skipping, we performed RT-PCR and western blotting analyses, focusing on the molecules indicated to be affected in D3-7 hiPSC-CMs by the above experiments.Notably, the ratio of TNNI3/TNNI1 increased after treatment with 4 mM in D3-7 hiPSC-CMs (Figures 6E and S10A).DES, CASQ2, SPARC, ALPK3, and ANKRD1 maintained low expression in D3-7 hiPSC-CMs for at least 2 weeks after treatment (Figure S10B).Channel genes, including SCN5A, KCNJ2, KCNJ4, KCND3, and RYR2, showed no significant changes (Figure S10C).The protein levels of a-DG, b-DG, desmin, cTnI, and cTnT and glycosylation of a-DG were not restored by the treatment (Figures S10D and S10E).
Next, we examined whether CaMKII activation was mitigated via exon skipping.Western blotting analysis showed that the protein level of T287 phosphorylated CaMKII was significantly decreased in D3-7 hiPSC-CMs by the administration of AOs at 4 mM (Figures 8A-8C), while the protein level of oxidized CaMKII was not changed by the treatment (data not shown).

DISCUSSION
This study investigated the validity and efficacy of ABD1 targeting exon skipping treatment to correct the reading frame to D3-9 using three isogenic hiPSC-CMs, including two models of dystrophinopathy: the DMD model caused by D3-7 and a very mild version of the BMD model caused by D3-9, along with the WT control.
In D3-7 hiPSC-CMs, Dp427m protein was absent, as expected.The protein level of internally truncated dystrophin was approximately 2-fold higher in D3-9 hiPSC-CMs than in WT hiPSC-CMs, implying a compensatory increase for the impaired function of shortened dystrophin or high tertiary structure stability; this may be partially attributable to the increased transcription rate resulting from array shortening.Following dystrophin coIP, we estimated that the truncated dystrophin lacking ABS2 and ABS3 can bind a-actin at the level of 48% of WT Dp427m.These results suggest that the dystrophin binding rate of a-actin in D3-9 hiPSC-CMs is approximately 90% of that in WT hiPSC-CMs, which might corroborate the reduced severity of phenotypes in BMD caused by D3-9.Notably, the protein level of the dystrophin shorter isoform Dp71 in D3-7 hiPSC-CMs was more than 2-fold higher than that in D3-9 hiPSC-CMs and WT hiPSC-CMs (Figures 1E-1G).In addition, Dp71 expression was decreased by AO treatment following the restoration of internally truncated dystrophin in a dose-dependent manner in a short period, which is a new discovery.Dp427m is a full-length dystrophin located in the sarcolemma and T-tubules in skeletal and cardiac muscles and is responsible for the pathogenesis of DMD and BMD.Dp71 is a ubiquitously expressed isoform, 63 and Dp116 is a Schwann cell-specific isoform that is also expressed in the human heart. 646][67] Genotypephenotype analyses of patients with DMD have suggested that the absence of Dp71 or Dp116 might be associated with less severe cardiac symptoms. 64The mechanism and pathophysiology of Dp71 upregulation in D3-7 hiPSC-CMs were not investigated in this study, and the expression of Dp71 in DMD hiPSC-CMs caused by other Desmin being significantly reduced in D3-7 hiPSC-CMs accompanied by reduced sarcomeric proteins, including cTnT and cTnI, was also a novel insight of this study.Desmin is a major intermediate filament of striated and smooth muscles, which plays an important role in the maintenance of muscle structural and cellular integrity, force transmission, and mitochondrial homeostasis by linking the contractile myofibrils to the sarcolemma and cellular organelles, constructing a network with various muscle component proteins, including dystrophin and dystrophin-associated proteins. 68,69Mutations in DES cause cardiomyopathy and skeletal myopathy, with prominent mitochondrial dysfunction. 70,71In the skeletal muscles of mdx mice, desmin expression is increased and plays a beneficial role in dystrophic muscles. 72However, the expression and role of desmin in cardiac muscle have not been studied in patients with DMD or animal or cell models.
Our study revealed highly reduced desmin expression at both the mRNA and protein levels in D3-7 hiPSC-CMs, whereas desmin protein was 2-fold higher in D3-9 hiPSC-CMs than in WT hiPSC-CMs.Furthermore, the binding ability of D3-9 truncated dystrophin to desmin was comparable with that of WT Dp427m, probably owing to the spared desmin-binding region in the cysteine-rich domain of dystrophin in D3-9 hiPSC-CMs. 4Binding to dystrophin might be necessary for desmin to be stably expressed in CMs, and desmin might reinforce the stability of cTnT and cTnI.Furthermore, decreased desmin might be involved in the pathogenesis of DMDassociated DCM, whereas increased desmin in D3-9 hiPSC-CMs might compensate for the impaired function of internally truncated dystrophin, which might partially explain the reduced severity in the phenotype of DCM in D3-9 patients.Further research is required to determine whether desmin modulates the DCM phenotype in patients with DMD.Although recovery of the expression of these proteins was expected through skipping therapy, no significant changes were observed in the evaluation during this short period.The extended culture or repeated administration of AOs might be required for the restoration of these proteins, despite the possibility that the decrease in these proteins might be an irreversible change caused by developmental defects caused by the absence of Dp427m.
D3-7 hiPSC-CMs specifically represented impaired adhesion, which may be explained by the decreased glycosylated a-DG, integrin, and ILK gene expression.These are major linkers between the ECM and cytoskeleton and are important in cell adhesion and mechano-transduction-related signaling pathways. 73tracellular calcium overload caused by disruption of the sarcolemma is a promising hypothesis for the pathogenesis of DCM in DMD. 746][77] This study recapitulated the increase in the activated form of CaMKII by auto-phosphorylation at T287 and oxidation at M281/M282, accompanied by increased phosphorylation of PLB at T17 downstream of activated CaMKII 78 in D3-7 hiPSC-CMs, indicating an intracellular calcium overload.The targets for activated CaMKII, including ion channels such as RyR2, SERCA2a, and Na + / Ca 2+ exchanger, and type II histone deacetylases, which regulate MEF2 signaling involved in myocardial hypertrophy, might also be enhanced, leading to aberrant excitation-contraction coupling and hypertrophy signaling in D3-7 hiPSC-CMs.AO-mediated exon skipping successfully decreased CaMKII auto-phosphorylation, which is speculated to be caused by the amelioration of calcium over-influx by the restoration of dystrophin.
Transcriptomic analysis of the three groups revealed the greatest similarity in gene expression profiles between D3-9 hiPSC-CMs and WT hiPSC-CMs by PCA, which might corroborate the mild cardiac phenotype in patients with D3-9 BMD. 23,31Enrichment analysis indicated that defective signaling pathways associated with mechano-transduction, in which multiple proteins and protein complexes from inside the sarcomere to outside the sarcolemma and ECM, including DAPC, might have a strong impact on the pathology of DCM in D3-7 hiPSC-CMs. 79,80The expression of the genes involved in these pathways was suggested to have the potential to approach the expression patterns in WT hiPSC-CMs and D3-9 hiPSC-CMs through exon skipping therapy.Among the genes involved in these pathways, however, NPPA, NPPB, CASQ2, ANKRD1, ACTA1, and FSTL3 were prominently downregulated not only in D3-7 hiPSC-CMs, but also in D3-9 hiPSC-CMs.5][86] DCM modeling of hiPSC-CMs with mutations in TNNT2 and frameshifting deletions (D44) in DMD have shown decreased NPPA and NPPB expression. 43,44The precise mechanism of impaired ANP/BNP production and its biological effect in patients with DMD/BMD remain to be elucidated.However, the impaired production of natriuretic peptides in the heart tissue might contribute to HF progression in patients with DMD/BMD.The isoform switching of sarcomeric genes, including TNNI, MYH, and MYL, was delayed in D3-7 hiPSC-CMs, consistent with previous studies, indicating maturation defects in DMD-hiPSC-CMs. 19mong these, the decrease in TNNI3/TNNI1, a later maturation marker, 87 was significantly improved via exon skipping in D3-7 hiPSC-CMs, indicating that dystrophin may play an important role in regulating the transcriptomic switch of TNNI.Remarkably, numerous genes involved in ribosome biogenesis were downregulated in both D3-7 hiPSC-CMs and D3-9 hiPSC-CMs compared with those in WT hiPSC-CMs.Alteration of YAP signaling downstream of Hippo signaling has been demonstrated to be associated with the impairment of actin dynamics, following the decreased proliferation ability in DMD (D48-54) hiPSC-CMs. 21Proteosome analysis of the myocardium from porcine DMD models showed a marked decrease in ribosome proteins with reduced heart weight and cell atrophy, suggesting deficient muscle protein synthesis caused by reduced translation activity due to impaired ribosome protein production. 88The function of D3-9 dystrophin might be insufficient in terms of Hippo/YAP signal transduction and ribosome biogenesis.
Regarding the electrophysiological aspects, the spontaneous beating rate was increased in D3-7 hiPSC-CMs, and variability in the interbeat interval was increased both in D3-7 hiPSC-CMs and D3-9 hiPSC-CMs, which might correspond with the susceptibility to ventricular and sinus tachycardia and accelerated beat rate variability in patients with DMD. 74Apparent arrhythmic transient was not detected in all groups in this study, which is inconsistent with previous studies on DMD modeling hiPSC-CMs by us and the other groups. 15,17,18This discrepancy might be due to the differences in cell lines, mutations, cardiac differentiation protocols, and calcium indicators used.The analysis of monolayered hiPSC-CMs with field pacing revealed a significantly prolonged duration of both calcium and potential signals, as well as a prolonged rising phase of calcium transient in D3-7 hiPSC-CMs.These findings suggest the presence of ion channel malfunction and abnormalities in calcium handling in DMD hiPSC-CMs, which is consistent with previous studies, 15,18,19,43,89 and are considered phenomena that can explain electrocardiographic abnormalities in patients with DMD. 90 AO treatment improved the electrophysiological properties following partial restoration of the internally truncated dystrophin in D3-7 hiPSC-CMs.As a potential mechanism, abnormalities in the expression of channel genes were initially suspected, and RNA-seq data revealed changes in the gene expression of multiple ion channels that contribute to the action potential and calcium transient.However, significant fluctuations in that gene expression did not accompany the rapid improvement in electrophysiology achieved through skipping therapy.Therefore, it was considered highly likely that changes in the expression of channel genes do not directly contribute to therapeutic effects.Previous studies on DMD hiPSC-CMs have failed to come to a consensus on channel gene expression. 89,91 of ion channels on the sarcolemma of CMs, including Na V 1.5, Kir2.1, and Kir2.2, by interacting with them. 91,92The dysfunction of DAPC is associated with cardiac conduction defects in mdx mice. 91,93Jimenez-Vaquez et al. ( 2022) demonstrated that electrophysiological alterations were ameliorated by the overexpression of SNTA1 encoding a1-syntrophin in hiPSC-CMs derived from patients with DMD. 4,89nother dystrophin-associated protein, AHNAK1, modulates b-adrenergic regulation of L-type calcium channel (LTCC) activity in CMs. 48,50The D3-9 dystrophin retains the binding domains for a1-syntrophin and AHNAK1 in its structure, and, for AHNAK1, dystrophin IP experiments have demonstrated that its binding ability in hiPSC-CMs is comparable to that in WT Dp427m.In addition, activated CaMKII accelerates the phosphorylation of the targets, including SERCA2a, RyR2, LTCCs, NCX, and PLN. 75Consequently, it is hypothesized that the aberrant post-translational modification of channels on the sarcolemma and endoplasmic reticulum membrane by DAPC and CaMKII may account for the electrophysiological abnormalities observed in D3-7 hiPSC-CMs.Another possible explanation is that increased Dp71 may adversely affect channel function or activity in D3-7 hiPSC-CMs.These hypotheses might explain the rapid electrophysiological amelioration by the partial restoration of dystrophin and the decrease in Dp71 following treatment with AOs.
This study has several limitations.First, we used hiPSC-CMs, but these cannot fully represent patients' heart tissue because they are immature, resembling neonatal ventricular cells in terms of gene expression and function.Second, since the analysis relied on isogenic disease model cells derived from a single cell line, evidence for the correlation between the mutation and pathophysiology is not sufficiently strong.Third, this study's exon skipping experiment focused solely on the short-term effects of a single administration.Although we observed rapid electrophysiological improvements alongside the restoration of dystrophin expression, the expression patterns of proteins and genes following exon skipping therapy did not tend to resemble the pattern of D3-9.This might be attributed to the time required for the stabilization and functional recovery of DAPC, as well as potential changes in the expression of other genes and proteins after the restoration of dystrophin expression.Determining the optimal skipping level for functional recovery was not possible in this study.Last, the study was carried out in vitro, meaning that the delivery of AOs to the heart and the skipping efficiency in the tissue could not be investigated.Further studies using an ABD1-deficient animal model exhibiting a severe DCM phenotype would be required to fully assess the clinical application of this system.
In summary, our findings revealed that internally truncated dystrophin lacking ABS2 and ABS3 in ABD1 was biologically stable with substantial actin-binding function in D3-9 hiPSC-CMs, even in comparison with WT Dp427m.Furthermore, D3-9 hiPSC-CMs showed little electrophysiological impairment without acceleration of CaMKII auto-phosphorylation and oxidation, contrary to D3-7 hiPSC-CMs, although some molecular pathologies, including alterations in ribosomal and mitochondrial biogenesis and impaired mechano-transduction-related hypertrophy and remodeling, were shared with D3-7 hiPSC-CMs.We demonstrated that exon skipping by AOs targeting exon 8 efficiently induced exons 8-9 skipping to produce functional dystrophin and ameliorated electrophysiological abnormalities in D3-7 hiPSC-CMs.In conclusion, exon skipping therapy targeting ABD1 to convert the reading frame to D3-9 may be promising for treating DMD cardiomyopathy.

Generation of a WT hiPSC line with a genetically encoded dual fluorescent indicator of voltage and intracellular calcium ions
All hiPSC experiments were performed using a genetically modified cell derivative from the 610B1 (RIKEN BRC, Tsukuba, Japan) hiPSC line, a commercially available WT control line derived from a healthy male.First, we developed a plasmid containing ASAP2s, a fluorescent green voltage indicator, and R-CaMP1.07, a fluorescent red calcium indicator.Briefly, a donor plasmid using the pSF-CAG-Ub-Puro (OGS600, Sigma-Aldrich, St. Louis, MO) cloning vector was generated, which consisted of approximately 800-bp-long homology arms flanking the AAVS1 gRNA (CCAATCCTGTCCCTAGTG GCCCC) cut site surrounded by an 8.8-kb insert with two elements: a cassette bearing the CAG promoter that drives both ASAP2s and subsequent R-CaMP1.07 expression using self-cleaved 2A peptide and a second cassette encoding for Ubc-driven expression of puromycin resistance gene surrounded by LoxP sites, as illustrated in Fig- ure 1A.We obtained the ASAP2s sequence from Addgene (#101274, Addgene, Watertown, MA) and the R-CaMP1.07 94 plasmid as a courtesy of Dr. Nakai.Plasmids expressing sgRNA and Cas9 were constructed using the Guide-it CRISPR-Cas9 system (Takara Bio, Kusatsu, Japan) following the manufacturer's protocols.
A day prior to electroporation, 610B1 hiPSCs were treated with 10 mM valproic acid and 10 mM Y27632 for 24 h.ASAP2s-RCaMP1.07 donor plasmid (6 mg), Cas9/sgRNA plasmid (3 mg), and RAD51-expressing plasmids 95 (1 mg) were co-electroporated using Nucleofector 2b (Lonza, Basel, Switzerland) with the B-16 program into 1 Â 10 6 hiPSCs.Two days after electroporation, transfected cells were selected with 1 mg/mL puromycin for 4 d.After expansion, PCR genotyping was performed to examine whether the clones were correctly targeted.The pCAG-iCre plasmid (10 mg; #89573, Addgene) was electroporated into the correctly targeted clones to remove the selection cassette.Genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Venlo, the Netherlands) following the standard protocol.The region around the cut site was amplified with PrimeSTAR GXL DNA polymerase (Takara Bio).The primers used are listed in Table S1.
Sanger sequencing of the predicted potential off-targets on undifferentiated cell-extracted genomic DNA revealed no sgRNA-mediated nonspecific cleavage by Cas9 for each cell line.

Cardiac differentiation and maturation
Undifferentiated hiPSCs were induced to differentiate into CMs using a previously reported protocol. 97Briefly, undifferentiated hiPSCs were plated on an MG (Corning, Corning, NY)-coated culture dish and cultured in Essential 8 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 1 mM CHIR99021 (Sigma-Aldrich).On day 0, the medium was replaced with RPMI 1640 medium (Thermo Fisher Scientific) supplemented with B27 (RPMI/B27) without insulin (RPMI/B27-ins) with 100 ng/mL activin A (R&D Systems, Minneapolis, MN) and MG.On day 1, the medium was replaced with 10 ng/mL BMP-4 (R&D Systems) and 1 mM CHIR99021.On days 3-4, the medium was replaced with 1 mM XAV939 (R&D systems), a Wnt inhibitor.On day 7, insulin was added to RPMI/B27 (RPMI/B27+ins); the medium was changed every 2 d.Spontaneous beating was observed on days 7-10.On day 9, the cells were exposed to a glucose-and glutamine-free medium supplemented with 4 mM lactate and StemFit medium AS501 (Ajinomoto, Tokyo, Japan) to enrich CMs. 36,39,98To promote cell proliferation, cells were re-plated as dissociated single cells with 2 mM CHIR99021 in 10% knockout serum replacement (Thermo Fisher Scientific) in RPMI/B27+ins onto an MG coated 10 cm culture dish at 4-8 Â 10 4 /cm 2 density on day 13.On day 15, the medium was replaced with 2 mM CHIR99021, and on day 17 the medium was replaced with 10 mM Y-27632 and 2 mM Wnt-C59, a Wnt inhibitor in RPMI/B27+ins. 38On day 18, the cells were replated in MG-coated dishes at a density of 3 Â 10 5 /cm 2 .To enhance CM maturation on days 21-35, the culture medium was refreshed daily with RPMI/ B27+ins supplemented with 100 nM T3 and 1 mM Dex. 37 Starting from day 35, hiPSC-CMs were maintained in RPMI/B27+ins, with the medium changed every 3-4 d until harvesting for characterization using immunofluorescence, mRNA expression, and protein analyses.Cardiac purity was determined by immunostaining for cTnT (clone CT3), followed by anti-mouse IgG 1 conjugated with phycoerythrin, using FACS Canto II (BD Biosciences, Franklin Lakes, NJ).Cell diameter was determined using the standard curve related to forward scatter and cell size generated using the SPHERO Particle Size Standard Kit (#PPS-6K, Spherotech, Lake Forest, IL).

RNA extraction and real-time qPCR
RNA was isolated from hiPSC-CMs using the RNeasy Mini Kit (Qiagen).Human fetal heart total RNA (#636532, Takara Bio) and human adult heart RNA (# 636583, Takara Bio) were purchased.Reverse transcription of RNA to cDNA was performed using the Superscript IV Reverse Transcriptase Kit (Thermo Fisher Scientific) following the manufacturer's instructions, using random hexamers.qPCR was performed using Fast SYBR Green Master Mix (Thermo Fisher Scientific) and QuantStudio 3 (Thermo Fisher Scientific) with the primers listed in Table S1.Thermal cycling conditions were as follows: initial denaturation at 95 C for 20 s, 40 cycles at 95 C for 3 s, and 60 C for 30 s.We calculated mRNA fold expression using the DDCT method with GAPDH as the housekeeping gene.All experiments were conducted in duplicate.

RNA-seq
RNA was isolated from day-48 hiPSC-CMs cultured in six-well plates using the RNeasy Mini Kit, following the manufacturer's instructions.All RNA was quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) to ensure RNA integrity (all samples had RIN scores R9.1).mRNA was enriched with poly(A) selection, and 50-bp paired-end RNA-seq was completed on the DNBSEQ platform at the Beijing Genomics Institute (BGI, Shenzhen, China).Raw reads were filtered using SOAP and SOAPnuke, 99 and clean reads were mapped to the transcriptome of the RefSeq database using Bowtie2. 100Gene expression was counted by RSEM 101 and normalized as transcripts per kilobase of exon model per million mapped reads.We used DESeq2 to evaluate the differential expression.DEGs were identified by a false discovery rate-adjusted p value < 0.05. 102All data were submitted to the online tool Dr. TOM software (BGI) for PCA and GO and pathway enrichment analyses.

Western blotting analysis and coIP
Proteins were extracted by directly lysing day 48 hiPSC-CMs in cell culture wells with M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) supplemented with Protease/ Phosphatase Inhibitor Cocktail (100Â) (Cell Signaling Technology, Danvers, MA).Protein samples were mixed with 4Â Laemmli sample buffer (Bio-Rad, Hercules, CA) and boiled for 5 min at 95 C. Samples containing 1.25-10 mg total protein were separated on an Any kD mini-Protean TGX gel (Bio-Rad) and transferred onto polyvinylidene Molecular Therapy: Nucleic Acids fluoride membranes.The membranes were blocked with Bullet Blocking One (Nacalai Tesque, Kyoto, Japan).The membranes were incubated with specific primary antibodies overnight at 4 C, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT.

Figure 1 .
Figure 1.Generation of isogenic cell lines with in-frame and out-of-frame deletions in the ABD1 coding region of DMD harboring genetically encoded dual indicator for voltage and calcium in AAVS1 (A) An expression cassette of genetically encoded action potential indicator ASAP2s (green) and calcium indicator R-CaMP1.07 (red) was engineered into intron 1 of AAVS1, a safe harbor in a healthy human male hiPSC by CRISPR-Cas9 editing.(B and C) Representative fluorescent images of spontaneously beating hiPSC-CMs (WT, on day 50) showing voltage (ASAP2s) (B, top) and intracellular calcium (R-CaMP1.07)(C, top), and representative traces of voltage (B, bottom) and intracellular calcium ions (C, bottom).Scale bar, 20 mm.(D) Generation of isogenic cell lines with the DMD D37 and D3-9 from WT hiPSCs carrying ASAP2s/RCaMP1.07 (A) by genome editing using gRNAs targeting deep introns.(E) Immunostaining of cTnT (green) and dystrophin (red) in WT, D3-7, and D3-9 hiPSC-CMs on day 48 after differentiation.DNA was counterstained with DAPI.Scale bar, 20 mm.(F) Western blot analysis showing protein levels of three isoforms of dystrophin protein, Dp427, internally truncated dystrophin, Dp116, and (legend continued on next page)
) genes in both D3-7 and D3-9 hiPSC-CMs compared with those in WT hiPSC-CMs, with the exclusion of genes showing a mitigating trend in D3-9 hiPSC-CMs compared with those in D3-7 hiPSC-CMs.As significantly enriched terms in the downregulated genes, ribosome was outstanding (Figures5I, S7B-e, and S7B-f), while mitochondria-associated terms ranked among the top enriched terms in upregulated genes (Figures 5K, S7D-a, and S7D-b).

Table S3 . Potential candidate off-target exonic loci with CFD off-target score more than 0.02.
Off-target sequence is shown with mismatch to sgRNA sequence shown in red.