Advances in Stem Cell Modeling of Dystrophin-Associated Disease: Implications for the Wider World of Dilated Cardiomyopathy

Pioner JM, Fornaro A, Coppini R, Ceschia N, Sacconi L, Donati MA, Favilli S, Poggesi C, Olivotto I, Ferrantini C.Front Physiol. 2020 May 12;11:368. doi: 10.3389/fphys.2020.00368. eCollection 2020.PMID: 32477154 Free PMC article. Review.



Familial dilated cardiomyopathy (DCM) is mostly caused by mutations in genes encoding cytoskeletal and sarcomeric proteins. In the pediatric population, DCM is the predominant type of primitive myocardial disease. A severe form of DCM is associated with mutations in the DMD gene encoding dystrophin, which are the cause of Duchenne Muscular Dystrophy (DMD). DMD-associated cardiomyopathy is still poorly understood and orphan of a specific therapy. In the last 5 years, a rise of interest in disease models using human induced pluripotent stem cells (hiPSCs) has led to more than 50 original studies on DCM models. In this review paper, we provide a comprehensive overview on the advances in DMD cardiomyopathy disease modeling and highlight the most remarkable findings obtained from cardiomyocytes differentiated from hiPSCs of DMD patients. We will also describe how hiPSCs based studies have contributed to the identification of specific myocardial disease mechanisms that may be relevant in the pathogenesis of DCM, representing novel potential therapeutic targets.


FIGURE 1 Cardiac magnetic resonance (CMR) cine imaging of a 24 years old DMD patient. The collection of representative images from patients was approved by the Ethical Committee of the Meyer Pediatric Hospital of Florence in the context of a project funded by Telethon Italy (grant GGP16191). Informed consent to patients was performed conform the declaration of Helsinki. This data does not contribute to any novel finding. (A) Late gadolinium-enhancement (B) left ventricular (LV) short-axis section images of a patient with Duchenne muscular dystrophy (DMD). Yellow arrows indicate the inferolateral subepicardial and midwall contiguous fibrosis; light blue arrows indicate the anterior segment and the red arrows the posterolateral right ventricle wall, both showing midwall fibrosis; CMR cine imaging (C) and late gadolinium-enhancement (D) LV long-axis section images of the same patient with DMD; yellow arrows indicate midwall fibrosis of the inferolateral segment.


FIGURE 2 Overview of full-length dystrophin in the context of other DCM-related proteins. The blow-up box, is a focus on full-length dystrophin structure and interactions. Full-length dystrophin is a large rod-shaped protein with a molecular weight of 427 kDa composed by 4 structural domains. The amino (N)-terminal domain has homology with α-actinin and binds, in particular, the F-actin; the central rod-domain contains 24–25 spectrin-like repeats the cysteine-rich domain intereacting with syntrophin and sarcoglycans; the last carboxy (C)-terminal domain associates at the C-terminal with β-dystroglican and several other proteins to form a major protein complex referred to as the dystrophin glycoproteic complex (DGC) (Hoffman et al., 1987; Ervasti and Campbell, 1993). The DGC consists of α- and β-dystroglycan subunits, α-, β-, δ-, γ-, and ε-sarcoglycans, sarcospan, α- and β-syntrophins, α-dystrobrevin, and neuronal nitric oxide synthase (nNOS) (Mosqueira et al., 2013). DGC related-pathways include Ca2+ homeostasis and E-C coupling, mitochondrial function, motor protein interaction (sarcomere/Z-band), and gene expression. For instance, the acetylcholine receptor, the skeletal and cardiac isoforms of the voltage-gated sodium channels (Nav1.4 and Nav1.5, respectively), the L-type Ca2+ channel, aquaporin, and stretch-activated channel or transient receptor potential (TRP) cation channels (Shirokova and Niggli, 2013) are closely associated with the DGC via syntrophins. In the cardiac tissue, dystrophin is also associated to: Cavin-1 and Caveolin-3 (responsible for caveolae/T tubule formation), Ahnak1 (modulates L-type Ca2+ channel), CryAB (involved in cytoprotection and antiapoptosis), and Cipher (plays a role in muscle contraction maintaining the Z-line integrity and signaling). Dystrophin can be also target of phosphorylation by Calmodulin-dependent kinase II (CaMKII) that modulates the affinity for F-actin and syntrophin (Madhavan and Jarrett, 1994). Other short isoforms of dystrophin come from spliced variants and are expressed in several other tissues. In particular, the Dp71 is expressed in cardiac muscle and likely present in T-tubular membranes (Kaprielian and Severs, 2000; Kaprielian et al., 2000).


FIGURE 3 Human induced pluripotent stem cell derived cardiomyocytes. (A) Generation of hiPSCs by cell reprogramming of somatic cells from patients or healthy donors or via CRISPR-Cas9 gene editing for the generation of isogenic pairs. (B) Current maturation strategies for hiPSC-CMs at cell level (2D strategies) or in multicellular model (3D strategies). (C) Possible methods for the assessment of electrophysiological and contractile properties.


FIGURE 4 Morphology and function altered in DMD-hiPSC-CMs from patient confirmed in a CRISPR-Cas9 gene edited cell line. Original data are modified from Pioner et al. (2019b) with the correct permission from the owner (Pioner et al., 2019a). A DMD-hiPSC-cell line from a patient (with Δ Exon 50 in DMD gene) and a CRISPR-Cas9 gene edited cell line (c.263 ΔG) targeting Exon 1 in healthy control cell line (Control) were generated and the cardiomyocytes were matured onto nanotopographic cues for 3 months. (A) DMD-hiPSC-CMs displayed aspect ratio similar to adult cardiomyocytes and Z-bands were observed across the entire cell width (diameter), suggesting DMD-CMs experienced hypertrophy and a greater number of myofibrils in parallel. (B) Despite similar myofibril alignment to control-hiPSC-CMs, sarcomere diameters (estimated from the length of Z-bands by transmitted electron microscopy) were significantly smaller, suggesting a possible reduction in the parallel assembly of myofilaments within individual myofibrils. (C) Meta-analysis on Calcium transient duration. (D) Representative traces of calcium transients (CaT), cell shortening and myofibril mechanics of DMD-hiPSC-CMs compared to controls. Simultaneous recordings at single cell level revealed slower CaT decay (estimated from time to peak to 50% of CaT decay, RT50, ms), slower cell relaxation (RT50, ms) at 37°C and external pacing (0.5 Hz reported). Compared to other studies reporting similar analysis of calcium transients, slower CaT duration might be a peculiarity of DMD-hiPSC-CMs. Single myofibrils showed lower isometric-tension generating capacity and slower myofibril relaxation (slow tREL and fast kREL). This study concluded that both calcium handling and myofibril abnormalities may contribute to prolong cell relaxation.