Therapeutic Exon Skipping via a CRISPR-guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy In Vivo

Background: Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adult. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, due to the lack of a suitable preclinical model. Methods: We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as Dmd E4* mice). Echocardiography, micro-CT, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo . AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal Dmd E4* mice, which were analyzed 2- or 12-month post treatment to evaluate the extents of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles. Results: Dmd E4* mice recapitulated many aspects of human DMD, including shortened lifespan (by ∼50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted over 50% targeted exon skipping in the Dmd transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased lifespan of the Dmd E4* mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least one year. Conclusions: Our study demonstrates the feasibility and efficacy to institute exon skipping via an enhanced TAM (eTAM) for therapeutic application(s).

Sarcolipin haploinsufficiency prevents dystrophic cardiomyopathy in mdx mice

First, reducing sarcopolin (SLN) expression improves sarco/endoplasmic reticulum Ca2+ uptake and intracellular Ca2+ handling and prevents cardiomyopathy in mdx mice. Second, reducing SLN expression prevents diastolic dysfunction and improves cardiac contractility in mdx mice Third, reducing SLN expression activates apelin-mediated cardioprotective signaling pathways in mdx heart.

Cardiac Pathophysiology and the Future of Cardiac Therapies in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease featuring skeletal muscle wasting, respiratory insufficiency, and cardiomyopathy. Historically, respiratory failure has been the leading cause of mortality in DMD, but recent improvements in symptomatic respiratory management have extended the life expectancy of DMD patients. With increased longevity, the clinical relevance of heart disease in DMD is growing, as virtually all DMD patients over 18 year of age display signs of cardiomyopathy. This review will focus on the pathophysiological basis of DMD in the heart and discuss the therapeutic approaches currently in use and those in development to treat dystrophic cardiomyopathy. The first section will describe the aspects of the DMD that result in the loss of cardiac tissue and accumulation of fibrosis. The second section will discuss cardiac small molecule therapies currently used to treat heart disease in DMD, with a focus on the evidence supporting the use of each drug in dystrophic patients. The final section will outline the strengths and limitations of approaches directed at correcting the genetic defect through dystrophin gene replacement, modification, or repair. There are several new and promising therapeutic approaches that may protect the dystrophic heart, but their limitations suggest that future management of dystrophic cardiomyopathy may benefit from combining gene-targeted therapies with small molecule therapies. Understanding the mechanistic basis of dystrophic heart disease and the effects of current and emerging therapies will be critical for their success in the treatment of patients with DMD.

Vamorolone targets dual nuclear receptors to treat inflammation and dystrophic cardiomyopathy

Cardiomyopathy is a leading cause of death for Duchenne muscular dystrophy. Here, we find that the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) can share common ligands but play distinct roles in dystrophic heart and skeletal muscle pathophysiology. Comparisons of their ligand structures indicate that the Δ9,11 modification of the first-in-class drug vamorolone enables it to avoid interaction with a conserved receptor residue (N770/N564), which would otherwise activate transcription factor properties of both receptors. Reporter assays show that vamorolone and eplerenone are MR antagonists, whereas prednisolone is an MR agonist. Macrophages, cardiomyocytes, and CRISPR knockout myoblasts show vamorolone is also a dissociative GR ligand that inhibits inflammation with improved safety over prednisone and GR-specific deflazacort. In mice, hyperaldosteronism activates MR-driven hypertension and kidney phenotypes. We find that genetic dystrophin loss provides a second hit for MR-mediated cardiomyopathy in Duchenne muscular dystrophy model mice, as aldosterone worsens fibrosis, mass and dysfunction phenotypes. Vamorolone successfully prevents MR-activated phenotypes, whereas prednisolone activates negative MR and GR effects. In conclusion, vamorolone targets dual nuclear receptors to treat inflammation and cardiomyopathy with improved safety.

Exacerbation of dystrophic cardiomyopathy by phospholamban deficiency mediated chronically increased cardiac Ca2+ cycling in vivo

Cardiomyopathy is a significant contributor to morbidity and mortality in Duchenne muscular dystrophy (DMD). Membrane instability, leading to intracellular Ca2+ mishandling and overload, causes myocyte death and subsequent fibrosis in DMD cardiomyopathy. On a cellular level, cardiac myocytes from mdx mice have dysregulated Ca2+ handling, including increased resting Ca2+ and slow Ca2+ decay, especially evident under stress conditions. Sarco(endo)plasmic reticulum Ca2+ ATPase and its regulatory protein phospholamban (PLN) are potential therapeutic targets for DMD cardiomyopathy owing to their key role in regulating intracellular Ca2+ cycling. We tested the hypothesis that enhanced cardiac Ca2+ cycling would remediate cardiomyopathy caused by dystrophin deficiency. We used a genetic complementation model approach by crossing dystrophin-deficient mdx mice with PLN knockout (PLNKO) mice [termed double-knockout (DKO) mice]. As expected, adult cardiac myocytes isolated from DKO mice exhibited increased contractility and faster relaxation associated with increased Ca2+ transient peak height and faster Ca2+ decay rate compared with control mice. However, compared with wild-type, mdx, and PLNKO mice, DKO mice unexpectedly had reduced in vivo systolic and diastolic function as measured by echocardiography. Furthermore, Evans blue dye uptake was increased in DKO hearts compared with control, mdx, and PLNKO hearts, demonstrating increased membrane damage, which subsequently led to increased fibrosis in the DKO myocardium in vivo. In conclusion, despite enhanced intracellular Ca2+ handling at the myocyte level, DMD cardiomyopathy was exacerbated owing to unregulated chronic increases in Ca2+ cycling in DKO mice in vivo. These findings have potentially important implications for ongoing therapeutic strategies for the dystrophic heart. NEW & NOTEWORTHY This study examined the effects of phospholamban ablation on the pathophysiology of cardiomyopathy in dystrophin-deficient mice. In this setting, contractility and Ca2+ cycling were enhanced in isolated myocytes; however, in vivo heart function was diminished. Additionally, sarcolemmal integrity was compromised and fibrosis was increased. This is the first study, to our knowledge, examining unregulated Ca2+ cycling in the dystrophin-deficient heart. Results from this study have implications for potential therapies targeting Ca2+ handling in dystrophic cardiomyopathy. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/unregulated-ca2-cycling-exacerbates-dmd-cardiomyopathy/ .

Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

ABSTRACTDirected differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

Dystrophin Cardiomyopathies: Clinical Management, Molecular Pathogenesis and Evolution towards Precision Medicine

Duchenne’s muscular dystrophy is an X-linked neuromuscular disease that manifests as muscle atrophy and cardiomyopathy in young boys. However, a considerable percentage of carrier females are often diagnosed with cardiomyopathy at an advanced stage. Existing therapy is not disease-specific and has limited effect, thus many patients and symptomatic carrier females prematurely die due to heart failure. Early detection is one of the major challenges that muscular dystrophy patients, carrier females, family members and, research and medical teams face in the complex course of dystrophic cardiomyopathy management. Despite the widespread adoption of advanced imaging modalities such as cardiac magnetic resonance, there is much scope for refining the diagnosis and treatment of dystrophic cardiomyopathy. This comprehensive review will focus on the pertinent clinical aspects of cardiac disease in muscular dystrophy while also providing a detailed consideration of the known and developing concepts in the pathophysiology of muscular dystrophy and forthcoming therapeutic options.