Duchenne cardiomyopathy: targeting ROS and NOX4 as a promising therapeutic strategy

KEYWORDS:

• Duchenne muscular dystrophy
• cardiomyopathy
• ROS
• NOX4
• therapeutics

1. Introduction

Duchenne muscular dystrophy (DMD; OMIM#310200) is a pediatric muscle disorder, affecting approximately 1 in every 5000 male births worldwide [1]. DMD is caused by mutations in the X-linked DMD gene, resulting in the absence of the functional form of its major protein product Dystrophin. The absence of Dystrophin results in several detrimental downstream effects, such as disruption of calcium (Ca²⁺) homeostasis, oxidative stress, inflammation, and mitochondrial dysfunction. Ultimately, these impairments make cardiac myocytes extremely vulnerable for degeneration, necrosis and thereby contributing to the progressive phase of cardiac fibrosis (Reviewed by [2]).

Adverse myocardial remodeling and chronic cardiomyopathy are major causes of morbidity and early mortality of most DMD patients. Initially, left ventricular (LV) dilation and hypertrophy develop to cope with the higher muscle wall stress, induced by pressure/volume overload. Subsequently, rhythm abnormalities (mainly supraventricular arrhythmias) and remodeling of the heart architecture occur that progress to dilated cardiomyopathy (DCM). In particular, repetitive mechanical stress causes apoptosis and myocardial fibrotic deposition. Eventually, heart failure and arrhythmias will develop as the disease progresses (Reviewed by [3]). Here, we focus on the increased oxidative stress as a major contributor to the development and progression of heart failure and more specifically in Duchenne cardiomyopathy.

2. Role of ROS and NOX4 in a stressed heart

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family enzymes (NOX1-5, DUOX1, and DUOX2) generate reactive oxygen species (ROS) in a highly regulated manner during cardiac adaptation to different types of physiological and pathophysiological stresses. ROS in turn regulates gene expression, posttranscriptional processing of proteins, cellular signaling, and cell differentiation. Four distinct NOX isoforms have been identified as the main source of ROS in the heart: NOX1, 2, 4, and 5. The NOX4 isoform is predominantly expressed in cardiac myocytes. In contrast to the inducible NOX2, NOX4 has constitutive mRNA transcripts, which are fine-tuned by epigenetic processes with micro-RNA-dependent posttranscriptional repression. Moreover, NOX4 exhibits alternative mRNA splice variants [4], which further complicates full understanding of the role of NOX4 under normal physiologic and pathologic circumstances (Reviewed by [5]).

Various studies described a cardioprotective role of Nox4 as unique inducible activator of myocardial angiogenesis in models of stress-induced cardiac remodeling or cardiac hypertrophy, first demonstrated by Zhang et al. [6,7] (Table 1). Furthermore, the importance of Nox4-derived ROS signaling has been demonstrated on the transcription factor Nrf2, which controls a network of cytoprotective genes. Endogenous Nox4 could regulate Nrf2 activation, mediating protective effects in the pressure-overloaded heart [8]. However, contradictory results were reported by other groups, showing detrimental effects of Nox4 in the overloaded heart due to increased mitochondrial ROS production and fibrosis [9,10]. Interestingly, the studies performed by Zhang et al. [6,7] and Kuroda et al. [9] may highlight the different function of Nox4 when genetically modified in a cardiomyocyte-targeted manner or in the entire heart, supported by the study of Cucoranu and colleagues in which NOX4-derived ROS has been shown to differentiate cardiac fibroblasts into myofibroblasts [11]. More specifically in Dystrophin-deficient cardiomyopathy in mouse [10,12] and human [13], detrimental effects of NOX4 were associated with LV fibrosis and altered functional parameters in the heart (significantly increased LV internal diameter and decreased posterior wall thickness), as well as significantly increased oxidative stress levels. In addition to the chronical pressure overloaded heart disease models, NOX4 involvement has also been described during ischemia/reperfusion (I/R) injury. For example, a study reported that Nox4 deletion did not influence myocardial reperfusion injury, while ablation of Nox1 and Nox2 conferred cardioprotection [14]. Another study showed less myocardial damage following I/R in cardiac-specific Nox4-deficient mice, associated with lower ROS production and reduced infarct size. However, myocardial injury was exacerbated in Nox2/Nox4-deficient mice, suggesting that a certain amount of ROS produced by either Nox2 or Nox4 is necessary for protection against I/R injury [15]. Taken together, these studies indicate that NOX enzymes, and more specifically NOX4, may be a novel and effective interesting target for therapeutic strategies.

Table 1. NOX4 in cardiac disease models.

Disease model	NOX4 intervention (animal model)	Phenotypic outcome	Reference
Chronic stress (PO)	Global deletion (Nox4 KO mice)	Normal basal cardiac function. Contractile dysfunction, hypertrophy and cardiac dilatation.	Zhang et al. [6]
Chronic stress (PO)	Cardiomyocyte-specific overexpression (CM-Nox4 Tg mice)	Normal basal cardiac function. Reduced interstitial fibrosis and hypertrophy. Enhanced angiogenesis.	Zhang et al. [6]
Chronic stress (PO)	Cardiac-specific deletion (c-Nox4 KO mice)	Improved cardiac function. Decreased mitochondrial ROS production. Reduced hypertrophy, fibrosis, and cell death.	Kuroda et al. [9]
Chronic stress (PO)	Cardiac-specific overexpression (c-Nox4 Tg mice)	Exacerbated cardiac dysfunction. Increased fibrosis and cell death.	Kuroda et al. [9]
Chronic stress (PO)	Inducible cardiomyocyte-specific deletion (iCM-Nox4 KO mice)	Hypertrophy and contractile dysfunction. Increased interstitial fibrosis. Decreased angiogenesis.	Zhang et al. [7]
Chronic stress (PO)	Inducible endothelial-specific deletion (iEndo-Nox4 KO mice)	Hypertrophy and contractile dysfunction. Increased interstitial fibrosis. Decreased angiogenesis.	Zhang et al. [7]
Chronic disease (mdx)	No genetic modification of Nox4. Nox4 upregulation in mdx hearts	Cardiac dysfunction. Increased LV fibrosis.	Spurney et al. [11]
Chronic disease (mdx)	Global deletion (Nox4 KO mice)	Decreased LV fibrosis.	Hammers [12]
Chronic disease (DMD)	No genetic modification of NOX4. NOX4 upregulation in human iPSC-CMs	Increased cell death and mitochondrial ROS production.	Duelen et al. [13]
I/R injury	Global deletion (Nox4 KO mice)	Does not influence myocardial reperfusion injury	Braunersreuther et al. [14]
I/R injury	Global deletion (Nox4 KO mice)	Reduced myocardial damage and infarct size/area. Decreased ROS production.	Matsushima et al. [15]
I/R injury	Cardiac-specific deletion (c-Nox4 KO mice)	Reduced myocardial damage and infarct size/area. Decreased ROS production.	Matsushima et al. [15]

Abbreviations: c-Nox4: cardiac-specific Nox4; CM-Nox4: cardiomyocyte-specific Nox4; iCM: inducible 3 cardiomyocyte; iEndo: inducible endothelial; iPSC-CMs: induced pluripotent stem cell-derived cardiomyocytes; 4 I/R: ischemia/reperfusion; KO: knockout; LV: left ventricular; PO: pressure overload; Tg: transgenic.

3. Emerging therapeutic approaches for DMD

Daily high-dose corticosteroids (prednisone and deflazacort) are considered the standard of care for DMD, and deflazacort is labeled for use for DMD in the USA. While corticosteroids result in relatively rapid improvement in skeletal muscle, the effects of corticosteroids on DMD heart function are not clear. However, long-term use of high doses of corticosteroids may increase a person’s chance of developing cardiovascular disease or exacerbating heart failure, for instance, by promoting remodeling through fibrosis, which might be accelerated by myofibroblast activation due to increased NOX4-derived ROS production in DMD hearts [4,11,13]. A safer alternative than long-term corticosteroids is vamorolone, which has been shown its efficacy in placebo-controlled double-blind trials in DMD [16], although cardiac outcomes were not included.

Several emerging therapeutic strategies focus on targeting the primary defect in DMD including the restoration of Dystrophin (Reviewed by [17]). Based on the DMD gene mutation type, stop codon read-through compounds, like gentamicin and ataluren (PTC124), have been tested to stimulate ribosomal read-through of premature stop codons (potentially applicable to all nonsense mutations, representing up to 10% of DMD patients). Ataluren has been approved by the EMA but has not yet shown clinical benefit. Another therapeutic approach for DMD is exon skipping therapy. This therapy originated from Becker muscular dystrophy (BMD; OMIM#300376), in which patients express a shorter isoform of Dystrophin (partially functional) and have a milder phenotype. Exon skipping targets mutated exons with predesigned antisense oligonucleotides (AONs) to produce a shorter but functional version (applicable for 55% of DMD patients). Among the AONs, four AONs have been approved in the USA (eteplirsen, casimersen, golodirsen, and viltolarsen) for treatment of subsets of DMD patients, but none demonstrated clinical efficacy in controlled clinical trials. Vector-mediated gene therapy is under clinical testing by multiple sponsors, where partly functional DMD gene (mini-/micro-Dystrophin) is delivered to the cells that lack the Dystrophin protein, but again no clinical benefit has been demonstrated. Utrophin (Utro), a structural paralogue of Dystrophin, might substitute the function of Dystrophin. Despite the encouraging results of using miniaturized Utrophin (μUtro) in preclinical animal models, nowadays no tests have been performed in DMD patients.

Recently, cell-based treatments with derivatives of induced pluripotent stem cells (iPSCs) are being tested in preclinical models. Results demonstrated that injected iPSC-derived myogenic progenitors are able to integrate into limb muscles and the heart of dystrophic mice, thereby ameliorating both skeletal and cardiac muscle function [18]. Finally, another emerging therapeutic approach, in preclinical setting, enabled the conversion of DMD-like to BMD-like mutations through CRISPR/Cas9-mediated gene editing and awaits testing in clinical trials.

Current treatments for DMD heart disease include eplerenone, a drug targeting the mineralocorticoid pathway, which has been demonstrated to benefit cardiac function in DMD patients. Interestingly, idebenone, a synthetic analogue of coenzyme Q₁₀ that maintains mitochondrial function and energetics, may be an important therapeutic strategy in the treatment of DMD-related heart failure since NOXs are critically involved in the mitochondrial electron transport chain (ETC)-derived ROS production. In a phase 3 clinical trial, idebenone showed beneficial effects on the respiratory function of previously used and glucocorticosteroid-naive DMD patients [19]. Recently, a similarly designed study was discontinued by the sponsor Santhera Pharmaceuticals because the primary endpoint, an increase in the percentage of predicted forced vital capacity (FVC) from baseline to 18 months of treatment, was not met [20]. However, an interesting therapeutic target to explore further are the ROS producing NOX family enzymes.

4. Expert opinion

Although the landscape of novel therapeutic strategies for noncardiac and cardiorespiratory failure in DMD is expanding, it is highly possible that not all DMD patients will respond to specific treatment options. This is due to an incomplete understanding of the complex nature of the disease etiology and the pathogenesis together with the variability in disease progression among patients. Growing knowledge of the pathologic signaling pathways continues to reveal new potential therapeutic targets, which may be relevant in certain DMD patient groups. Emerging data continue to implicate ROS and NOX family enzymes as central mediators in the pathogenesis of DMD and Duchenne cardiomyopathy. However, it is clear that controlled NOX4 production is crucial for cardiac homeostasis, since deletion of NOX4 in cardiomyocytes impairs heart function and NOX4 upregulation promotes adaptive cardiac remodeling in a disease setting [9].

In a recent work published by Duelen and colleagues [13], evidence was found of significantly increased NOX4 expression and ROS-producing activity in DMD patient-derived iPSC cardiomyocytes (iPSC-CMs), contributing to death of these cells (Figure 1). This elevated ROS production from hyperactive NOX4 in the mitochondria of DMD iPSC-CMs could be reduced by idebenone administration by stimulating ATP generation through the mitochondrial ETC, which in turn reduced NOX4-mediated ROS production by binding to an ATP-binding motif within NOX4. Whether the abnormally increased expression and rate of ROS production of NOX4 are a direct or indirect consequence of the absence of Dystrophin protein is currently unknown and needs further investigation. However, due to the dual mode of action of idebenone (detoxifying ROS by donating electrons to produce nontoxic reaction products, and donating electrons directly to the ETC of mitochondria, ultimately restoring ATP production), idebenone seems an interesting strategy to target both the excessive ROS production and dysfunctional mitochondria for the treatment of Duchenne cardiomyopathy.

Figure 1. Human induced pluripotent stem cells (hiPSCs) from Duchenne muscular dystrophy (DMD) patients were differentiated to cardiac myocytes to model dilated cardiomyopathy (DCM). Due to the absence of Dystrophin protein expression, cardiac myocytes are more vulnerable to contraction-induced ruptures of the cell membrane, leading to increased calcium (Ca²⁺) entry. Duelen and colleagues [13] demonstrated that hiPSC-derived cardiac myocytes (hiPSC-CMs) from patients with a genetic mutation in the DMD gene were subjected to significantly increased oxidative stress, partially as a result of increased expression and activity of ROS-producing NOX4 enzymes. Therefore, DMD hiPSC-CMs showed accelerated cell death. By administration of idebenone, beneficial effects were observed on the mitochondrial membrane potential, as well on the expression and the ROS-producing activity of NOX4, resulting in an increased cell survival and function of DMD iPSC-CMs. Abbreviations – DCM: dilated cardiomyopathy; DMD: Duchenne muscular dystrophy; hiPSCs: human induced pluripotent stem cells; NOX4: NADPH oxidase 4; ROS: reactive oxygen species. Created with BioRender.com.

Idebenone treatment on patients with previous steroid use or steroid-naive patients had a similar attenuated decline on peak expiratory flow [17]. Although here the cardiac function was not a primary endpoint, a beneficial effect could not be excluded on cardiomyocyte survival and function. Since the life-threatening complications of DMD patients are cardiorespiratory in nature, prolonging treatments that improve cardiac function could have a beneficial effect later on the respiratory outcomes, improving the patient quality of life.

Several questions remain unanswered regarding ROS-producing NOX family enzymes, and more specifically NOX4. The function and regulatory mechanism of NOX4 in various subcellular localizations is an important aspect to be clarified (Figure 1). Since the various roles of NOX4 may depend on its localization, location-specific regulation of NOX4 for the treatment of Duchenne cardiomyopathy may be required. Moreover, it is crucial to maintain the optimal ROS concentration in order to avoid excess oxidative stress or reductive stress. Therefore, it is likely important to optimize the ROS levels and selectively inhibit NOX4 in the mitochondria of cardiac myocytes. Given the controversies in the NOX4 research field, deep RNA sequencing of the heart transcriptome in biopsy heart samples from patients suffering heart failure has shown alternative splicing, giving rise to differential expression of NOX4 isoforms. Therefore, they may also contribute to the different DMD disease phenotypes, and represent an important factor to consider as drug candidates may have differential (unwanted) effects on the spliced variants [4]. In conclusion, therapeutic approaches targeting specific NOXs have not yet been established for heart failure in a clinical setting, but development and systemic screening of effective NOX inhibitors in validated in vitro iPSC-derived cellular disease models is now feasible and urgently needed.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions

Conception and design: Duelen, Janssens, Sampaolesi. Drafting the editorial article: Duelen. Critically revising the editorial article: Duelen, Janssens, Sampaolesi. Reviewed submitted version of the manuscript: Duelen, Janssens, Sampaolesi. Approved final version of the manuscript on behalf of all authors: Duelen. Article supervision: Sampaolesi.

Additional information

Funding

This manuscript was funded by the Research Foundation Flanders (FWO; #G066821N), Small Research Infrastructure KU Leuven – BioAssemblyBot 400 (KA/20/088), Interreg – Euregio Meuse-Rhine (GYM, Generate Your Muscle; 2020-EMR116), and the Italian Ministry of Health – Ricerca Finalizzata (RF-2019-12369703). RD was supported by KU Leuven Rondoufonds voor Duchenne Onderzoek (EQQ-FODUCH-O2010) and KU Leuven Postdoctoral Mandate Type 1 (PDMT1/21/037). MS is a recipient of the Hercules Foundation grant (AKUL/19/34).