Potential Therapeutic Targets for Hypotension in Duchenne Muscular Dystrophy

Abstract

Duchenne Muscular Dystrophy (DMD) is marked by genetic mutations occurring in the DMD gene, which is widely expressed in the cardiovascular system. In addition to developing cardiomyopathy, patients with DMD have been reported to be susceptible to the development of symptomatic hypotension, although the mechanisms are unclear. Analysis of single-cell RNA sequencing data has identified potassium voltage-gated channel subfamily Q member 5 (KCNQ5) and possibly ryanodine receptor 2 (RyR2) as potential candidate hypotension genes whose expression is significantly upregulated in the vascular smooth muscle cells of DMD mutant mice. We hypothesize that heightened KCNQ5 and RyR2 expression contributes to decreased arterial blood pressure in patients with DMD. Exploring pharmacological approaches to inhibit the KCNQ5 and RyR2 channels holds promise in managing the systemic hypotension observed in individuals with DMD. This avenue of investigation presents new prospects for improving clinical outcomes for these patients.

Graphical Abstract

Exploring Hypotension Treatment Targets in Duchenne Muscular Dystrophy (DMD): This study utilizes snRNA-seq analysis to pinpoint possible treatment targets for hypotension in DMD. We hypothesize that heightened KCNQ5 and RyR2 expression, in particular, contributes to decreased arterial blood pressure in DMD patients (Created in BioRender).

Introduction

Duchenne muscular dystrophy (DMD) arises due to a mutation in the DMD gene on the X chromosome, disrupting the production of the dystrophin protein(1). Dystrophin is crucial for maintaining the integrity of the glycoprotein complex during muscle contraction and relaxation. The impact of this condition is particularly pronounced in early childhood, progressively affecting the neuromuscular system(1). The consequences of DMD are far-reaching, affecting multiple tissues including musculoskeletal, respiratory, and cardiovascular functions. Over the course of the first three to four decades of life, patients endure persistent muscle degeneration, leading to profound weakness(1). Additionally, respiratory complications arise, further impairing their quality of life. Notably, mortality risks are significantly heightened by the combination of musculoskeletal and cardiovascular issues. Dilated cardiomyopathy, arrhythmias, and congestive heart failure are the most prevalent cardiac manifestations of DMD(1, 2). Tragically, these heart-related issues are the primary drivers of mortality in individuals with DMD(3).

Dystrophin is not only confined to skeletal muscle and heart but also is expressed in vascular smooth muscle cells (VSMCs) and non-VSMC. Petkova et al. (4) developed a DMD (eGFP) reporter mouse, which incorporated the eGFP coding sequence after the final DMD exon and confirmed DMD transcription in smooth muscle cells. Shen et al.(5) further validated the presence of dystrophin protein in VSMCs through immunofluorescent staining.

Numerous instances in medical literature have pointed to a link between dystrophin and changes in blood pressure among individuals affected by DMD. A study conducted by Ryan et al. (6) delved into this connection by examining 43 patients with DMD. The researchers discovered a notable reduction in central systolic blood pressure when compared to a control group of healthy individuals. However, there is no difference in central diastolic blood pressure between DMD patients and control patients. In another investigation by Masood S.A. et al. (7) at Rush University’s cardiology clinic, the goal was on the occurrence of unusually low resting blood pressure in DMD patients. This study unveiled a significant proportion of DMD patients with diminished resting blood pressure, with predictors of hypotension being pediatric age group and Hispanic ethnicity in the stepwise multiple regression analysis. Interestingly, the study also found that the use of heart failure therapy did not correlate with low blood pressure in this patient group. This suggests a critical requirement for healthcare professionals to be more vigilant about monitoring blood pressure in individuals with DMD. The renin angiotensin aldosterone system (RAAS) is well-known for its role in regulating blood pressure and fluid balance. In the context of DMD and Becker Muscular Dystrophy, studies have shown that RAAS hyperactivity can exacerbate myocardial fibrosis, which is a consequence of dystrophin deficiency. This understanding has led to the use of ACE inhibitors, angiotensin receptor blockers, or aldosterone antagonists in treating cardiomyopathy in DMD and BMD patients(8). Importantly, there has been a case reported by Li J. et al. (9) involving a 15-year-old male with DMD who experienced severe hypotension after receiving sacubitril/valsartan, an angiotensin-receptor/neprilysin inhibitor. This case indicates that individuals afflicted with DMD exhibit heightened sensitivity to antihypertensive medications. It also underlines the complexity of managing blood pressure in DMD patients and the need for cautious therapeutic approaches.

Supporting these clinical findings, research involving Dmd^mdx mouse (referred to as mdx), which serves as a model for Duchenne muscular dystrophy (10), revealed that these mice demonstrated lower systolic, diastolic, and mean blood pressure compared to their healthy counterparts (5). These combined clinical and experimental results point to the existence of a hypotensive trait associated with DMD, possibly attributed to disruptions in dystrophin expression within VSMCs.

Single-cell RNA sequencing (scRNA-seq) is an advanced technique that enables the analysis of RNA sequences at the individual cell level (11, 12). Unlike conventional bulk RNA sequencing, scRNA-seq can uncover cellular heterogeneity that is obscured in aggregate sample measurements (13). By precisely identifying which cells express specific genes, scRNA-seq offers deeper insights into gene transcriptional profile at the cellular level (5, 14).

The underlying molecular mechanisms connecting DMD mutations to hypotension are yet to be fully understood. In the pursuit of identifying gene networks influenced by DMD in VSMCs, Shen et al.(5) analyzed a publically available snRNA-seq dataset to systematically compare gene expression profiles in VSMCs from DMD^mut and control mice. This approach identified a number of differentially expressed genes (DEGs), with noteworthy emphasis on the upregulation of potassium voltage-gated channel subfamily Q member 5 (KCNQ5) and ryanodine receptor 2 (RyR2).

KCNQ5, a member of the KCNQ gene family, predominantly generates M-type currents and plays a regulatory role in the voltage-gated K+ channel KV7.5. KCNQ genes encode transmembrane segments, including the pore module S5–6 and the voltage-sensing domain VSD, S1–4, which assemble into functional potassium channels (15). In VSMCs, KCNQ5 expression is pivotal for maintaining normal vascular tone regulation (16, 17). KCNQ5 interacts with KCNQ4 to modulate arterial tone at rest, and its inhibition leads to vasoconstriction (18). Notably, KCNQ5 has been implicated as a hypertension-associated gene in spontaneously hypertensive rat mesenteric arteries (19). Selective activation of KCNQ5 has been demonstrated to lower blood pressure through vasodilation, achieved by compounds such as celecoxib and aloperine (20–22).

Conversely, RyR2 contributes to L-type Ca (2+) currents, orchestrating calcium release and signaling(23, 24). This receptor also plays a significant role in systemic blood pressure control and vascular adaptations to pressure changes (25). Pritchard et al. (26) examined the effects of DMD mutations on VSMC contractility. Their findings revealed augmented spontaneous and transient Ca²⁺-release events via SR RyR2s (“Ca2+ sparks”) in VSMCs of DMD^mut mice. This elevated activity correlated with increased Ca²⁺-dependent BK channel activity, indicating that larger RyR2 protein clusters in DMD^mut mice lead to heightened Ca²⁺ sparks and BK channel functionality, thereby contributing to cerebral microvascular dysfunction. They also uncovered how DMD mutations impede spontaneous myogenic tone in cerebral pial arteries and parenchymal arterioles by enhancing RyR2 and BK channel activity, consequently reducing pressure-induced arterial constriction. These findings collectively suggest that the upregulation of KCNQ5 and RyR2 genes in DMD mutant VSMCs may be pivotal in causing hypotension.

Dantrolene is a prototypical RyR2 inhibitor, thereby reducing calcium leakage through RyR2 and elevating the threshold for spontaneous calcium release (27). Dantrolene enhanced mean arterial blood pressure in chronic canine models of propofol-induced hypotension(28) and increased arterial blood pressure in chloralose-anesthetized swine (29). Moreover, dantrolene elevated LV diastolic pressure in mineralocorticoid-salt-induced hypertensive rats (30). Nevertheless, the effect of dantrolene on blood pressure regulation remains contentious. Some studies have demonstrated no influence on blood pressure or even blood pressure-lowering effects. For instance, dantrolene alone failed to alter blood pressure in age-matched sham rats(31), but when combined with nimodipine, it significantly lowered blood pressure in rat models (31). Moreover, dantrolene reduced systolic blood pressure in patients with cerebral vasospasm after a subarachnoid hemorrhage while inhibiting cerebral vasoconstriction(32). Like other pharmacological agents, dantrolene may have off-target effects that could potentially contribute to discrepant results.

The potential of other RyR2 inhibitors to alleviate hypotension remains largely unexplored. Compound Ro 90–7501, for instance, inhibits RyR2 channels by enhancing intracellular binding between calmodulin (CaM) and the ryanodine receptor RyR2(33) via a serine phosphorylation-dependent mechanism(34). However, research on Compound Ro 90–7501’s capacity to raise blood pressure is limited.

While RyR2 inhibition is a theoretically attractive approach to treating hypotension in DMD patients, much more preclinical research is required to assess its potential efficacy and tolerability. Until such research is conducted, advancing RyR2 inhibition toward translational research in patients with DMD and hypotension is not feasible.

By amalgamating insights garnered from snRNA-seq analysis and the clinical literature, a compelling rationale emerges for devising a targeted strategy to mitigate hypotension in DMD patients via pharmacological inhibition of KCNQ5.

In Table 1, we present a comparative analysis of linopirdine, XE9991, vasopressin, and diclofenac – compounds that inhibit KCNQ5 in blood pressure regulation. This analysis includes inhibition efficiency, dosage, delivery routes, and limitations of each compound. It is worth mentioning that the most chemical inhibitors carry the potential for off-target effects. Currently, there is no available research paper that offers a comparison of the specificity of linopirdine, XE991, Vasopressin, and Diclofenac in inhibiting KCNQ5. Therefore, it would be more prudent to utilize a siRNA-based gene silencing approach to validate the observed phenotypes.

Table 1:

Comparative Analysis of KCNQ5 Inhibitors in Blood Pressure Regulation.

	Inhibition efficiency	Effect on blood pressure	Delivery Route	Limitation
Linopirdine	Approximately 65% inhibition with a concentration of 300 nM (35)	10μM counteracted kynurenine-induced hypotension in adult rats (36) Improved arterial blood pressure in adult male rats (37) Improved MAP, SBP, and DBP in combination with normal saline (NS) in a lung ischemia-reperfusion injury model (38) Restored MAP in male rats after hemorrhagic shock (39)	Administered intravenously. (36, 37, 40)	Did not improve left ventricular developed pressure (LVDP) as effectively as XE991 (41)
XE991	Sensitive to inhibition with concentrations over 3 μM (42)	Perfusion of XE991 raised the arterial baroreceptor threshold in young rats (43) Increased MAP in adult male rats. Improved MAP in anesthetized rats with acute myocardial infarction (AMI) (44) Improved recovery of LVDP during reperfusion and reduced postischemic left ventricular end-diastolic pressure in adult rats (41) Same dosage (10 μM) as linopirdine results in greater inhibition of channels (45)	Administered systemically (44)	Less effective than linopirdine in restoring MAP in male rats after hemorrhagic shock at equivalent dose of 1 mg/kg (39)
Vasopressin	Around 74% inhibition with a concentration of 100 μM after 10 minutes of exposure between −44 and −14 mV voltage range (46)	Improved the MAP in hypotensive, low birth weight infants at concentrations of approximately 0.01 to 0.04 units/kg/h (47) Improved blood pressure in ACEi-induced hypotensive patient with a loading dose of 1 unit (48) Increased mean, systolic, and diastolic blood pressure in infants with hypertrophic obstructive cardiomyopathy (HOCM) (49) Well-established research on infants (47, 49)	Administered intravenously (49)	Ineffective at treating dilated cardiomyopathy (DCM), one of the major cardiac pathologies in DMD patients (50) Lacks specificity for KCNQ5
Diclofenac	Approximately 53% inhibition at a concentration of 100 μM (51)	Increased blood pressure in hypertensive male and female rats (52) Elevated blood pressure in elderly hypertensive patients by 4.1 mmHg (53) Increased mean blood pressure in adults undergoing elective coronary artery bypass grafting (CABG) (54)	Administered subcutaneously (52)	Enhanced channel output at negative voltages (35) It is more effective in the elderly than in younger children (35, 53) Lacks specificity for KCNQ5

Hypothesis

By inhibiting the KCNQ5 and RyR2 channels, we can potentially treat the hypotension observed in individuals with DMD, thus alleviating associated symptoms of weakness, dizziness, etc., and potentially improving shock in critically ill patients.

Testing of the Hypotheses

Our hypothesis introduces an innovative therapeutic paradigm with regard to DMD-associated hypotension. A comprehensive testing approach involving in vitro, ex vivo, and in vivo experiments will be required to test this hypothesis.

To confirm the inhibitors’ KCNQ5 channel blockade, we will use cell patch-clamping assay, we could consider using a mouse VSMC line like Movas. To target the DMD gene, we will use siRNA to knock it down, and for control purposes, employ scramble siRNA. Alternatively, we have the option to isolate primary VSMCs from both DMD mutant mice (e.g., Mdx mice) and control mice. However, it’s important to note that the dedifferentiation of primary VSMCs from the contractile to the synthetic phenotype can be a challenging aspect of this approach. There are several references that have conducted patch clamping experiments on smooth muscle cells. For instance, Quinn K et al(55) detailed a method for direct patch-clamp recording from smooth muscle cells embedded in functional brain microvessels, which involves several key steps: (1) Isolation of small blood vessels from the rabbit brain using enzymatic and mechanical procedures, followed by the identification of arterioles under a microscope; (2) Establishing patch-clamp seals on smooth muscle cells within arterioles, recording membrane potentials using patch pipettes containing amphotericin-B, and voltage-clamping short arteriolar segments; (3) Inducing constriction through the injection of depolarizing current and conducting cell-attached patch, inside-out patch, and outside-out patch recordings to study K+ channel unitary currents. This method offers the advantage of avoiding single-cell isolation, minimizing enzymatic treatment, and allowing the study of cells within their natural environment. We expect the patch-clamping experiments can confirm the elevation of KCNQ5 activity in DMD^mut mice and demonstrate the efficacy of KCNQ5 inhibitors in VSMC of these mice, and establish specificity (i.e., lack of effect of the inhibitors on other potassium channels).

Ex vivo wire myography will next be utilized to investigate the contractile responses of the isolated arterioles under isometric conditions (56). This entails isolating an arteriole segment and affixing it within a myograph chamber (57), whereby contractile and relaxation responses can be measured (58). Such experiments will provide comparative data on maximal contractile responses to depolarizing concentrations of KCl and dose-dependent constriction to agonists such as phenylephrine and thromboxane mimetics in blood vessels from DMD^mut and control mice. Importantly, these experiments will establish whether the KCNQ5 inhibitors can increase baseline vasoconstriction and/or potentiate vasoconstrictor responses to agonist compounds in blood vessels from DMD^mut mice.

Next, we will assess blood pressure responses in DMD^mut and control mice. Arterial blood pressure measurement will be accomplished through both non-invasive and invasive techniques, such as tail-cuff methods or arterial catheters. Notably, cuff-less approaches like implanted telemetric transducers yield more precise blood pressure readings by minimizing the stress linked with traditional cuff-based methods (44). Blood pressure can be assessed at baseline and after perturbations known to cause hypotension, such as volume depletion and administration of sub-lethal doses of endotoxin. The impact of the KCNQ5 inhibitors on hypotension severity and subsequent organ dysfunction can be established. We would also need to conduct safety studies in the mice to ensure tolerability and exclude serious adverse effects.

Significance and Outcomes

Investigating hypotension in DMD is vital due to its complex link with cardiovascular issues. While conditions such as dilated cardiomyopathy, arrhythmias, and congestive heart failure are prevalent in DMD, the concern for hypotension in these patients is increasingly recognized. Existing research primarily focuses on the heart and respiratory complications due to muscle degeneration in DMD, often neglecting the significant role played by VSMCs. These cells, expressing the dystrophin protein crucial for blood pressure control, are a key aspect often missed in DMD studies. The literature consistently demonstrates a correlation between dystrophin levels and blood pressure variations in DMD patients. This highlights the need for cautious prescription and management of common cardiac treatments like ACE inhibitors and angiotensin receptor blockers in DMD and BMD patients. The use of standard anti-hypotensive drugs in DMD-related hypotension may increase blood pressure, but it might not be the most effective approach. This is partly due to the unique pathophysiology and molecular mechanisms involved in DMD-associated hypotension. This study’s importance lies in its aim to bridge the research gap in understanding blood pressure regulation in DMD. By doing so, it seeks to enhance patient care and evolve treatment methodologies for those affected by this challenging condition. To solve this critical issue, we propose employing advanced single-cell technologies to pinpoint potential genetic targets. This approach aims to develop more tailored hypotheses for future testing, representing a different strategy towards the same objective of effectively managing hypotension in DMD patients.

Establishing that KCNQ5 channel inhibitors effectively and safely elevate arterial blood pressure in DMD^mut mice would support their evaluation in patients with DMD. If our results are successfully translated, they could have important implications for the acute management of DMD patients who are critically ill due to sepsis, hypovolemic shock, etc. Moreover, chronic KCNQ5 channel inhibition may mitigate symptomatic orthostatic hypotension in selected patients with DMD. It is important to point out that patients with DMD often develop cardiomyopathy and increases in systemic vascular resistance induced by KCNQ5 channel inhibitors could potentially worsen left ventricular systolic function. Thus, patients would need to be monitored closely for signs and symptoms of heart failure.

There are potential risks associated with targeting the KCNQ5 gene. It has been observed that de novo missense variants in KCNQ5 can lead to developmental and epileptic encephalopathy (DEE) or intellectual disability (ID)(59). Additionally, variants in KCNQ2, KCNQ3, and KCNQ5 that affect their activity, predominantly causing loss of function but occasionally gain of function, have been linked to a spectrum of electrical disorders. These range from benign familial neonatal convulsions to more severe conditions, including epileptic encephalopathy with intellectual disability(60). Furthermore, haploinsufficiency of KCNQ5 has been associated with severe intellectual disability and epileptic encephalopathy(61). We need to differentiate the side effects of KCNQ5 inhibitor from intellectual impairment, a recognized aspect of DMD. Mutations in the dystrophin gene, which are central to DMD, have been linked to impaired intellectual functioning in patients(62). Approximately 20% of boys with DMD have an IQ below 70, and a more pronounced intellectual impairment is often seen in those with mutations affecting the brain-specific dystrophin isoform(63). Furthermore, it’s notable that many patients with congenital muscular dystrophies (CMD) exhibit mental retardation and brain pathology. About 30% of DMD patients are affected by intellectual impairment(64). This context emphasizes the broader impact of DMD beyond its physical manifestations.

Whether inhibition of RyR2 channels might serve as an alternative approach to treating hypotension in patients with DMD remains unclear and may merit further investigation. As mentioned above, patients with DMD often develop dilated cardiomyopathy and heart failure, with an associated increased risk of ventricular arrhythmias (65), which can escalate to sudden cardiac death (SCD) (66). In this context, while pharmacological RyR2 channel inhibition might not significantly elevate blood pressure, it may offer a preventive measure against ventricular arrhythmias (67). For instance, inhibition of RyR2 channels with dantrolene (33) effectively suppressed arrhythmic episodes in an ex vivo study involving isolated mouse hearts (68) (69), underscoring its potential as a therapeutic agent for managing ventricular arrhythmias in DMD patients.

Implications

scRNA-seq allows for the detailed examination of cellular responses at the individual cell level, such as identifying significant changes in gene expression profiles within specific cell types like VSMCs during disease conditions. This approach facilitates the comparison of gene expression in individual cells across different populations in healthy and diseased conditions, unveiling genes that are distinctly altered due to the disease. Identifying these dysregulated genes opens the door to potential therapeutic interventions. However, a major challenge lies in selecting the critical genes from many genes identified by scRNA-seq as differentially expressed between normal and diseased cell populations. This selection process is vital for identifying biomarkers and therapeutic targets. Integrating known gene functions with specific symptoms observed in diseased cell populations can help us filter potential candidate genes. Prioritizing these genes requires evaluating their relevance and known roles in disease mechanisms or their direct impact on disease phenotypes. Subsequent experimental validation, such as gene knockdown by siRNAs or overexpression studies, is essential to confirm the significance of these candidates. By targeting these genes or their associated pathways, we can explore novel therapeutic interventions. Additionally, scRNA-seq’s capacity to profile gene expression at the single-cell level in diseased tissues heralds a new era of personalized medicine. It enables the identification of unique biomarkers linked to various cell types, allowing for customized treatment based on the molecular profile of an individual’s disease.

Conclusions

Our study, incorporating insights from clinical reports and single-cell RNA sequencing analysis, hypothesizes that targeting the KCNQ5 and RyR2 channels could be a viable strategy for alleviating symptomatic hypotension in patients with Duchenne Muscular Dystrophy.

Highlights:

1. There is a link between genetic mutations in the DMD gene and the development of symptomatic hypotension in patients with DMD.

2. The findings from single-cell RNA sequencing data identified two genes, KCNQ5 and RyR2, as potential candidates influencing hypotension in DMD due to their upregulated expression in VSMC of DMD mutant mice.

3. Therapeutic Hypothesis: Inhibiting the KCNQ5 and RyR2 channels could potentially treat hypotension observed in DMD patients.

4. Pharmacological approaches to inhibit KCNQ5 and RyR2 channels: the potential of various compounds, their efficiency, dosage, and limitations in blood pressure regulation.

Abbreviations:

snRNA-seq

single-nuclear RNA-sequencing

VSMC

Vascular smooth muscle cells

KCNQ5

Potassium Voltage-Gated Channel Subfamily Q Member 5

RyR2

Ryanodine Receptor 2