Ryanodine receptor 2 inhibition reduces dispersion of cardiac repolarization, improves contractile function, and prevents sudden arrhythmic death in failing hearts
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eLife assessment
This important study examined the use of dantrolene, a Ryanodine Receptor stabilizer, in slowing pathological progression of pressure-overload heart failure in a guinea pig model and reducing arrhythmias. Convincing data were collected and analyzed using validated methodology and can be used as a starting point for future studies of dantrolene in Ca2+ handling in ROS production and further deterioration of cardiac function in chronic heart failure.
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Abstract
Sudden cardiac death (SCD) from ventricular tachycardia/fibrillation (VT/VF) is a leading cause of death, but current therapies are limited. Despite extensive research on drugs targeting sarcolemmal ion channels, none have proven sufficiently effective for preventing SCD. Sarcoplasmic ryanodine receptor 2 (RyR2) Ca 2+ release channels, the downstream effectors of sarcolemmal ion channels, are underexplored in this context. Recent evidence implicates reactive oxygen species (ROS)-mediated oxidation and hyperactivity of RyR2s in the pathophysiology of SCD. We tested the hypothesis that RyR2 inhibition of failing arrhythmogenic hearts reduces sarcoplasmic Ca 2+ leak and repolarization lability, mitigates VT/VF/SCD and improves contractile function. We used a guinea pig model that replicates key clinical aspects of human nonischemic HF, such as a prolonged QT interval, a high prevalence of spontaneous arrhythmic SCD, and profound Ca 2+ leak via a hyperactive RyR2. HF animals were randomized to receive dantrolene (DS) or placebo in early or chronic HF. We assessed the incidence of VT/VF and SCD (primary outcome), ECG heart rate and QT variability, echocardiographic left ventricular (LV) structure and function, immunohistochemical LV fibrosis, and sarcoplasmic RyR2 oxidation. DS treatment prevented VT/VF and SCD by decreasing dispersion of repolarization and ventricular arrhythmias. Compared to placebo, DS lowered resting heart rate, preserved chronotropic competency during transient β-adrenergic challenge, and improved heart rate variability and cardiac function. Inhibition of RyR2 hyperactivity with dantrolene mitigates the vicious cycle of sarcoplasmic Ca 2+ leak-induced increases in diastolic Ca 2+ and ROS-mediated RyR2 oxidation, thereby reducing repolarization lability and protecting against VT/VF/SCD. Moreover, the consequent increase in sarcoplasmic Ca 2+ load improves contractile function. These potentially life-saving effects of RyR2 inhibition warrant further investigation, such as clinical studies of repurposing dantrolene as a potential new therapy for heart failure and/or SCD.
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Author Response
The following is the authors’ response to the original reviews.
We thank the reviewers for their positive feedback and very helpful comments. We agree that this manuscript focuses primarily on functional outcomes and phenotypes. The studies were designed to address an important clinical question, i.e., repurposing dantrolene for the treatment of ventricular tachyarrhythmias and the prevention of sudden cardiac arrest. Thus, the current manuscript emphasizes in vivo studies over in vitro studies.
However, we also acknowledge the need for additional mechanistic studies. We are in the final stages of submitting a second manuscript in which we dissect the underlying mechanisms through detailed in vitro studies of mitochondrial antioxidant capacity, reactive oxygen species, phosphorylation of ryanodine receptors, autonomic …
Author Response
The following is the authors’ response to the original reviews.
We thank the reviewers for their positive feedback and very helpful comments. We agree that this manuscript focuses primarily on functional outcomes and phenotypes. The studies were designed to address an important clinical question, i.e., repurposing dantrolene for the treatment of ventricular tachyarrhythmias and the prevention of sudden cardiac arrest. Thus, the current manuscript emphasizes in vivo studies over in vitro studies.
However, we also acknowledge the need for additional mechanistic studies. We are in the final stages of submitting a second manuscript in which we dissect the underlying mechanisms through detailed in vitro studies of mitochondrial antioxidant capacity, reactive oxygen species, phosphorylation of ryanodine receptors, autonomic dysfunction, beta-adrenergic signaling, etc. that are beyond the scope of the current manuscript.
Additionally, a third manuscript in progress focuses on the mechanistic link between ion channels, dispersion of repolarization, and sudden cardiac death. We previously reported the preliminary results in abstract form (Circulation Research. 2019;125:A102). Briefly, current-voltage relationships from patch clamp studies of isolated LV myocytes revealed that pressure-overload stress strongly reduced K currents, including IK1, IKs, and IKr. These changes were driven by downregulation of K channels and their components at the mRNA level. As expected, the reduced K currents destabilized the resting membrane potential, especially in phases II and II of the cardiac action potential, and reduced repolarization reserve. Scavenging mitochondrial ROS stabilized repolarization, suggesting mROS is the upstream driver of K channel downregulation. However, we have not specifically tested whether dantrolene stabilizes repolarization via the same mechanism. As such, we agree that "lability" or "dispersion" are more precise terms than "reserve" for the phenomenon reported in the present manuscript, and we have made these changes. Thank you for pointing this out. We have also changed the title accordingly.
The present study investigates the effect of dantrolene on male animals. We agree that we need to evaluate the effect on females, especially because females have historically been underrepresented in studies of sudden cardiac arrest. Based on our preliminary studies, female animals exhibit increased variability in their phenotypic response to pressure-overloaded stress. Given the importance of this issue, we will examine the sex differences in carefully controlled future experiments, including the effect of dantrolene in females controlled for hormonal effects (e.g., with and without oophorectomy).
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eLife assessment
This important study examined the use of dantrolene, a Ryanodine Receptor stabilizer, in slowing pathological progression of pressure-overload heart failure in a guinea pig model and reducing arrhythmias. Convincing data were collected and analyzed using validated methodology and can be used as a starting point for future studies of dantrolene in Ca2+ handling in ROS production and further deterioration of cardiac function in chronic heart failure.
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Reviewer #1 (Public Review):
The current study tests the hypothesis that inhibition of ryanodine receptor 2 (RyR2) in failing arrhythmogenic hearts reduces sarcoplasmic Ca leak, ventricular arrhythmias and improves contractile function. A guinea pig model of nonischemic heart failure (HF) was used and randomized to receive dantrolene (DS) or placebo in early or chronic HF. The authors show that DS treatment prevented ventricular arrhythmias and sudden cardiac death by decreasing dispersion of repolarization. The authors conclude that inhibition of RyR2 hyperactivity with DS mitigates the vicious cycle of sarcoplasmic Ca leak-induced increases in diastolic Ca and reactive oxygen species-mediated RyR2 oxidation. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function.
In general, the study is well …
Reviewer #1 (Public Review):
The current study tests the hypothesis that inhibition of ryanodine receptor 2 (RyR2) in failing arrhythmogenic hearts reduces sarcoplasmic Ca leak, ventricular arrhythmias and improves contractile function. A guinea pig model of nonischemic heart failure (HF) was used and randomized to receive dantrolene (DS) or placebo in early or chronic HF. The authors show that DS treatment prevented ventricular arrhythmias and sudden cardiac death by decreasing dispersion of repolarization. The authors conclude that inhibition of RyR2 hyperactivity with DS mitigates the vicious cycle of sarcoplasmic Ca leak-induced increases in diastolic Ca and reactive oxygen species-mediated RyR2 oxidation. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function.
In general, the study is well designed and the findings are likely to be of interest to the field.
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Reviewer #2 (Public Review):
Joshi et al. investigated the use of dantrolene, an RyR stabilizing drug, in improving contractile function and slowing pathological progression of pressure-overload heart failure. In a guinea pig model, they found that dantrolene treatment reduced cytosolic Ca2+ levels, improved contractility, reduced the incidence of arrhythmias, reduced fibrosis, and slowed the progression of heart failure. Importantly, delaying treatment until 3 weeks after aortic banding (when heart failure was already established) also resulted in improvements in function and decreased arrhythmogenesis. While some of the mechanistic details remain to be worked out, the data suggest that improving intracellular Ca2+ handling can break the vicious cycle of sympathetic activation, ROS production, and further deterioration of cardiac …
Reviewer #2 (Public Review):
Joshi et al. investigated the use of dantrolene, an RyR stabilizing drug, in improving contractile function and slowing pathological progression of pressure-overload heart failure. In a guinea pig model, they found that dantrolene treatment reduced cytosolic Ca2+ levels, improved contractility, reduced the incidence of arrhythmias, reduced fibrosis, and slowed the progression of heart failure. Importantly, delaying treatment until 3 weeks after aortic banding (when heart failure was already established) also resulted in improvements in function and decreased arrhythmogenesis. While some of the mechanistic details remain to be worked out, the data suggest that improving intracellular Ca2+ handling can break the vicious cycle of sympathetic activation, ROS production, and further deterioration of cardiac function.
The functional ECG and echo data are convincing, and very clearly demonstrate the positive effects of dantrolene in heart failure. This is important because dantrolene is already FDA-approved to treat malignant hyperthermia and muscle spasms, so repurposing this drug as a heart failure therapeutic might have a straightforward path to clinical implementation. This also highlights the non-specific nature of dantrolene to interact with RyR1, and therefore, potential side effects. However, this does not detract from the main proof-of-concept demonstrated here.
The guinea pig model employed here is also a strength, as the guinea pig has intracellular Ca2+ handling and ionic currents that are much more similar to human (vs. a murine model, for example).
One weakness is the exclusion of female animals from the study. The authors report more heterogeneity in the progression of HF in the female guinea pig model, however it will be very important to determine effects of dantrolene in the female heart, as there are considerable known sex differences in intracellular Ca2+ handling and contractility. Therefore, it is possible that dantrolene could have sex-dependent effects.
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eLife assessment
This valuable study examined the use of dantrolene, a Ryanodine Receptor stabilizer, in slowing pathological progression of pressure-overload heart failure in a guinea pig model and reducing arrhythmias. The data were collected and analyzed using solid and validated methodology and can be used as a starting point for future studies of dantrolene in Ca2+ handling in ROS production and further deterioration of cardiac function in chronic heart failure.
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Reviewer #1 (Public Review):
The current study tests the hypothesis that inhibition of ryanodine receptor 2 (RyR2) in failing arrhythmogenic hearts reduces sarcoplasmic Ca leak, ventricular arrhythmias and improves contractile function. A guinea pig model of nonischemic heart failure (HF) was used and randomized to receive dantrolene (DS) or placebo in early or chronic HF. The authors show that DS treatment prevented ventricular arrhythmias and sudden cardiac death by decreasing dispersion of repolarization. The authors conclude that inhibition of RyR2 hyperactivity with DS mitigates the vicious cycle of sarcoplasmic Ca leak-induced increases in diastolic Ca and reactive oxygen species-mediated RyR2 oxidation. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function.
In general, the study is well …
Reviewer #1 (Public Review):
The current study tests the hypothesis that inhibition of ryanodine receptor 2 (RyR2) in failing arrhythmogenic hearts reduces sarcoplasmic Ca leak, ventricular arrhythmias and improves contractile function. A guinea pig model of nonischemic heart failure (HF) was used and randomized to receive dantrolene (DS) or placebo in early or chronic HF. The authors show that DS treatment prevented ventricular arrhythmias and sudden cardiac death by decreasing dispersion of repolarization. The authors conclude that inhibition of RyR2 hyperactivity with DS mitigates the vicious cycle of sarcoplasmic Ca leak-induced increases in diastolic Ca and reactive oxygen species-mediated RyR2 oxidation. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function.
In general, the study is well designed and the findings are likely to be of interest to the field. The characterization of the phenotypes is comprehensive, however, the study appears relatively weak in terms of the proposed mechanisms. Only in vivo functional analyses were presented with no in vitro analyses. The rationale for only using the male animals remains unclear. Data presented in Supplemental Figure 1 lacks the HF with DS group. As presented, the manuscript appears relatively descriptive in nature.
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Reviewer #2 (Public Review):
Joshi et al. investigated the use of dantrolene, an RyR stabilizing drug, in improving contractile function and slowing pathological progression of pressure-overload heart failure. In a guinea pig model, they found that dantrolene treatment reduced cytosolic Ca2+ levels, improved contractility, reduced the incidence of arrhythmias, reduced fibrosis, and slowed the progression of heart failure. Importantly, delaying treatment until 3 weeks after aortic banding (when heart failure was already established) also resulted in improvements in function and decreased arrhythmogenesis. While some of the mechanistic details remain to be worked out, the data suggest that improving intracellular Ca2+ handling can break the vicious cycle of sympathetic activation, ROS production, and further deterioration of cardiac …
Reviewer #2 (Public Review):
Joshi et al. investigated the use of dantrolene, an RyR stabilizing drug, in improving contractile function and slowing pathological progression of pressure-overload heart failure. In a guinea pig model, they found that dantrolene treatment reduced cytosolic Ca2+ levels, improved contractility, reduced the incidence of arrhythmias, reduced fibrosis, and slowed the progression of heart failure. Importantly, delaying treatment until 3 weeks after aortic banding (when heart failure was already established) also resulted in improvements in function and decreased arrhythmogenesis. While some of the mechanistic details remain to be worked out, the data suggest that improving intracellular Ca2+ handling can break the vicious cycle of sympathetic activation, ROS production, and further deterioration of cardiac function.
The functional ECG and echo data are convincing, and very clearly demonstrate the positive effects of dantrolene in heart failure. This is important because dantrolene is already FDA-approved to treat malignant hyperthermia and muscle spasms, so repurposing this drug as a heart failure therapeutic might have a straightforward path to clinical implementation. This also highlights the non-specific nature of dantrolene to interact with RyR1, and therefore, potential side effects. However, this does not detract from the main proof-of-concept demonstrated here.
The guinea pig model employed here is also a strength, as the guinea pig has intracellular Ca2+ handling and ionic currents that are much more similar to human (vs. a murine model, for example).
One weakness is the exclusion of female animals from the study. The authors report more heterogeneity in the progression of HF in the female guinea pig model, however it will be very important to determine effects of dantrolene in the female heart, as there are considerable known sex differences in intracellular Ca2+ handling and contractility. Therefore, it is possible that dantrolene could have sex-dependent effects.
The title and parts of the discussion of the manuscript focus on 'repolarization reserve'. This term is often used in the realm of safety pharmacology, and 'reserve' refers to the fact that blocking a single K+ channel (for example) may not impact action potential duration because there may be enough other K+ currents to ensure proper repolarization. The repolarization reserve refers to this overall balance of depolarizing and repolarizing currents and potential redundancies to ensure proper repolarization. Although the present study clearly demonstrates QT shortening with dantrolene (thus, there must be a change in the balance of depolarizing and repolarizing currents), the study does not definitively demonstrate changes in any membrane currents. While this may seem like a minor point of terminology, it may mislead readers as to the main focus of the study, which is not at all on ionic currents, but on functional outcomes.
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