A Randomized Controlled Trial of RyR2 Inhibition With Dantrolene and Susceptibility to Ventricular Arrhythmias in Patients With Structural Heart Disease.
Phase: PHASE2
Conditions: Ventricular Tachycardia
Sponsor: Vanderbilt University Medical Center

 
 
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A randomized controlled trial of RyR2 
inhibition with dantrolene and susceptibility to 
ventricular arrhythmias in patients with 
structural heart disease 
 
 
 
Principal Investigator: 
 
William G. Stevenson, MD 
Professor of Medicine 
Arrhythmia Section 
Division of Cardiovascular Medicine 
Vanderbilt University Medical Center 
 
 
Co-Principal Investigator: 
 
M. Benjamin Shoemaker, MD, MSCI 
Assistant Professor of Medicine 
Division of Cardiovascular Medicine, Arrhythmia Section 
Vanderbilt University Medical Center 
 
 
 
 

 
 
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Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 
Statistical Considerations 
11.0 
Privacy/Confidentiality Issues 
12.0 
Follow-up and Record Retention 
 
Appendices 
Appendix A Study Procedure Calendar 

 
 
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1.0 
BACKGROUND 
 
1.1 
Structural 
heart 
disease 
causes 
calcium leak from the cardiac ryanodine 
receptor 2 channel (RyR2) 
This is a randomized controlled trial that aims to 
study whether RyR2 inhibition with dantrolene 
reduces susceptibility to ventricular arrhythmias 
and improves hemodynamics in patients with 
structural heart disease.  Figure 1 is the 
concept diagram of our proposed trial. The idea 
to be tested is that structural heart disease 
results in post-translational modifications to 
RyR2, which is the intracellular Ca channel, 
causing Ca to leave the sarcoplasmic reticulum 
and raising the concentration of intracellular Ca. 
This causes arrhythmias through delayed after 
depolarizations 
(DADs) 
that 
can 
trigger 
ventricular tachycardia (VT) and ventricular 
fibrillation (VF) and also are believed to contribute to changes in refractoriness and conduction that promote 
scar-related reentrant VT. Intracellular Ca overload also reduces cardiac Ca cycling which reduces cardiac 
contractility. Both conditions promote VT and VF which are the major causes of sudden cardiac death (SCD) in 
patients with structural heart disease.  
 
1.2 
Ventricular arrhythmias due to structural heart 
disease are a major cause of sudden cardiac death 
An estimated 150,000 to 450,000 individuals suffer SCD per 
year in the U.S, and approximately 90% are estimated to 
have underlying coronary artery and/or structural heart 
disease (Figure 2).1-4 Recent reports estimate VT or VF are 
detected as the initial rhythm in ~20% of SCD cases, 
however when there is a shorter time between collapse and 
initial rhythm detection this estimate increases to 75-80%.2,4 
Along with improved strategies to identify at-risk individuals 
and increase the rate of successful resuscitation, new 
therapies are desperately needed to prevent the recurrence 
of VT/VF. Implantable cardioverter defibrillators (ICDs) can 
successfully terminate VT/VF, but do not prevent it. ICD 
shocks are associated with increased mortality, heart 
failure, post-traumatic stress disorder, and healthcare utilization.5-10 Furthermore, once VT occurs, it is likely 
to recur, thus therapies to prevent recurrent VT/VF (e.g. antiarrhythmic drugs [AADs]) are of great 
importance.11-13   
 
1.3 
New pharmacologic therapies are needed to prevent VT/VF in structural heart disease.  
In 2018 there are fewer AADs available for clinical use than 20 years ago.14 Discouraging the development 
of new agents are the problems associated with AADs that target ion channels on the cell surface. They 
possess a narrow therapeutic window with higher drug levels leading to pro-arrhythmia that is worse in 
structural heart disease.  Currently available AADs target the cardiac sodium (Na) current, rapidly-activating 
potassium (K) current, beta-adrenergic stimulation, or multiple currents (e.g. amiodarone) and all have 
significant limitations. Class IC agents that predominantly block Na channels, (e.g. flecainide, propafenone) 
increase mortality in patients with structural heart disease and are relatively contraindicated in this 
population (CAST).15 K channel blockers prolong the QT interval, can cause polymorphic VT and have 
disappointing efficacy for VT. The risk of drug-induced torsades-de-pointes, a life-threatening VT, is 
Figure 2: Structural heart disease due to CAD, 
cardiomyopathy, and valvular heart disease is a 
major cause of SCD. Image: Hayashi et al. (2015) 
Figure 1: Concept Diagram for a Future Phase II Clinical Trial.  

 
 
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increased in structural heart disease due to down regulated K channels and abnormal calcium (Ca) 
homeostasis.16,17 Amiodarone is the most effective AAD for preventing recurrent VT/VF, but 10% of 
participants treated with amiodarone still have recurrent VT within one year and ~20% discontinue it due to 
potentially severe non-cardiac toxicities.12 Furthermore, intolerance to amiodarone is more common at the 
doses used for refractory VT/VF.13 
 
1.4 
Mechanisms promoting monomorphic and polymorphic VT in structural heart disease.  
In structural heart disease, monomorphic VT is most often due to reentry in regions of scar comprised of 
myocyte bundles and fibrosis. As Dr. Stevenson and others showed in early landmark studies, the 
arrhythmia substrate is often complex, such that numerous reentrant circuits are present giving rise to 
multiple different monomorphic VTs (pleomorphism) or polymorphic VTs.18-23 In addition to reentry, 
automaticity and triggered activity are potentially important mechanisms contributing to VT in structural heart 
disease as they can provide the triggers necessary to initiate reentry, or in some cases may be the dominant 
mechanism.24-26 A major mechanism for triggered activity are DADs due to intracellular Ca overload. 
Abnormal Ca handling is well recognized in models of heart failure and hypertrophy. The diminished cell-
to-cell coupling associated with fibrosis in areas of scar may also increase susceptibly to triggered 
automaticity resulting in an automatic VT or initiating a reentrant VT.24-26 Spontaneous Ca leak also 
inactivates Na channels which would be predicted to slow conduction velocity27 and activates SK channels 
which shortens refractoriness28-32, both of which may extend the excitable gap to promote scar-related 
reentry. Conditions that increase intracellular Ca are common in heart failure, especially β-adrenergic 
stimulation and increased heart rate. Relevant to our proposal is that dantrolene at clinically achievable 
doses has been found to reduce spontaneous Ca leak and DADs in ventricular cardiomyocytes isolated 
from failing human hearts without changing actional potential duration or reducing contractility.33 
A major knowledge gap we seek to address is the clinical potential of selectively targeting 
intracellular Ca to treat VT/VF in structural heart disease. Currently available AADs that reduce intracellular 
Ca are also potent Na channel blockers (e.g. flecainide, propafenone). Na channel blockade reduces 
conduction velocity and promotes scar-related reentry, thereby negating the benefit of RyR2 inhibition for 
reducing triggered activity in structural heart disease. Dantrolene inhibits RyR2-mediated Ca leak but does 
not block the Na channel.27 By reducing spontaneous Ca leak, dantrolene may reduce Ca mediated Na 
channel inactivation thereby reversing slowed conduction in abnormal tissues. It also may reduce SK 
channel activation thereby reversing shortened refractoriness in abnormal tissues. Accordingly, an 
overarching goal of our work is to investigate the ability of RyR2 inhibition with dantrolene to speed 
conduction and prolong refractoriness in diseased (low voltage) tissue, and to assess whether it has a 
similar effect in higher voltage tissue that may have less fibrosis. Taken together, we seek to test the idea 
that selective RyR2 inhibition with dantrolene will 
reduce VT/VF in structural heart disease by reversing 
proarrhythmic 
changes 
in 
conduction 
and 
refractoriness that promote scar-related reentry.  
 
1.5 
Antiarrhythmic drugs and VT ablation: 
complementary approaches for refractory VT.  
Presently ICDs are the major protection for prevention 
of SCD in patients with structural heart disease.  Over 
90,000 are implanted in the U.S. annually and within 2 
years of implant up to 30% of patients will experience 
an ICD shock for an arrhythmia.34  ICD shocks are 
associated with increased risk for heart failure and 
mortality despite the presence of the ICD and are 
associated with increased health care resource 
utilization.5-7 Shocks also reduce quality of life and 
cause post-traumatic stress disorder.8-10 Available 
Figure 3: Ventricular arrhythmias induced using 
programmed stimulation following VT ablation. Image: 
Fujii, Stevenson and colleagues (2017). 

 
 
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AADs can reduce VT but have limited efficacy and significant toxicity12, therefore ablation is an alternative 
therapy used to prevent VT.  
The efficacy of ablation is also limited. In multicenter trials approximately ½ of patients experience a 
recurrence of VT during follow-up, although the VT frequency is reduced for many. VT ablation is superior 
to escalating AAD therapy in patients with a prior myocardial infarction who have VT during AAD therapy, 
however, many continue to require AADs following ablation. 11,13,35 The acute effect of catheter ablation is 
typically assessed using programmed stimulation to induce VT at the end of the procedure.  Figure 3 
presents the breakdown of ventricular arrhythmias that were acutely inducible following VT ablation by Dr. 
Stevenson and colleagues (N=165 patients).36 These data support the design of a clinical trial proposing to 
study ventricular arrhythmias that remain inducible in patients following a VT ablation procedure.  
Monomorphic VT that lasted >30 seconds was induced in 38 patients, VF was induced in 9 patients, and 
non-sustained VT (NSVT) was induced in 34 patients. These arrhythmias are not ablated because an 
ablation target site could not be found or they do not match a VT that had been observed clinically (e.g. 
NSVT, VF, monomorphic VT that is much faster than spontaneously occurring VT), and therefore predict a 
lower risk of VT recurrence.37 However, a major finding of this study was that inducible NSVT was a marker 
for VT recurrence.  Similarly, inducible polymorphic VT/VF, and rapid monomorphic VTs are associated with 
an increased risk of spontaneous VT in other studies.37,38 Thus inducibility of these arrhythmias indicates 
the continued presence of an arrhythmia substrate which lends itself to the electrophysiologic assessment 
of therapy, particularly in paired, blinded assessments. In our proposal we selected a duration of 10 seconds 
as our definition for an inducible ventricular arrhythmia, as this has been used in other studies of drug 
assessment and reduces the likelihood of VT/VF requiring cardioversion.39   
 
1.6 
The effect of dantrolene on muscle strength and respiratory mechanics. 
A side effect of dantrolene is mild muscular weakness40,41. The impact of dantrolene, specifically 
Ryanodex, on respiratory mechanics, however, is unclear. Dantrolene is typically given to patients with 
malignant hyperthermia who are intubated, who have been given neuromuscular blocking drugs, and 
who are under general anesthesia, since the triggers for malignant hyperthermia require general 
anesthesia or paralytic administration. We seek to measure the effect of the administration of 
dantrolene/Ryanodex on incidence of interventions to support respiration (bag/mask ventilation, CPAP, 
BiPAP, LMA placement, tracheal intubation) in the perioperative period. We will also compare additional 
markers of respiratory mechanics and strength (secondary endpoints) between groups including 
negative inspiratory force, dynamometer measured grip strength, accelerometer measured twitch 
strength and train-of-four, ventilation (respiratory rate, tidal volume, minute ventilation), markers of gas 
exchange (arterial pH, pCO2, pO2, base excess), and electrolytes. 
 
2.0 
RATIONALE AND SPECIFIC AIMS 
Sudden cardiac death (SCD) accounts for up to 15-20% of all deaths per year in the U.S. and ventricular 
tachycardia (VT) and ventricular fibrillation (VF) from structural heart disease are major causes.2,42 To 
prevent these deaths, approximately 90,000 implantable cardioverter defibrillators (ICDs) are placed 
annually in the U.S. Up to 30% of these patients will have VT/VF terminated by the ICD within 2 years, often 
by a shock.34,43 Unfortunately, ICDs terminate VT/VF after it occurs, but do not prevent it. The occurrence 
of VT/VF is associated with increased risk of heart failure hospitalizations, and death, and once VT occurs, 
it is likely to recur.11-13  
For decades chronic therapy with antiarrhythmic drugs (AADs) has been the major option to prevent 
recurrence of VT/VF. Currently available AADs target the cardiac sodium (Na) current, rapidly-activating 
potassium (K) current, beta-adrenergic stimulation, or multiple currents (e.g. amiodarone). Through clinical 
trials (CAST15) and clinical experience, it was recognized that Na channel blockers increased mortality in 
patients with structural heart disease through scar-related VT. Similarly, QT-related proarrhythmia is a risk 
with K channel blockers. Amiodarone is the most effective AAD for preventing recurrent VT/VF, but 10% of 
participants treated still have recurrent VT within one year and ~20% discontinue it due to pulmonary toxicity, 

 
 
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thyroid toxicity and symptomatic bradycardia (OPTIC Trial).12 Taken together, a major unmet need is the 
development of a new class of AADs to prevent VT/VF in structural heart disease.  
Research by our group and others has focused on the role of diastolic calcium (Ca) leak via the 
RyR2 channel as a cause of delayed afterdepolarizations (DADs) and polymorphic VT in rare genetic 
disorders such as catecholaminergic polymorphic VT (CPVT).44,45 This led to the discovery that drugs which 
stabilize the RyR2 channel eliminate DADs and polymorphic VT in CPVT; this includes not only the 
experimental probe drug ent-verticilide but also clinically used agents such as flecainide and its derivatives 
and dantrolene, which is being studied here.46,47 The recognition that structural heart disease results in post-
translational modification of RyR2 and diastolic Ca leak is consistent with the idea that Ca leak is also 
involved in generating VT/VF in more common diseases.48-51 Accordingly, the overarching hypothesis to 
be tested is that RyR2 hyperactivity in patients with structural heart disease drives proarrhythmic changes 
in refractoriness and conduction, and decreases cardiac contractility, which promotes VT/VF (Figure 1).  
Despite the success of flecainide to treat CPVT, it is relatively contraindicated in patients with 
structural heart disease because of its Na-channel inhibition. Dantrolene, a currently available drug that 
inhibits RyR2, but has no Na or K channel activity, will be used as a tool to study RyR2 modulation. Here, 
we propose a randomized controlled trial of dantrolene versus placebo in patients with structural heart 
disease referred for VT ablation to evaluate electrophysiologic, hemodynamic, and arrhythmia prevention 
endpoints. Dantrolene’s inhibition of RyR1 will also be studied to define its effect on muscle and respiratory 
strength in this clinical population, which will be important if dantrolene is to be considered for repurposing 
as an antiarrhythmic drug. Accordingly, we propose the following Specific Aims:  
 
Aim 1: To conduct a randomized, placebo-controlled trial of dantrolene to study the effect of RyR 
inhibition on cardiac electrophysiology, hemodynamics, arrhythmia inducibility, muscle 
strength, and respiratory mechanics in patients with structural heart disease referred for VT 
ablation.  
 
Aim 2: To explore the pharmacokinetic/pharmacodynamic relationship of I.V. dantrolene and its 
short-term effect on cardiac electrophysiology, hemodynamics, and muscle and respiratory 
strength. 
 
3.0 
ANIMAL STUDIES AND PREVIOUS HUMAN STUDIES 
 
3.1 
Experimental evidence: RyR2 inhibitors treat VT/VF and improve hemodynamics 
Experimental evidence from animal models of VF and scar-related VT demonstrates the efficacy of RyR2 
inhibition with dantrolene to treat and prevent VT/VF.52 In a swine model, treatment with I.V. dantrolene 
administered during VF decreased the number of shocks required for successful defibrillation, the time to 
return of spontaneous circulation (defined as systolic blood pressure >60 mmHg), and the number of 
episodes of refibrillation.52 Dantrolene (2 mg/kg) was administered during CPR 3 minutes prior to attempted 
defibrillation, suggesting the myocardial uptake and pharmacodynamic effect of dantrolene on cardiac 
electrophysiology is rapid. Refibrillation was initiated by a premature ventricular complex (PVC) in 96% of 
cases and the proportion of animals refibrillating was 71% (5/7) in the control group compared to 27% (3/11) 
in the dantrolene group, supporting the ability of dantrolene to reduce spontaneous ventricular ectopy.  

 
 
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In preliminary data 
generated by Dr. Bjorn 
Knollmann (unpublished), 
mouse 
infarct 
models 
were 
generated 
by 
coronary ligation which 
resulted 
in 
a 
chronic 
transmural 
infarct 
involving ~50% of the LV. 
A wildtype model and a 
transgenic heterozygous 
calsequestrin-2 (Casq2+/-
) knockout model were 
used. 
Heterozygous 
Casq2 mice have hyperactive RyR2 Ca release, but do not exhibit catecholaminergic polymorphic VT 
(CPVT) as seen in homozygous Casq2 mice.  3-4 weeks after coronary ligation, mice underwent 
programmed stimulation using transesophageal pacing. Monomorphic VT was inducible. Treatment with 
dantrolene (Ryanodex) or ent-verticilide (an investigational RyR2 specific inhibitor) significantly reduced 
inducible VT and ventricular ectopy (premature ventricular complexes) compared to vehicle (placebo) 
(Figure 4). These data support the idea that RyR2 inhibition with dantrolene reduces scar-related 
monomorphic VT in experimental models and supports our plans to advance this research into clinical 
investigation.   
 
3.2 
Single dose and chronic oral exposure to dantrolene in humans 
In an unpublished Phase 1 study conducted to support the FDA application for Ryanodex (approved in 
2014), 30 healthy volunteers were given a single I.V. dose of Ryanodex (1.0, 1.75, 2.0, 2.25, and 2.5 mg/kg). 
Adverse reactions were dose dependent and the most common were flushing (27% of subjects) and 
somnolence (13%). All adverse events were classified as mild/moderate and spontaneously resolved 
without sequelae. There were no severe or serious adverse events and no dose limiting toxicity. There were 
no significant changes from baseline in vital signs or ECG assessments. Recently, the experience of 
patients prescribed chronic oral dantrolene for the treatment of muscle spasticity was reported.53 In 20 
patients with a mean of 10.9 years of follow-up on oral dantrolene there were no major cardiac events, no 
QTc or QRS prolongation, and no change in cardiac function on serial echocardiograms. 
 
3.3 
A pilot study to demonstrate the feasibility of I.V. dantrolene to study the effects of RyR 
inhibition in patients with structural heart disease undergoing VT ablation 
In preparation for our randomized controlled trial of dantrolene, an open label pilot study was recently 
completed. Five participants were enrolled and dantrolene (1 mg/kg I.V) was given. The study protocol was 
successfully performed. Evidence of a pharmacodynamic effect was observed as muscle strength 
measured by twitch height was found to decrease by 60%. Cardiac electrophysiology and hemodynamic 
measurements were feasible. No serious adverse events (SAEs) occurred. Two adverse events (AEs) 
occurred. One participant experienced a possibly drug-related exacerbation of pre-existing sinus node 
dysfunction, which required a temporary period of ventricular pacing and an increase in vasopressor dose, 
which resolved by the time the participant left the EP lab. As a result of this AE, the requirement of a 
permanent pacemaker or implantable cardioverter defibrillator was added to the inclusion criteria. The other 
AE was not related to the study drug and was an oropharnygeal laceration that likely occurred during 
laryngeal mask airway (LMA) placement prior to study drug administration. It required intubation overnight 
for airway protection. The results of the pilot study were reviewed by the data safety monitoring board on 
April 15th, 2020, which recommended proceeding with the RCT phase of the study and remaining at the 1 
mg/kg dose of dantrolene. 
 
 
 
Figure 4: RyR2 inhibition with dantrolene (Ryanodex) and ent-verticilide reduces 
inducible VT and ventricular ectopy compared to vehicle (placebo) in a Casq2+/- 
chronic infarct mouse model (unpublished data courtesy of Bjorn Knollmann, MD/PhD). 

 
 
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4.0 
INCLUSION/EXCLUSION CRITERIA (TABLE 1) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.0 
ENROLLMENT AND 
RANDOMIZATION 
 
Recruitment and informed consent: Eligible subjects will be identified by either: 1) Arrhythmia Clinic nurses or 
physicians will contact a member of the study team if they are scheduling an eligible subject for VT ablation or 
PVC ablation, or 2) the study coordinator will review the EP Lab schedule for VT and PVC ablation cases and 
screen their medical record for eligibility. The study coordinator will then request the clinical staff to obtain 
permission from the patient to contact them about research. If the patient agrees, a member of the study team 
will approach the patient in person in Arrhythmia Clinic, Vanderbilt University Hospital, or by phone prior to 
ablation. Information describing the study, why the research is being done and what will be learned will be 
provided. The risks as described in this protocol will be delineated. The benefits will be described as general 
scientific knowledge, along with potential treatment advances. No direct immediate benefit to the subjects is 
anticipated. The patient will be given either a hard or electronic copy of the informed consent document. At least 
24 hours will be given for the patient to consider participation after which the study coordinator will recontact the 
patient. If the patient desires to participate, informed consent will be documented with the subject’s signature 
using the IRB-approved consent form for this protocol. This will occur in Arrhythmia Clinic or Vanderbilt University 
Hospital. If participation is being discussed with the patient by phone, a tentative agreement to participate will be 
obtained and the study coordinator will meet the patient on the morning of the procedure at Vanderbilt University 
Hospital to sign the informed consent document. 
 
Randomization: 84 participants will be randomly assigned in a 2:1 ratio to treatment (dantrolene) or control 
(placebo). A computer-generated randomization list will be prepared by the study statistician using the 
stratified permuted block randomization, where block size varies randomly from 4 or 6 to ensure overall 
balance across treatment arms. Randomization will be stratified by 1) amiodarone use within the past 21 
days defined as chronic oral use or >5 grams cumulative dose including ongoing use and 2) planned PVC 
vs. VT ablation. The study statistician will generate the allocation schedule but will remain blinded to the 
treatment assignment. 
Inclusion 
 ≥18 years of age 
Able to give written, informed consent 
Referred for catheter-based VT ablation or PVC ablation 
Structural heart disease (LVEF < 50%, history of prior 
myocardial infarction, cardiac sarcoid or valvular heart 
disease) 
Permanent pacemaker or implantable cardioverter defibrillator 
Exclusion 
Mechanical ventricular support (e.g. LVAD, ECMO) 
NYHA class IV heart failure 
LVEF <20% 
Severe renal insufficiency (GFR<30 mL/min) 
Morbid obesity (BMI≥ 40 kg/m2) 
Chronic liver disease (Child Pugh class A-C) 
Current use of calcium channel blockers  
Neuromuscular disorder (e.g. muscular dystrophy) 
Chronic obstructive pulmonary disease or restrictive lung 
disease requiring oxygen therapy or history of intubation 
Pregnant or nursing 
History of dysphagia 
 
Table 1. Eligibility Criteria 
 

 
 
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Dropouts and replacement: To ensure adequate statistical power, we will account for up to a 20% rate of 
patient withdrawal from the study prior to randomization (for reasons such as non-study related procedural 
complications, hemodynamic instability, prolonged case duration), therefore up to 105 participants may 
need to be enrolled for 84 participants to complete the study.  
 
Study Drugs: The investigational compound is dantrolene sodium (RYANODEX, Eagle Pharmaceuticals Inc, 
Woodcliff Lake, NJ). Ryanodex is supplied in a single dose vial by the manufacturer and it is reconstituted 
with 5 mL of sterile water prior to use. Each vial contains 250 mg of Ryanodex. Based on results from the 
pilot study, Ryanodex will be administered as an I.V. push over 3 minutes at a dose of 1 mg/kg. Ryanodex 
was tested in a Phase I study and safely tolerated in doses ranging from 1 mg/kg to 2.5 mg/kg. It is approved 
for use in adults with malignant hyperthermia at a recommended dose of 2.5 mg/kg. The calculated dose is 
based on measured body weight with a maximum dose of 250 mg used in our trial. 
 
6.0 
STUDY PROCEDURES 
 
6.1 
VT or PVC Ablation will be Performed According to Standard-of-Care Clinical Practice  
Duration 
 (mins) 
STANDARD CLINICAL PROTOCOL 
RESEARCH PROTOCOL 
EP team 
Anesthesia team 
--- 
Pre-procedure holding room 
 
Pre-drug muscle strength 
(grip and NIF) 
60-140* 
Clinical ablation procedure completed 
 
5 
Post-ablation EPS & inducibility 
Pre-drug EPS & inducibility 
NIF, tidal volume, minute 
ventilation, ABG 
5 
Post-ablation ICE survey 
LVERP & conduction time 
testing 
5 
Post-ablation hemodynamics 
Pre-drug hemodynamics 
3 
 
Study drug given 
10 
PK/PD blood sampling with 
measurement of ECG intervals 
and Fick cardiac output 
 NIF, tidal volume, minute 
ventilation, ABG 
5 
5 
5 
LV ERP & conduction time 
testing 
 
2 
Post-drug hemodynamics 
5 
Post-drug inducibility testing 
--- 
Procedure completed 
 
5 
Post-procedure care unit 
2-hour post-drug muscle 
strength (grip and NIF) and ABG 
 

 
 
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This is a description of the clinical activities for VT or PVC ablation. 
Table 2 provides an overview of the timeline for clinical and research 
activities. VUMC is a high-volume PVC and VT ablation center that in 
2018 performed greater than 250 VT ablations. Dr. Stevenson directs 
the VT ablation research program at VUMC and has been a pioneer in 
the field. He, along with other international experts, authored the 2019 
Heart Rhythm Society’s expert consensus statement on catheter 
ablation for VT. 54 VT or PVC ablation will be performed according to 
our standard clinical practice. When feasible, amiodarone is held for 48 
hours prior to ablation. Other oral AADs will be held 24 to 48 hours prior 
to the procedure and continuous I.V. AADs will be held 4 to 6 hours 
prior to the beginning of the procedure. Sedation during PVC or VT 
ablation and study protocol will be per usual care and remain at the 
attending anesthesiologist’s discretion. Typically, PVC or VT ablations 
are done under monitored anesthesia care (MAC) with varying levels 
of sedation from moderate to deep sedation, although general 
anesthesia with or without a secured airway can occur. Propofol and 
dexmedetomidine infusions are typically used to achieve the desired level of anesthesia. The procedure 
will be performed with sterile preparation. The left ventricle will be accessed via either retrograde aortic 
or trans-septal access. Sheaths are placed in the femoral veins and artery for central vascular access. 
Hemodynamics are monitored with a radial or femoral arterial line, and a PA catheter. Standard EP 
catheters include: 1) a quadripolar catheter placed in the right ventricle, 2) a multipolar mapping array, 
and 3) an irrigated-tip radiofrequency ablation catheter. An intracardiac echocardiogram (ICE) catheter 
is also placed. A pre-ablation survey with an intracardiac echocardiogram (ICE) is performed to inspect 
the heart for baseline abnormalities (e.g. thrombus, pericardial effusion). A 3D mapping system is used 
to create an electroanatomic voltage map where low voltage areas (bipolar electrograms <1.5 mV) 
identify regions of fibrosis/scar to help localize the potential VT substrate. An EP study is performed, 
which includes programmed ventricular stimulation to induce VT. The programmed ventricular 
stimulation protocol is pacing at a fixed rate for 8 beats at a cycle length of 400-600 ms followed by 
early extrastimuli. In most cases, the clinical VT, which resembles spontaneous VT recorded by an ICD 
or on ECG, can be induced. Non-clinical ventricular arrhythmias are also often encountered which are 
usually monomorphic VTs that differ from the spontaneously observed VT (typically having a faster rate 
than the clinical VT) or polymorphic VT. The clinical VT and nonclinical monomorphic VTs that have a 
cycle length slower or only 20 ms shorter than clinical VT are targeted for ablation starting in low voltage 
areas.  The reentry circuit may be identified by mapping during VT (activation mapping).  More 
frequently ablation targets are identified during sinus rhythm as areas in the scar where pacing 
replicates the VT morphology (pace mapping), or electrograms have characteristics of abnormal 
conduction consistent with reentry substrate. When it is suspected that ablation has eliminated the 
targeted VTs, programmed ventricular stimulation is repeated. If any sustained monomorphic VTs are 
induced, additional ablation may be performed if there are additional regions that can be identified for 
those VTs.  Acute procedural success is defined as 1) absence of any inducible sustained monomorphic 
VT, or 2) modification of the arrhythmia substrate if the clinical VT is no longer inducible, but other VTs 
are induced. Acute procedural failure is defined as continued inducibility of the clinical VT. Following 
completion of ablation, post-ablation hemodynamics are recorded, and an ICE survey is performed. 
For stable patients undergoing elective VT ablation, such as our study population, post-anesthesia 
recovery is performed in the EP Lab and the patient leaves awake, extubated, and hemodynamically 
stable. 
Table 2. List of peri-procedural clinical and research activities. NIF: negative inspiratory force; EPS: electrophysiology study; 
ICE: intracardiac echocardiogram; LVERP: left ventricular effective refractory period; ABG: arterial blood gas; PK/PK: 
pharmacokinetics/pharmacodynamics.  
 
Figure 5: A voltage map of the LV 
endocardial surface. From a patient 
undergoing VT ablation at Vanderbilt who 
met our proposed eligibility criteria, the 
pacing catheter is positioned in the scar 
border zone. Pacing for conduction time 
and ERP measurements was performed 
from the distal bipolar electrodes (A) and 
a more proximal electrode (B). 

 
 
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6.2 
List of Encounters Detailing the Clinical and Research Activities 
Encounter 1: Enrollment and Informed Consent 
• 
Performed by the study team in the Vanderbilt Arrhythmia Clinic or Vanderbilt University Hospital.  
 
Encounter 2: Time of Ablation 
• 
The participant will arrive the morning of ablation to the EP pre-op holding room and undergo standard 
preparations for ablation according to the clinical protocols (e.g. labs, vitals, exam, procedural consent). 
• 
The participant will undergo baseline muscle (grip) and respiratory (NIF) strength testing in the pre-op area. 
• 
The participant is then transported to the operating room (EP lab) where standard of care ablation is 
performed under the direction of the clinical team. See section 6.1 for full details.  
• 
Research activities will begin after completion of the ablation portion of the ablation procedure. Research 
activities occur concurrently with clinical activities and are performed by a dedicated physician and nurse 
team to avoid extending overall case time. 
• 
The clinical post-ablation EP study with programmed ventricular stimulation will serve as the baseline (pre-
dantrolene) assessment of cardiac electrophysiology parameters (conduction and refractoriness) and 
ventricular arrhythmia inducibility. Data analyses are performed offline following the case to avoid adding 
procedural time. See sections 6.3 to 6.6 for full details.  
• 
Hemodynamic monitoring is routinely performed during ablation with an arterial line and pulmonary artery 
(PA) catheter (aka. Swann-Ganz Catheter). See sections 6.7 to 6.10 for full details. The pre-dantrolene 
arterial blood pressure mean right atrial, PA systolic, PA diastolic, mean PA pressure, and pulmonary artery 
wedge pressures will be recorded.  
• 
Blood samples will also be drawn for calculation of a Fick cardiac output and for the pharmacokinetic 
(PK)/pharmacodynamic (PD) analysis. See section 6.12 for full details of the PK/PD analysis. A 2 mL sample 
from the femoral venous, arterial, and distal PA catheter will be collected.  
• 
I.V. dantrolene will be administered over 3 minutes. Peak drug levels occur rapidly allowing the data collection 
protocol to proceed without delay. 
• 
The post-dantrolene hemodynamics will be recorded. 
• 
The post-dantrolene EP study with programmed ventricular stimulation will be repeated. 
• 
A 2 mL sample from the femoral venous, arterial, and distal PA catheter will be collected at 5, 10, 15, and 20 
minutes post-dantrolene. The total amount of blood collected during the ablation is 30 mL. 
• 
Respiratory mechanics (tidal volume, rate, minute ventilation) and an arterial blood gas (ABG) will be 
measured at 0, 10, and 20 minutes, and need for additional respiratory support will be assessed continuously 
as is standard care (see Section 6.13). 
• 
The clinical and research activities will be complete, and the participant will leave the operating room awake, 
extubated, and hemodynamically stable. 
• 
The participant will undergo baseline muscle (grip) and respiratory (NIF) strength testing in the post-op 
recovery area and an ABG will be measured 120 minutes after study drug was initiated 
 
Encounter 3: Post-ablation Day 0-2. 
• 
Following ablation, the participant will be returned to the EP holding room. It is clinical standard-of-care for 
patients to remain in the hospital at least overnight for monitoring. They will be under nursing observation 
and on continuous cardiac telemetry and oximetry. They will be on strict bedrest until post-operative Day #1 
and they will be ambulated with assistance the morning after the procedure. 
• 
2 mL blood samples will be drawn at intervals of 4 to 12 hours for up to 48-hours during the post-ablation 
period. No more than 15 mL total will be drawn. We will use indwelling catheters whenever possible for blood 
collections. All patients will have indwelling catheters throughout the duration of study, and we do not 
anticipate needing to perform phlebotomy with a fresh stick during the study. See section 6.12 for full details. 

 
 
12 
12/13/23 
• 
The research protocol is complete when the participant is discharged from the hospital. 
 
6.3 
EP Catheter Placement and Pacing 
Protocol  
The scar and its border zone (defined by 
peak to peak bipolar voltage of 1 to 2 mV) will 
have been defined during the clinical 
procedure (Figure 5).  Following ablation a 
10-pole 
catheter 
with 
2-8-2 
mm 
interelectrode spacing is positioned in a 
ventricular area remote from the scar (peak 
to peak bipolar electrogram voltage > 2 mV) 
ensuring that electrograms of adequate 
quality for measurement are present on at 
least two of the electrode pairs, and that the 
pacing threshold on one of these pairs is less 
than 10 ma.  The position of this catheter is 
marked on the 3D-electroanatomic map so 
that it can be positioned in the same location 
when the pacing protocol is repeated 
following administration of the study drug. 
Pacing 
threshold 
is 
determined, 
and 
stimulation is performed at 2-times the 
threshold. An electrode that has stable 
capture with pacing is selected for pacing 
and bipolar signals are recorded from the 
other electrodes. Pacing is performed at a basic cycle length (CL) of 600 ms (100 beats per minute [bpm]) for 8 
beats with the addition of a single extrastimulus followed by a 3 second pause. The initial coupling interval of the 
extrastimulus is 400 ms. It is then decremented by 10 ms down to loss of capture on two consecutive attempts 
to define the effective refractory period. The 10-pole catheter is then repositioned to the scar borderzone. The 
above steps are then repeated at this location. The electrograms from the EP study are exported for offline 
measurement and analysis. A conduction curve will be plotted to define the conduction time (primary endpoint) 
and conduction restitution (secondary endpoint). 
 
6.4 
Refractoriness  
Ventricular Effective Refractory Period (VERP) 
is defined as the longest stimulus coupling 
interval which fails to capture the ventricle twice 
(Figure 6). The drive train for ERP testing will be 
600 ms. 
 
6.5 
Conduction  
All measurements of conduction are performed 
off-line following completion of the procedure 
and therefore do not prolong case time. They 
are performed by members of the investigator 
team who are blinded to drug administration. 
Conduction Time is measured as the time from 
the: 1) stimulus artifact to the local electrogram 
on the electrode most distant from the site of 
pacing that has a consistent reliable electrogram 
(Figure 7).  The dominant peak of the bipolar 
electrogram will be used for measurement of 
conduction times.  If multiple similar amplitude 
Figure 6: Ventricular effective refractory period (ERP) is 
displayed for the same patient and catheter position shown in 
Figure 5. Pacing is from the distal electrode (labeled A in Fig. 5).  
Figure 7: A Conduction Curve for the patient and catheter position in 
Figure 5. (Inserted panel shows data from the point at the red arrow). 
At the coupling interval of 290 ms the time from the stimulation artifact 
to the electrogram at a proximal electrode is 226 ms and is plotted on 
the conduction curve. The basal conduction time is 140 ms. 

 
 
13 
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signals are present, as may be encounted in or near scar, the latest sharp peak will be used for measurement.  
Multiple measurements for conduction time will be obtained and are specific to a given location (scar border 
zone or remote higher voltage site). Conduction Restitution55: from plots of conduction time versus coupling 
intervals the following parameters are determined and compared pre-post dantrolene administration: 1) The 
paced conduction time at basic cycle length; 2) Onset of the paced conduction time increase (the longest 
coupling interval at which the paced conduction time exceeded that of the basic cycle length by 5 ms and 
continued to increase); and 3) The maximum paced activation time. 
 
6.6 
Ventricular Arrhythmia Induction Protocol 
A standardized induction protocol is performed using programmed ventricular stimulation from the RV apex. The 
protocol is based on a prior study evaluating a range of induction protocols used to test inducibility before and 
after AAD administration in the EP lab.39 Sedation and vasopressor infusion is maintained at constant levels 
throughout the study protocol and, any changes warranted for clinical care will be documented.  The pacing cycle 
length is 8 beats followed by extrastimuli (ES) and a 3 second rest period. The basic cycle length of the pacing 
drive train (S1 – S1) is 600 ms (100 bpm). The first extrastimulus (S2) is introduced at 300 ms and decremented 
by 10 ms to VERP or 200 ms. S2 is then set to a coupling interval (S1 – S2) of VERP (or 200 ms) + 20 ms and 
a second extrastimulus (S3) added at a coupling interval of S1-S2 + 50 ms and scanned to refractoriness or a 
minimum coupling interval of 200 ms.  The second extrasimulus is then set to its VERP + 20 ms and a third 
extrastimulus (S4) added at an initial coupling interval (S3 – S4) of S2 – S3 + 50 ms and scanned in to 
refractorness or 200 ms. Thus up to 3 extrasimuli are added and the minimum coupling interval will not be shorter 
than 200 ms.  The induction protocol is stopped when: 1) the primary endpoint is met, or 2) the induction protocol 
has been completed. The primary endpoint is an episode of sustained VT that lasts ≥10 seconds or two episodes 
of non-sustained ventricular arrhythmia defined as VT or VF that lasts 3-9 seconds.  
 
6.7 
Pulmonary Artery (Swan-Ganz) Catheter Placement 
The participant is supine and under deep sedation or general anesthesia during the procedure. Due to the 
creation of a complex electroanatomic map, it is critical that patients do not move during an ablation procedure. 
This is also ideal for comparative hemodynamic assessments. The transducer is placed midway between the 
level of the anterior and posterior surfaces of the patient’s chest and zeroed to atmospheric pressure. The 
balloon-tipped PA catheter is placed via a femoral venous sheath and advanced to the right atrium (RA). A 
combination of pressure wave form interpretation and fluoroscopy is used to guide the PA catheter through the 
RV to the distal PA. The PA catheter remains in the same position during the pre and post-drug hemodynamic 
measurements.  
 
6.8 
Pressure Measurement 
Recordings are obtained to measure the following pressures: mean RA, PA systolic, PA diastolic, and mean PA. 
All pressures are recorded as the average of 3 measurements obtained at end expiration. 
 
6.9 
Arterial Blood Pressure Measurement 
As part of an ablation procedure, invasive arterial blood pressure is monitored via an arterial pressure catheter 
(femoral and/or radial artery). As clinical endpoints, systolic, diastolic, mean and pulse pressures are recorded 
immediately prior to study drug administration, and then every 2 minutes after drug administration during the 
research protocol (except during pacing maneuvers). Changes in vasopressor infusion rate and boluses are also 
recorded.    
 
6.10 
Cardiac Output, RV Function, and Vascular Resistance Assessment 
Cardiac output is measured using the Fick method. A pre-study drug cardiac output is recorded, and then cardiac 
output is sampled 4 times post-study drug for the purposes of Aim 2 and for PK/PD analysis. Indices of RV 
function are calculated including RV stroke volume index and PA pulsatility index. Systemic and pulmonary 
vascular resistance are also calculated. Note, if the pre-study drug mixed venous oxygen saturation (SvO2) is 
<50%, the research protocol will be stopped, and the study drug will not be administered.  
 
6.11 
ECG Intervals 

 
 
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ECG intervals (RR, PR, QRS, QT) are recorded from a continuous 12-lead tracing obtained prior to study drug 
administration and then for 20 minutes after study drug. Measurements are made offline at a sweep speed of 
100 mm/s. Measurements are made every minute during the post-study drug period. Measurements are 
recorded as the median of 3 beats.  
 
6.12 
Pharmacokinetic (PK)-Pharmacodynamic (PD) Analysis 
PK/PD analysis will be performed by Scott Akers (Lipscomb University). Ryanodex is a new formulation of 
dantrolene (nanoparticle suspension for injection) with enhanced aqueous solubility designed for rapid I.V 
administration. The FDA determined that our proposal is exempted from requirements for an Investigational New 
Drug application (see accompanying letter from FDA). The PK of Ryanodex have been investigated in healthy 
volunteers at a dose range of 1 to 2.5 mg/kg and demonstrated a dose-proportional increase in dantrolene 
exposure with a peak concentration observed 1 minute after drug administration.  Mean PK parameter estimates 
are independent of dose with a mean half-life of 10 hours, volume of distribution of 36.4 L, and systemic 
clearance of 2.5 L/hr.  One major determinant of the short-term effects of many cardiac drugs is the concentration 
of drug within the myocardial tissue.  Previous in-vivo studies evaluating the myocardial uptake of drugs in 
humans using a transcoronary blood sampling technique have demonstrated that lipophilic drugs like dantrolene 
exhibit rapid uptake into myocardial tissue with peak myocardial tissue content (1-4% of dose administered) 
being achieved within 0.5-5 minutes after I.V. bolus.56,57 To define the PK/PD relationship of dantrolene and its 
short-term effect on the specific hemodynamic (PA wedge pressure, Fick cardiac output) and EP parameters 
(electrocardiogram intervals [RR, PR, QRS, QT]), femoral venous, arterial, and PA distal blood samples will be 
collected simultaneously from indwelling sheaths in all subjects at the pre-dantrolene timepoint and at 5, 10, 15, 
and 20 minutes post-dantrolene. Arterial drug concentrations will be assumed to represent dantrolene 
concentrations present in the ascending aorta and reflect concentrations entering the coronary circulation.  As 
such, the temporal relationship between dantrolene concentrations (arterial and venous) and peak hemodynamic 
and EP effects will be evaluated using PK/PD modeling approaches to determine direct response correlations 
or a potential time lag in response. 
Following the PK/PD phase of the study, PK sampling will be performed by collecting up to seven blood 
samples per subject that are drawn at variable time intervals.  Blood samples will be 2 mL each and no more 
than a total of 15 mL of blood will be drawn post-operatively. PK will be analyzed using both an intensive and 
sparse (population) PK approach.58 Dantrolene plasma concentrations in all blood samples will be determined 
using liquid chromatography coupled with tandem mass spectrometry (LCMS-MS) with an assay threshold of 5 
ng/ml.  Plasma concentration-time data from individual subjects will be imported into Phoenix WinNonlin® 8.0 
software (Certara USA, Inc., Princeton, NJ) to estimate the PK parameters of dantrolene from population PK 
approaches. 
 
6.13 
Measurement of Respiratory Mechanics and Ventilation   
To enhance the safety of the study protocol and enable research on the effect of dantrolene on respiratory 
mechanics, the study team will include cardiac anesthesiologists (Drs. Siegrist, Mantinan, Billings, Pretorius, and 
Shotwell) and will specifically study the effect of dantrolene on respiratory mechanics. Prior to the procedure, a 
baseline measurement of respiratory muscle strength will be obtained with a negative inspiratory force test (NIF), 
which is a quick, non-invasive bedside breathing test. During the procedure, to monitor adequacy of ventilation, 
maintain safety, and measure the effect of dantrolene administration on ventilation, the anesthesiologist will 
monitor respiration oxygenation as is standard care for all patients during procedures and surgeries.  If the patient 
is already under general anesthesia with controlled ventilation (either with laryngeal mask airway [LMA] or 
endotracheal tube [ETT]), attempts will be made to have the patient spontaneously breathing with neuromuscular 
blockers reversed. These measurements include respiratory rate, minute ventilation, tidal volume, as well as 
arterial blood gas measurements (pH/pO2/pCO2/BE/Na/K/Cl/Ca). These measurements will be recorded prior 
to study drug administration and at 10 and 20 minutes after study drug administration. Finally, two hours and 
again at 24 hours after study drug administration, a NIF will be repeated to assess whether the respiratory muscle 
strength has returned to baseline. An arterial blood gas will also be repeated at 2 hours after study drug 
administration (sample obtained from the radial arterial line). 
 
6.14 
Measurement of Muscle Strength 

 
 
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To monitor the effect of dantrolene on muscle strength, a baseline assessment of handgrip strength will be 
obtained prior to the procedure using a dynamometer. Finally, two hours and again at 24 hours after study drug 
administration, an assessment of grip strength using the dynamometer will be repeated to assess whether the 
respiratory muscle strength has returned to baseline.  
 
7.0 
RISKS 
Patients with structural heart disease and PVCs or VT who are undergoing ablation are at risk for complications 
by nature of their underlying disease process and the procedure. These risks are inherent to the patient 
population studied here. The risks of obtaining blood samples from phlebotomy of peripheral veins and existing 
venous lines are minimal. There are risks associated with inducing ventricular arrhythmias, however the pacing 
maneuvers used in this protocol are commonplace during ablation and have a long history of being used both 
clinically and for research purposes.59,60 There are risks associated with dantrolene (Ryanodex). A 
comprehensive list of adverse reactions from the Ryanodex brochure include skeletal muscle weakness, 
dyspnea, respiratory muscle weakness, dysphagia, somnolence, lightheadedness, tissue necrosis (only if 
extravasated into surrounding tissue), nausea, thrombophlebitis, urticaria, and anaphylaxis. Drug interactions 
include calcium channel blockers, therefore patients using calcium channel blockers are excluded from the trial. 
Precautions also include muscle relaxants, antipsychotic and antianxiety agents as they may potentiate 
weakness, somnolence, and dizziness with Ryanodex. The risk of adverse reactions are dose-dependent and 
we propose here to use the lowest approved dose of Ryanodex (1 mg/kg). Furthermore, a one-time dose of I.V. 
Ryanodex will be given in the EP lab while under the care of both the Cardiac EP and Anesthesiology teams. 
Collaborating investigators on this study include cardiac anesthesiologists who will be monitoring respiratory 
status and measuring respiratory mechanics, ventilation, and muscle strength which will add additional safety 
precautions to the protocol. According to standard clinical protocol following ablation, patients are on strict 
bedrest until post-operative Day #1 with continuous cardiac and respiratory telemetry. Participants will be 
ambulated with assistance for the first time on post-operative Day #1. This minimizes the risks associated with 
the potential for skeletal and respiratory muscle weakness and lightheadedness. Furthermore, the eligibility 
criteria for this trial will exclude participants with a history of skeletal muscle disorders (e.g. muscular dystrophy), 
pulmonary disease requiring supplemental oxygen or prior intubation, morbid obesity, or a history of dysphagia.  
According to the Ryanodex brochure, patients must not drive 48 hours after Ryanodex administration. However, 
the vast majority of patients undergoing ablation are on pre-existing driving restrictions because they have 
experienced recent ICD shocks and/or ventricular arrhythmias. 
 
Table 3. Anticipated risks associated with dantrolene, PVC or VT ablation procedures, and the study protocol 
 
Risks 
from 
Study 
Drug 
(Ryanodex) 
Risks from ablation procedure 
Flushing 
Acute myocardial infarction 
Heart block 
Somnolence 
Anemia 
Hematoma 
Dysphonia 
Arterial dissection 
Hypotension 
Dysphagia 
Atrial fibrillation/flutter/tachcyardia 
Hypoxia-volume overload 
Nausea 
Back pain 
Heparin-induced thrombocytopenia and disseminated intravascular coagulation 
Feeling abnormal 
Bleeding 
ICD lead malfunction/ICD shock 
Headache 
Cardiac or valvular injury 
Incessant VT/Recurrent VT/VT storm 
Vomiting 
Cardiac insufficiency 
Mitral valve regurgitation 
Vision blurred 
Cardiac perforation 
Pleural effusion 
Pain in extremity 
Cardiogenic shock 
Pneumonia 
Muscular weakness/asthenia 
Cerebrovascular accident 
Pericarditis 
Bradycardia 
(AV 
block, 
sinus 
node 
dysfunction) 
Transient ischemic attack 
Pulmonary edema 
Tachycardia 
Cardiac ischemia 
Respiratory distress/insufficiency 
Infusion site pain/necrosis if extravasated 
Chest pain 
Pseudoaneurysm 
Dizziness/lightheadedness 
Congestive heart failure 
Urinary retention 
Dyspnea, respiratory muscle weakness 
Death 
 
Urticaria, skin erythema 
Electromechanical dissociation 
 
Thrombophlebitis 
Epistaxis secondary to anticoagulation 
 
Anaphlaxis 
Headache 
 
 
 
8.0 
REPORTING OF ADVERSE EVENTS AND UNANTICIPATED PROBLEMS INVOLVING RISK TO 
PARTICIPANTS AND OTHERS 
 
8.1 
Data and Safety Monitoring Plan 

 
 
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The Data Safety Monitoring Board (DSMB) reviews safety data, study progress, and data quality. The DSMB is 
chaired by Dr. Prince Kannankeril, MD (Professor of Pediatrics, Division of Pediatric Cardiology, Vanderbilt 
Children’s Hospital) who is a clinical investigator and practicing cardiac electrophysiologist. Other members 
are Dr. Gregory Michaud, MD (Professor of Medicine, Director of the Division of Cardiac Electrophysiology, 
VUMC) who is a clinical researcher and has expertise in complex ablation including VT ablation; Dr. Chris 
Hughes who is an Anesthesiologist and clinical researcher (Associate Professor of Anesthesiology); and G. 
Dan Ayers, MS (Senior Associate, Department of Biostatistics, VUMC) who has extensive prior experience 
as a DSMB statistician. The DSMB will review all serious adverse events (AEs). Any serious AE will be reported 
to the DSMB and IRB within 7 days from the investigators’ awareness of the event. Any untoward medical event 
will be classified as an AE, regardless of its causal relationship with the study. The DSMB reviewed the results 
of the pilot study and provided input for the Phase II trial and recommended to proceed.  
 
8.2 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others 
 
Adverse event reporting will utilize the NIH Common Criteria for Adverse Event Reporting (CTCAE version 5.0, 
published November 2017). It defines an adverse event (AE) as any unfavorable and unintended sign (including 
an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medical treatment 
or procedure that may or may not be considered related to the medical treatment or procedure.  
 
Adverse events include: 
o Worsening (change in nature, severity or frequency) of conditions present at the onset of the trial 
o Patient / subject deterioration due to the primary illness 
o Intercurrent illnesses 
o Drug interactions 
o Events related or possibly related to concomitant medications 
o Abnormal laboratory values or changes of vital signs, as well as significant shifts from baseline within the 
range of normal, which the Investigator considers clinically significant. 
 
Unexpected Adverse Drug Reaction: an unexpected Adverse Drug Reaction is “an adverse reaction, the nature 
or severity of which is not consistent with the applicable product information (e.g., Investigator’s Brochure for an 
unapproved investigational medicinal product)”. Definitions of serious adverse events or serious adverse drug 
reaction: during clinical investigations, adverse events may occur which, if suspected to be drug-related (adverse 
drug reactions), must be significant enough to lead to important changes in the way the medicinal product is 
developed (e.g., change in dose, population, needed monitoring, consent forms). This is particularly true for 
reactions, which, in their most severe forms, threaten life or function.  
 
A serious adverse event/experience (SAE) or reaction is any untoward medical occurrence that at any dose: 
 
1. results in death 
2. is life-threatening 
3. requires inpatient hospitalization or prolongation of existing hospitalization 
4. results in persistent or significant disability/ incapacity (as per reporter’s opinion) 
5. is a congenital anomaly/birth defect 
6. is another medically important condition 
7. The term "life-threatening" in the definition of "serious" refers to an event in which the patient is at risk of 
death at the time of the event; it does not refer to an event which hypothetically might have caused death 
if it were more severe. 
 
Important medical conditions that may not result in death, be life-threatening, or require hospitalization may be 
considered as SAEs when, based upon appropriate medical judgment, they may jeopardize the patient or may 
require intervention to prevent one of the outcomes listed in the definition above. Examples of such events are 
intensive treatment in an emergency room or at home for allergic bronchospasm; blood dyscrasias or 
convulsions that do not result in hospitalization; or development of drug dependency or drug abuse.  

 
 
17 
12/13/23 
 
Definition of severity of adverse events: 
 
Grades: refer to the severity of the AE. The CTCAE displays Grades 1 through 5 with unique clinical descriptions 
of severity for each AE based on this general guideline:   
 
• 
Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; intervention not 
indicated.   
 
• 
Grade 2 Moderate; minimal, local or noninvasive intervention indicated; limiting age appropriate instrumental 
activities of daily living (ADL).   
 
• 
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization or prolongation 
of hospitalization indicated; disabling; limiting self-care ADL. 
 
• 
Grade 4 Life-threatening consequences; urgent intervention indicated.   
 
• 
Grade 5 Death related to AE.   
 
Definition of adverse event causality: 
 
The Investigator will determine causality of each adverse event by using the classification criteria: unlikely, 
likely, or not assessable. 
 
Unlikely: 
The AE is considered by the Investigator to be due to a pre-existing condition, a known 
manifestation of the target disease, a recurrent condition, or is likely explained by environmental 
or diagnostic therapeutic factors or was pre-existing and did not deteriorate. 
 
Likely: 
The AE occurred during or after administration of the study treatment or a pre-existing event 
worsened within an appropriate period of time, and at least one of the following criteria is 
applicable:  
o the event could not be explained by the clinical condition or history of the subject, 
environmental or toxic factors, or other diagnostic or therapeutic measure; 
o was an expected ADR associated with study treatment or a class-labeled drug effect; 
o AE subsided or disappeared after withdrawal or dose reduction of study treatment; or 
o AE recurred after re-exposure to study treatment. 
 
Not assessable: There is insufficient or conflicting evidence for classifying the causality of the AE as likely or 
unlikely. Lack of information may apply for this situation. 
 
Note: AEs with causality 'likely' or 'not assessable' are considered to be 'possibly drug-related.' 
 
Adverse event reporting 
 
Any adverse events (AEs) will be reported to the PI within 72 hours of notification of the event. The PI will notify 
the DSMB of any major adverse events. Any unanticipated problems involving risk to the participants or others 
will be discussed with the PI and DSMB. Non-serious AEs and incidences of noncompliance with the protocol 
will be reported to the IRB at the time of annual review. 
 
Serious Adverse Events (SAEs) will be reported according to the following procedure: 
 
The occurrence of serious adverse events will be reported to the Investigator within 24 hours after notification of 
their occurrence. The Investigator will report SAEs to the DSMB and the Vanderbilt University Medical Center 
Institutional Review Board within 7 days of the Investigator’s notification of the event. 

 
 
18 
12/13/23 
 
In an unanticipated event of prolonged side effect, requiring prolongation of hospital stay, patients will be retained 
in the hospital until side effects have resolved. For minor side effects, where inpatient care is deemed 
unnecessary, follow- up will be maintained via phone or as outpatient if necessary. Patient and their families will 
be given the PI’s contact number for reporting any other effects of medication following discharge. 
 
Any newly discovered information which may affect the subject or their caregiver’s decision to continue to 
participate in the study will be passed on to them as soon as possible. This may also result in a change to the 
consent form and review by the IRB. 
 
9.0 
STUDY WITHDRAWAL AND DISCONTINUATION 
A participant may withdraw from the study at any time by informing the study staff verbally or in writing. If an 
individual withdraws their consent, we will withdraw the participant. Contact information for the PI and study staff 
will be made available to the participant upon enrollment in the consent document.  Any remaining biological 
samples and data will be destroyed. Any data or biological samples that have been used for research prior to 
their withdrawal request will not be withdrawn and destroyed. 
A participant may be withdrawn from the study by the PI if any of the following occurs: 
i. 
The participant, for any reason, does not undergo VT or PVC ablation. 
ii. 
Prior to administration of dantrolene, the participant experiences a complication during the ablation 
procedure and/or is not considered by the clinical or research team to be clinically stable enough to 
undergo the research protocol 
iii. 
Prior to administration of dantrolene, if any of the following criteria are met on the pre-study drug 
assessment, the research protocol will be stopped, and the study drug will not be administered: 
a. Mixed venous oxygen saturation (SvO2) is <50% 
b. Criteria for RV failure (RA pressure >15 mmHg and CI <2.0 L/min/m2) 
c. Criteria for severe pulmonary hypertension (PA systolic pressure ≥60 mmHg) 
iv. 
The participant experienced an adverse event related to the study drug/protocol. 
 
10.0 
STATISTICAL CONSIDERATIONS 
 
Statistical Considerations: The statistical analysis plan was formulated and will be performed by Dr. Fei Ye, 
Co-investigator and Associate Professor of Biostatistics. Analysis Sets: 1) Full analysis set (FAS): All randomized 
patients. The FAS will be used in the analysis of all efficacy endpoints. Participants will be included in FAS 
according to the treatment to which they are randomized. 2) Safety analysis set (SAS): All randomized 
participants who have received dantrolene. The SAS will be used in the analyses of all safety endpoints. 
Participants will be analyzed according to the treatment they received. 
 
General Statistical Strategies: Baseline characteristics will be summarized descriptively by treatment group and 
by stratification factors. Categorical data will be presented as frequencies and percentages and analyzed with 
Fisher’s exact test and chi-squared test. For continuous data, summary statistics will be presented. For single 
time-point data, between-group differences will be assessed with t-test or ANOVA. Nonparametric counterparts, 
Wilcoxon rank sum test and Kruskal-Wallis test, will be used when assumptions for parametric methods are not 
met. For correlated data (e.g., repeated measurements) will be analyzed with mixed-effect models (LMM). Multiple 
comparison issues will be corrected using the Bonferroni’s. Analyses will be performed using R (latest version).  
 
Modified “Intention-to-Treat” (mITT) Set: all randomized patients who underwent VT/PVC ablation and received 
any study treatment (dantrolene or placebo) will be analyzed according to the study arm they were originally 
assigned, regardless of what treatment they received. We chose this population because eligibility is defined with 
respect to ablation, and all trial endpoints are postoperative outcomes. 
 

 
 
19 
12/13/23 
 
 
EP Endpoints: The primary hypothesis in Aim 1 tests whether RyR2 block with dantrolene significantly prolongs 
refractoriness and speeds conduction within low voltage areas adjacent to dense fibrosis and scar but has no 
significant effect within remote areas of relatively higher voltage.  
Primary Analysis (Aim 1): All FAS will be included in the primary analysis of conduction time and ERP. The 
primary analysis will be restricted to measurements performed in the LV endocardium. 
Conduction Time: is a continuous measurement. To most accurately measure conduction time, it is the mean 
value of multiple measurements along the horizontal aspect of the conduction curve (Figure 7). Conduction time 
is analyzed separately for the low voltage and higher voltage areas. Multivariable linear regression models will 
be built. Simple linear regression models will be used to test whether treatment with dantrolene is associated 
with a statistically significant decrease in mean post-drug conduction time when adjusted for mean pre-drug 
conduction time.  Multivariable linear regression models will include additional adjustment for age, sex, mean 
bipolar voltage, amiodarone exposure (yes/no), change in beta-agonist exposure, and time from start of drug to 
post-drug EPS.  Mean bipolar voltage will be the mean of the peak electrogram measured on the recording 
electrode for each of the extrastimuli.  
Effective Refractory Period: is a continuous measurement. The ERP is measured at within the low voltage area 
and higher voltage areas at a drive train of 600 ms. It is measured pre-study drug and post-study drug. 
Multivariable linear regression models will be built. Separate models will be built for the low and higher voltage 
areas. Simple linear regression will test whether treatment with dantrolene is associated with a statistically 
significant increase in mean post-drug ERP when adjusted for mean pre-drug ERP.  Multivariable analysis will 
be conducted using the same methods as described for conduction time. 
Secondary Analyses Aim 1: activation time and conduction restitution are additional metrics for conduction. They 
are analyzed with the same statistical approach as described for conduction time.  
Sample Size and Power Aim 1: The primary endpoints are conduction time and ERP. Pre- and post-drug 
difference in average conduction time and ERP will be compared between the dantrolene and the placebo group. 
We expect the conduction time to decrease and ERP to increase substantially after drug in the treatment arm, 
while no change is expected in the placebo arm. With 56 patients treated with dantrolene and 28 placebos, there 
will be 80% power to detect a 0.6xSD difference in the average conduction time and the average ERP change 
between the two study arms. 
Hemodynamic Endpoints: The primary hypothesis in Aim 2 tests whether RyR2 inhibition increases cardiac 
output and decreases PA diastolic pressure in patients with structural heart disease. The primary endpoints are 
cardiac index and mean PA diastolic pressure. Secondary endpoints are RA and PA pressures, RV stroke 
volume index, PA pulsatility index, and systemic and pulmonary vascular resistances.  
Primary Analysis (Aim 2): Our primary analyses use multivariable linear regression to test whether our primary 
determinant (dantrolene vs. placebo) is associated with: 1) post-drug mean cardiac index and 2) post-drug mean 
PA diastolic pressure. Adjustment is made for pre-study drug measurements. Additional adjustment in the 
multivariable model includes: age, sex, LV ejection fraction (continuous), change in beta-agonist exposure, 
ventricular arrhythmia induced (yes/no), and time from start of study drug to post-drug hemodynamic 
measurements. Mixed-effect models with linear regression will be built for the repeated measurements to 
account for the correlation structure in the data, where patient ID will be treated as a random effect. Fixed effect 
factors include treatment arm, measurement time (1,2,3, etc.), and all covariates. The cardiac index and PA 
diastolic pressure adjusted for pre-drug measurements is presented for both treatment arms, together with 
corresponding 95% confidence intervals (CIs) and p-values are graphically interpreted using boxplots, partial 
effect plots, residual plots, etc.  
Secondary Analysis (Aim 2): Secondary endpoints are RA and PA pressures, PA pulsatility index, and systemic 
and pulmonary vascular resistances. They are analyzed with the same statistical approach as described for 
output and PA diastolic pressure. 
 Sample Size and Power Aim 2: The pre- and post-drug difference in average cardiac index and PA diastolic 
pressure will be compared between the dantrolene and the placebo group. We expect the cardiac output to 
increase and PA diastolic pressure to decrease substantially after drug in the treatment arm, while no change is 
expected in the placebo arm. However, to allow for unexpected effects (e.g. negative inotropy), two-sided tests 
will be performed. With 56 patients treated with dantrolene and 28 with placebo, the study will provide 80% power 
to detect a 0.6xSD difference in the average cardiac index and the average PA diastolic pressure change 

 
 
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between the two study arms. For instance, if the pre-drug PA diastolic pressure measurements have a SD of 4 
mmHg, we will have 80% power to detect a difference of +/- 2.4 mmHg in PA diastolic pressure. If the pre-drug 
average PA diastolic pressure is 12 mmHg, this means a 20% change in the treatment arm is detectable 
assuming there is no change in the control arm. Each primary endpoint will be tested at the two-sided 5% 
significance level. 
Inducibility Endpoints: The primary hypothesis in Aim 3 tests whether RyR2 inhibition with dantrolene reduces 
the susceptibility to ventricular arrhythmias. Susceptibility to ventricular arrhythmias is measured by testing the 
inducibility of VT/VF using programmed ventricular stimulation.  
 Primary Analysis Aim 3:  
 Inducibility 
of 
Ventricular 
Arrhythmias: 
Ventricular 
arrhythmias are defined as monomorphic or polymorphic 
VT, or VF. The primary endpoint is either a sustained 
hemodynamically stable ventricular arrhythmia of ≥10 
seconds duration or an unstable ventricular arrhythmia 
requiring termination (with pacing or defibrillation), or two 
episodes of non-sustained ventricular arrhythmia defined as 
an episode lasting 3-9 seconds. To maximize statistical 
power, the primary analysis utilizes an ordinal endpoint that 
measures the relative inducibility based on the stage of the 
induction protocol and the arrhythmia induced (Table 5). 
Multivariable regression models are built.  Univariable 
logistic regression modeling will be used to test whether 
treatment with dantrolene is associated with the category of inducibility (ordinal endpoint) measured post-study 
drug with adjustment for the stage of inducibility measured pre-study drug. Multivariable modeling includes 
additional adjustment for age (continuous), sex, amiodarone exposure (yes/no), change in beta-agonist 
exposure, and time from start of drug to post-drug stimulation protocol. Models will be evaluated for goodness-
of-fit, and internally validated for calibration/discrimination with bootstrap.  
 
 Secondary Analyses Aim 3: A binary endpoint of inducibility 
(yes/no) is analyzed using logistic regression with the same 
covariate adjustment described for the primary analysis. Another 
secondary analysis tests the idea that RyR2 inhibition with 
dantrolene may alter inducible ventricular arrhythmias due to 
changes in conduction and refractoriness and hemodynamic 
improvement. An ordinal variable defining the cardiac rhythm 
stability class is analyzed (Table 6) using multivariable ordinal 
logistic regression. A secondary analysis will explore all the ventricular arrhythmia susceptibility endpoints for an 
interaction between cardiomyopathy type (ischemic versus non-ischemic) and treatment with dantrolene. 
Definitions:  1) Stable monomorphic VT is mean arterial pressure at 10 seconds (MAP10) or immediately prior to 
termination of >50 mmHg; 2) Unstable VT is MAP10 or prior to termination of <50 mmHg; 3) Pleormorphic VT is 
more than one QRS morphology for 3 or more consecutive beats during the same VT episode; and 4) 
Polymorphic VT is a continually changing QRS morphology.54Sample Size and Power Aim 3:  Primary Endpoint: 
The sample size selected for this trial was calculated to adequately power the analysis of the inducibility endpoint 
in Aim 3 which is an ordinal variable (Table 5). With N=84 patients (randomized to placebo and treatment in 1:2 
ratio) with an expected 50% of patients in the placebo arm non-inducible, and 10% in each of the other 
categories, the study will achieve 80% power to detect an odds ratio (OR) of 0.3 at the two-sided significance 
level of 5%. With an anticipated 20% withdrawal rate prior to study drug, up to a total number of 105 patients will 
be enrolled in the study to ensure statistical power. Secondary Endpoints: 1) The binary status of inducibility 
(yes/no). With an effective sample size of 84, the study provides 80% power to detect an OR of 0.25, and a 70% 
power to detect an OR of 0.3 at the two-sided significance level of 5%.  
Assessment of Safety (Aim 3): An interim analysis will be performed by the DSMB when 50% of the target 
enrollment is reached to test for an association between dantrolene or placebo and significant adverse 
events/harm. In order to preserve statistical power for the secondary analyses, the interim analysis will not 
include early stopping rules for efficacy. Although none of our Phase I pre-clinical or clinical data have raised 
Table 5. Ordinal Inducibility Endpoint 
 
Category 
 
Least 
inducible 
↓ 
↓ 
↓ 
↓ 
↓ 
↓ 
Most 
inducible 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
No sustained VT/VF or NSVT 
1 episodes of NSVT with S4 
1 episodes of NSVT with S3 
1 episodes of NSVT with S2 
2 episodes of NSVT with S4* 
2 episodes of NSVT with S3* 
2 episodes of NSVT with S2* 
Sustained VT/VF with S4 
Sustained VT/VF with S3 
Sustained VT/VF with S2 
*the category assigned for 2 episodes of NSVT is based 
on the extrastimulus used to induce the second episode of 
NSVT. 
Table 6. Secondary analysis of rhythm stability 
 
 
Category 
 
Least 
Stable 
↓ 
↓ 
Most 
Stable 
1 
2 
3 
4 
5 
6 
Ventricular Fibrillation 
Polymorphic VT 
Pleomorphic VT 
Unstable Monomorphic VT 
Stable Monomorphic VT 
No Inducible VT/VF 

 
 
21 
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safety concerns, and no adverse events have been observed, we planned the assessment of safety, which will 
be based on the frequency of adverse events. Other safety data such as blood pressure will be considered. All 
adverse events recorded during the study will be summarized. See Ethical Aspects of the Proposed Research 
for DSMB.  
 
Respiratory Mechanics and Ventilation Endpoints: To monitor adequacy of ventilation, maintain safety, and 
measure the effect of dantrolene administration on ventilation, the anesthesiologist will monitor respiration and 
oxygenation as is standard of care for all patients during procedures and surgeries. The primary endpoint of the 
Respiratory Mechanics substudy is the incidence of the addition of respiratory support, including bag/mask 
ventilation, CPAP, BiPAP, LMA, or tracheal intubation, from the time of study drug administration until 120 
minutes after study drug administration. Placement of airway support devices is at the discretion of the attending 
anesthesiologist and will be based on standard safety parameters such as oxygen saturation, adequacy of 
ventilation, and airway obstruction. Secondary endpoints include additional markers of respiratory mechanics 
and strength including negative inspiratory force, dynamometer measured grip strength, accelerometer 
(respiratory rate, tidal volume, minute ventilation), markers of gas exchange (arterial pH, pCO2, pO2, base 
excess), and electrolytes. Tidal volume, respiratory rate, and minute ventilation parameters will be continuously 
collected (recorded every minute) as available. We will summarize these data prior to study drug administration 
and at 10 and 20-minutes after dantrolene administration. 
Prior to the ablation procedure and administration of anesthesia, a baseline negative inspiratory force 
(NIF) will be collected x3 in the supine position. A repeat NIF measurement will be again collected in the PACU 
2 hours post dantrolene administration and then again at 24 hours. We will summarize NIF data prior to and 
following dantrolene administration. 
Arterial blood gas (ABG) sampling will be performed at four time points: intraoperatively just prior to 
dantrolene administration, 10- and 20-minutes post dantrolene administration, and 2 hours post dantrolene 
administration in the PACU. ABG sampling will allow sampling of respiratory mechanic parameters including pH, 
pCO2, pO2, and base deficit/excess. We will summarize ABG data at these four timepoints. 
 
Muscle Strength Endpoints: We will assess grip strength as measured with a handheld dynamometer x3 prior 
to the ablation procedure and administration of anesthesia and 2 hours following dantrolene administration and 
then again at 24 hours. We will summarize these data. 
11.0 
PRIVACY AND CONFIDENTIALITY ISSUES 
All records are retained on password-protected computers accessible only to members of the study team. 
Computers containing these records are only connected to networks if they include appropriate firewalls and 
security measures. Deidentified records and DNA samples may be shared with other investigators who have 
IRB-approved protocols and who agree to comply with the protections provided at this institution. These research 
materials are transferred only by secure methods. The identity of any individuals and their families are not to be 
revealed in any publication without their written informed consent. 
 
12.0 
FOLLOW-UP AND RECORD RETENTION 
The expected duration of this study is estimated to be 6 months. The study results will be retained for at least 
six years after the study is completed. At that time, the research information not already in the medical record 
will be destroyed.  
 
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