A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

MAJOR ISSUES 1. In the abstract it is stated that Compound 1 effectively rescues MH and heat stroke in several mouse models relevant to MH, however there is only evidence for this effect in the R2509C mice. The ability of the drug to normalize cytoplasmic Ca2+ (R163C) and decrease sarcolemmal Ca2+ influx in the G2435R model is not sufficient evidence that Compound 1 can also treat MH in those models. The idea of testing Compound 1 on multiple MH models provides strong evidence for the efficacy of the drug, but this needs to be tested more thoroughly 2. Functional analyses from the literature show that different mutations have variable effects on RyR1 and may result in different effects on cytoplasmic Ca2+, SR load etc. Since this mouse model has not been characterized before it should be shown whether it has increased resting cytoplasmic Ca2+ (like the R163C muscle) or increased sarcolemmal Ca2+ influx as in the G2435R muscle, rather then extrapolating the results obtained in those two mouse models to the new mouse model.
3. In the discussion it is proposed that continuous IV infusion of Compound 1 may be necessary due to the rapid clearance and transient effect of the drug during MH episodes. Has plasma clearance, liver metabolism or muscle function been analyzed following longer term IV administration or after repeated IP injections? These studies would be valuable in assessing the safety of the drug, and should also be repeated in female mice. 4. Has this drug been tested in intact skeletal muscle before? Has there been any work done to confirm that in skeletal muscle the drug is specifically targeting RyR1? For example, have you repeated the caffeine contracture study with 4-CMC or ryanodine? 5. The grip strength tests shown in Figure 6 should be repeated in mutant mice. It should also be stated whether the investigators who did grip strength tests were blinded.
6. How long were muscles and fibers treated with Compound 1? Also, it is unclear whether the sodium salt was used for all experiments (including isolated muscles and fibers) or just the in vivo experiments. If two different forms were used it should be clearly stated which form was used for each experiment. Ideally the same form of drug that was used to prevent MH in vivo should also be used in the in vitro experiments. 7. How long was mouse survival followed? It is only stated that mice treated with 10mg/kg Compound 1 survived for 60 min, but in patients and mouse models of MH death may occur at a later stage after drug/heat exposure. Did you ever monitor the mice for a 24hr period after isoflurane or heat exposure? 8. The data demonstrate that the R2509C model has enhanced heat sensitivity however there was no in vitro work done to show that the same cellular responses (calcium release, muscle contraction) that are triggered by isoflurane are also triggered by heat. The evidence for Compound 1 being an effective therapy for heat sensitivity in these mice is limited at the moment. While the drug does appear to delay death when given to mice already experiencing heat-induced hyperthermia, by the end of the 60 min exposure rectal temperature was clearly increasing. Therefore, these results would be more convincing if mouse survival and rectal temperature was monitored after the completion of the 60 min heat exposure.
MINOR ISSUES: 1. Figure 1 -Time to death is compared for male and female mice, but max rectal temperature is only shown for males. Was there a difference between males and females? 2. Compound 1 causes a significant decrease in soleus force production from het mice. Were there any effects of Compound 1 on WT muscle force production?
3. What age mice are used? This should be stated in the methods and/or results section.
4. The correct nomenclature should always be used when referring to the mouse strain instead of using the terms "het" and "hom", especially since multiple mouse models are used. There's also inconsistent use of "Wt" versus "WT". 5. Line 121 -provide a citation for halothane stimulation of RyR1 and its use in MH testing.
6. Figure 2 -individual data points should be shown for panel D.
7. Figure 5 -Individual data points should be shown for panel D.
8. Line 379 -change "x" to "X" 9. There are many examples of incorrect wording and grammar throughout the text. Please proofread carefully and correct so that the text is easier to read. Some examples: a. Line 160 -change "inducing" to "induction of" b. Line 173 -change "of existing" to "in other" c. Line 176 -change "was maintained a relatively" to "was maintained at a relatively" d. Line 182 -add "the" after tested e. Line 188 -change "at" to "in" f. Line 419 -add "at" after achieved g. Line 487 -change "points" to "point". h. Line 667 -change "fully" to "full" Reviewer #2: Remarks to the Author: Comments for Authors: Malignant hyperthermia (MH), a life-threatening disorder triggered by administration of volatile anesthetics (halothane, isofluorane, etc.), has been linked to mutations in the type-1 ryanodine receptor (RyR1), the Ca2+ release channel in skeletal muscle. Similar hyperthermic reactions (known as heat stroke; HS) can be triggered by exposure to high environmental temperature. Dantrolene is the only approved drug for acute treatment of MH crises (though, not used in case of HS). Nevertheless, dantrolene has some disadvantages for clinical use: a) poor water solubility which makes its rapid preparation difficult in emergency situations; b) long plasma half-life, which causes side effects in patients (such as prolonged muscle weakness). No alternative drugs have been developed over 50 years to replace Dantrolene.
In this paper the authors created and characterized a new MH mouse model RYR1-p.R2509C; and identified in a oxolinic acid derivative (Compound 1, which was already reported as an inhibitor of RyR1 in 2019) a drug that effectively prevents and rescues MH and HS crisis triggered by isoflurane and heat in mice.
The results are important as they characterize a new drug which has the potential to replace dantrolene.
However, the paper may need some adjustments in the way data are currently presented.
Main Comments: 1. Fig. 2d and e: could the authors explain why they used Soleus muscle (a slow twitch muscle) for these IVCTs? Muscles in mice are mostly fast-twitch. Title: A novel saline-soluble, rapidly-metabolized RyR1 inhibitor rescues volatile anesthesia induced death and environmental heat stroke in a mouse model relevant to malignant hyperthermia. Title could be improved. Suggestion 1: …. rescues volatile anesthesia-induced death and… Suggestion 2: A novel RyR1 inhibitor prevents and rescues sudden death in a mouse model of malignant hyperthermia and heat stroke.

Introduction:
Page 3, line 55: Correct as follow: This MECHANISM is referred to as….. Page 3, line 56: The RyR1 channel can also be directly activated by Ca2+…. I was under the impression that Ca2+ induced Ca2+ release was a mechanism used primarily by RyR2 and RyR3, while RyR1 is less sensitive to it.  We carried out additional in vivo experiments about the effect of Compound 1 (Cpd1) on MH model mice; isoflurane-induced MH episodes with R163C mice (Fig. 7) and heat stroke by environmental heat stress with G2435R mice (Fig. 8) (Fig. 2) and sarcolemmal Mn 2+ influx ( Supplementary   Fig. 2) of skeletal muscles from R2509C mice using fura-2. We found that [Ca 2+ ] i in R2509C muscle was significantly higher than that of WT and Cpd1 reduced it to the level of WT. Unexpectedly, Mn 2+ influx was not different than WT in R2509C muscle.

In the discussion it is proposed that continuous IV infusion of Compound 1 may be necessary due to the rapid clearance and transient effect of the drug during MH
episodes. Has plasma clearance, liver metabolism or muscle function been analyzed following longer term IV administration or after repeated IP injections? These studies would be valuable in assessing the safety of the drug, and should also be repeated in female mice.
We repeated plasma clearance, liver metabolism and muscle function of Cpd1 after single injection in female mice and found no sex differences (Supplementary Fig. 5).

Has this drug been tested in intact skeletal muscle before? Has there been any work done to confirm that in skeletal muscle the drug is specifically targeting RyR1? For example, have you repeated the caffeine contracture study with 4-CMC or ryanodine?
We show in the current manuscript that Cpd1 reduces resting Ca 2+ (Figs. 2 and 7,   Supplementary Fig. 8) and prevents halothane-and isoflurane-induced Ca 2+ release ( Fig. 2) in MH susceptible skeletal muscle. These findings strongly suggest that Cpd1 inhibits RyR1. We cannot completely exclude the possibility that Cpd1 also targets other proteins. However, the fact that there are no apparent side effects of Cpd1 other than muscle weakness in mice strongly suggests that Cpd1 specifically targets RyR1 in skeletal muscle. According to the reviewer's suggestion, we carried out contracture study with 4-CmC (figures for reviewers). Although 4-CmC elicited muscle contracture, the effect was not different between WT and R2509C muscles. This is consistent with the previous report that 4-CmC cannot discriminate MH status (Weigl et al., Anesth Analg 99:103-107, 2004). Figure 6 should be repeated in mutant mice. It should also be stated whether the investigators who did grip strength tests were blinded.

The grip strength tests shown in
We carried out the grip strength tests in R2509C mice (Supplementary Fig. 6). We found that muscle weakness was also observed in 10 min but recovered within 60 min after administration of Cpd1. These observations were described in Results (p. 10, lines 233-234). We stated in Methods that the investigators who performed the tests were blinded (p. 22, line 541-543). We monitored the mice for at least 24 h after isoflurane and heat exposure and found that all the survivors behaved normally. These observations were described in Results (p. 7,p. 8,[183][184]p. 9,.

How long were muscles and fibers treated with
8. The data demonstrate that the R2509C model has enhanced heat sensitivity however there was no in vitro work done to show that the same cellular responses (calcium release, muscle contraction) that are triggered by isoflurane are also triggered by heat.
The evidence for Compound 1 being an effective therapy for heat sensitivity in these mice is limited at the moment. While the drug does appear to delay death when given to mice already experiencing heat-induced hyperthermia, by the end of the 60 min exposure rectal temperature was clearly increasing. Therefore, these results would be more convincing if mouse survival and rectal temperature was monitored after the completion of the 60 min heat exposure.
To address the cellular responses triggered by heat, we carried out additional experiments of muscle contraction. We demonstrated a larger contracture of soleus muscles in R2509C muscle than in WT muscle when the bath temperature was raised to 42 o C and this was inhibited by Cpd1 (Fig. 3c, 3d). These observations were described in Results (p. 7,. Heat-induced contracture is consistent with the previous finding with Y522S MH model mice (Chelu et al., 2004). In the in vivo experiments all the survivors behaved normally for at least 24 h after being removed from the heat stress environment, and regaining consciousness. These observations were described in Results (p. 9, lines 209-211).

MINOR ISSUES:
1. Figure 1 -Time to death is compared for male and female mice, but max rectal temperature is only shown for males. Was there a difference between males and females?
We added maximum rectal temperature of female mice in Fig. 1. By including data for additional experiments, we re-evaluated sex differences. There was no significant sex difference in maximum rectal temperature (Fig. 1g) and time to death (Fig. 1h, 1i).
Therefore, we used male mice for studies of the preventive effect of Cpd1 on isoflurane-induced MH crisis. These observations were described in Results (p. 5, lines 104-107; p. 7, lines 166-167).

Compound 1 causes a significant decrease in soleus force production from het mice.
Were there any effects of Compound 1 on WT muscle force production?

What age mice are used? This should be stated in the methods and/or results section.
The ages of mice were described in methods section (p. 17, lines 405-406; p. 18, line 436; p. 19, lines 453, 467, and 475; p. 20, line 483).
4. The correct nomenclature should always be used when referring to the mouse strain instead of using the terms "het" and "hom", especially since multiple mouse models are used. There's also inconsistent use of "Wt" versus "WT".
We now refer to the mouse strain as R2509C, R163C, and G2435R, instead of het or hom. We apologize mistake in expression of wild type. In the revised version WT was consistently used.

Line 121 -provide a citation for halothane stimulation of RyR1 and its use in MH testing.
We cited an article (Hopkins, Br J Anaesth, 2011) for the effect of halothane and its use in MH testing (p. 6, line 126).

Figure 2 -individual data points should be shown for panel D.
We added individual data points in the figure (Fig. 3b).

Figure 5 -Individual data points should be shown for panel D.
Since data points in this figure (Fig. 8d) are too many (n=39~78), data were given as box and whisker plots without individual data points.

There are many examples of incorrect wording and grammar throughout the text.
Please proofread carefully and correct so that the text is easier to read. Some examples: a. Line  c. Line 176 -change "was maintained a relatively" to "was maintained at a relatively" d. Line 182 -add "the" after tested e.  h. Line 667 -change "fully" to "full" Thank you very much for your kind editing. We corrected all the points you suggested.  Fig. 2d and e: could the authors explain why they used Soleus muscle (a slow twitch muscle) for these IVCTs? Muscles in mice are mostly fast-twitch.
We examined the caffeine contracture of EDL muscles, but no apparent contracture was detected up to 20 mM caffeine (see the attached figure for reviewers). Soleus and diaphragm muscles, not EDL muscle, were used for contraction experiments of Y522S mutant mice (Chelu et al, 2004). It seems that mouse fast muscle is difficult to elicit caffeine contracture. Fig 2b and c, but Isoflurane in Fig. 3?

Figs. 2 and 3: could the authors explain why they used Halothane in
We carried out additional experiments of isoflurane on [Ca 2+ ] i in skeletal muscle cells (Fig. 2e, 2f). We found that isoflurane also increased [Ca 2+ ] i and Cpd1 completely prevent it. These observations were described in Results (p. 6, lines 133-135).

Comparison between Figs. 3 and 4: a. compound 1 was administered in one case
when rectal temperature reached 39oC, but at 38oC in the second case: can the authors explain why?
According to reviewer's suggestion, we presented a figure in which Cpd1 was administered when rectal temperature reached 39°C at heat stress experiments ( Fig.   5e-5g). The original Fig. 4 (administration at 38°C) was moved to Supplementary Fig.   4 (p. 9, lines 203-209). Fig. 4 is INCOMPLETE, as in panels a-c the dose 3mg/kg is missing….. ??

b. while Fig 3 is complete, it looks like
In preliminary experiments, we found no effect of 3 mg/kg dose in heat stress experiments. So, we omitted the 3 mg/kg data. Since preventive effect is still weak even at 10 mg/kg, it is easily predicted that lower doses may have no or only minor effects. To show the same sets of data, we added maximum rectal temperature in heat stress experiments (Fig. 5b).

Minor Comments:
Title could be improved. Thank you very much for your suggestion. We changed the title to "A novel RyR1 inhibitor prevents and rescues sudden death in a mouse model of malignant hyperthermia and heat stroke".

Introduction:
Page 3, line 55: Correct as follow: This MECHANISM is referred to as…..