A novel RyR 1 inhibitor prevents and rescues sudden death in a mouse model of malignant 1 hyperthermia and heat stroke 2 3

33 Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, 34 hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe 35 heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very 36 poor water solubility and long plasma half-life. We show here that a novel RyR1-selective 37 inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), 38 effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. 39 Cpd1 reduced resting intracellular Ca2+, inhibited halothaneand isoflurane-induced Ca2+ 40 release, suppressed caffeine-induced contracture in skeletal muscle, reduced sarcolemmal 41 cation influx, and prevented or reversed the fulminant MH crisis induced by isoflurane 42 anesthesia and rescued animals from heat stroke caused by environmental heat stress. 43 Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over 44 dantrolene. Cpd1 has the potential to be a promising new candidate for effective treatment of 45 patients carrying RyR1 mutations. 46 47 Introduction 48 The type 1 ryanodine receptor (RYR1; MIM# 180901) is a Ca2+-release channel in the 49 sarcoplasmic reticulum (SR) of skeletal muscle that plays a central role in muscle contraction1,2. 50

6 clinical MH testing 37 . Whereas halothane up to 0.1% (v/v) did not change [Ca 2+ ]i in WT cells,126 it caused increase in [Ca 2+ ]i in R2509C cells in a dose-dependent manner (Fig. 2b, 2c). 127 Fig. 1a, 1b) and completely abolished halothane induced increases in [Ca 2+ ]i (Fig. 2b, 2c). We 129 then used the ratiometric Ca 2+ indicator fura-2 to estimate the unstimulated resting [Ca 2+ ]i and 130 whether or not it could be modified by Cpd1. At rest fura-2 F340/F380 ratios were significantly 131 higher in R2509C cells than in WT and Cpd1 reduced [Ca 2+ ]i in R2509C cells in a dose-132 dependent manner but had no significant effect on WT (Fig. 2d). Similar to the effects of 133 halothane, exposure to isoflurane increased [Ca 2+ ]i in R2509C but not in WT cells and Cpd1 134 completely prevented this effect. (Fig. 2e, 2f). 135

Pretreatment of R2509C cells with Cpd1 (100 nM) reduced resting [Ca 2+ ]i (Supplementary 128
It has been shown that sarcolemmal cation influx is accelerated in skeletal muscle of 136 several MH model mice compared with WT mice 38, 39 . This is due to enhanced sarcolemmal 137 Ca 2+ entry in response to chronic reduced SR Ca 2+ load. We therefore measured resting Mn 2+ 138 quench of fura-2 fluorescence. Unexpectedly, rate of Mn 2+ quench in R2509C was not different 139 from WT and in this model Cpd1 had no effect (Supplementary Fig. 2a, 2b). 140 141

Effect of Cpd1 on contraction force in isolated muscles 142
The In Vitro Contracture Test (IVCT) using muscle biopsies is widely used for 143 diagnosis of MH susceptibility in humans 40 . A subject is considered MH positive if the muscle 144 exhibited a greater contracture at a lower dose of caffeine or halothane. We therefore tested the 145 effect of Cpd1 on caffeine-induced contracture in soleus muscles isolated from RYR1-p.R2509C 146 mice. WT muscles showed only minimal contracture in responses to caffeine up to 20 mM (Fig.  147   3a). In contrast, R2509C muscles exhibited dose-dependent contracture during exposure to 148 caffeine and pretreatment with 3 µM Cpd1 decreased caffeine-induced contracture tension of 7 It has been reported that soleus muscles from MH model mice exhibit heat-induced 151 increase in the basal force at temperatures of 35°C or higher 33 . Basal tension at 42°C was 152 significantly increased in R2509C solei compared to WT muscles (Fig. 3c, 3d) and pretreatment 153 with 3 µM Cpd1 significantly reduced the heat-induced contracture in R2509C muscles. 154 Dantrolene is known to suppress twitch and tetanic contractions in both normal and 155 MH muscles 41,42 . We tested whether Cpd1 affects twitch and tetanic contractions and whether 156 or not there was a difference in the Cpd1 effect between MH and WT. Untreated soleus muscles 157 from both WT and R2509C mice showed similar twitch and tetanic contraction force and 158 treatment with 3 µM Cpd1 significantly reduced both twitch and tetanic contraction force in 159 both groups ( Fig. 3e-3g). The effect was stronger on twitch (72% inhibition) (Fig. 3f) than on 160 tetanic (35% inhibition at 100 Hz) ( Fig. 3g) contractions, which is similar to dantrolene 41 . 161 162

Effect of Cpd1 on in vivo isoflurane challenge 163
We next tested whether Cpd1 can prevent or treat MH episodes in R2509C mice in 164 vivo. We first tested the preventive effect of Cpd1 on MH episodes in response to isoflurane 165 exposure. We used male mice for the experiments, since male mice were more sensitive to 166 isoflurane than female mice (see Fig. 1f). Doses of either 3 mg/kg or 10 mg/kg of Cpd1 167 solubilized in normal saline were administered i.p. 10 min before isoflurane challenge. The 3 168 mg/kg dose did not prevent rise in rectal temperature and only 1 out of 6 mice survived (Fig.  169 4a-4c). Time to death for mice treated at 3 mg/kg (47±15 min, n=5) was not significantly 170 different than that for control mice (53±24 min, n=9). However, with the 10 mg/kg dose, Cpd1 171 successfully prevented any rise in rectal temperature (Fig. 4a, 4b). All the treated mice survived 172 90 min after induction of anesthesia (Fig. 4c) and they behaved normally for at least 24 h after 173 recovery from anesthesia. 174 We next tested whether Cpd1 could rescue mice from MH episodes. R2509C mice when rectal temperature reached 39°C. The 3 mg/kg dose was able to reduce rectal temperature 177 by -0.03±0.81°C at 10 min after administration, compared to an increase of 1.79±0.82°C in 178 controls, but rectal temperature started to rise again ~30 min after administration of the drug in 179 some mice (Fig. 4d, 4e). The 10 mg/kg dose decreased body temperature by -0.64±0.29°C at 180 10 min after administration and maintained it at that level until the experiment was terminated 181 60 min after administration of the drug. At that time 60% of the mice in the 3 mg/kg group and 182 100% of the mice in the 10 mg/kg group survived (Fig. 4f) . 3a). Almost all the mice responded to heat and died (Supplementary Fig. 3b). Although 193 the time from start of heat stress to death largely varied substantially among individual mice, 194 the variation in time to death after reaching body temperatures of 38°C (36±13 min, n=13) or 195 39°C (22±8 min, n=12) became smaller (Supplementary Fig. 3c). 196 Using this model, we initially tested the preventive effect of Cpd1 on the heat stroke.

5d). 202
We next tested whether Cpd1 could rescue mice from environmental heat stroke. The 203 R2509C mice were incubated in the test chamber and the drug was administered i.p. when their 204 rectal temperature reached 39°C. All the untreated mice died within 30 min of the time of 205 injection (time to death, 22±8 min, n=13). The 3 mg/kg Cpd1 dose slowed rise in the body 206 temperature post injection (Fig. 5e, 5f) and 3 out of 5 mice survived 60 min after administration 207 (Fig. 5g). The 10 mg/kg Cpd1 dose showed more pronounced effect: it suppressed rise in the 208 body temperature and 10 out of 11 mice survived for 60 min after administration. After 209 removing from the heat stress environment, the survivors behaved normally for at least 24 h 210 after being and regaining consciousness. When the drug was administered at body temperature 211 of 38°C, after which most untreated mice died within 60 min (time to death, 36±13 min, n=13), 212 both the 3 and 10 mg/kg Cpd1 doses strongly suppressed the continued rise in the body 213 temperature (Supplementary Fig. 4a, 4b) and almost all mice survived 60 min after its 214 administration (Supplementary Fig. 4c). 215 216

Pharmacokinetics of Cpd1 217
We observed transient effects with the 3mg/kg dose of Cpd1 in rescue experiments in both in 218 vivo isoflurane and heat stress challenges with R2509C mice (see Fig. 4d, 5e). This implies fast 219 metabolism or excretion of the drug in mice. We therefore measured plasma concentration of 220 Cpd1 after i.p. administration. Cpd1 rapidly declined in mouse blood following first order 221 kinetics with t1/2 of ~8 min for both the 3 mg/kg and 10 mg/kg dose (Fig. 6a). There was no sex 222 difference in the kinetics of Cpd1 (Supplementary Fig. 5a, 5b). Since Cpd1 has hydrophobic 223 moiety of octyl group (see Fig. 2), it can be metabolized in the liver. In vitro drug metabolism 224 assay using mouse liver microsomes demonstrated that Cpd1 was reduced to 20% and 5% of 225 the original amount at 10 min and 60 min, respectively (Fig. 6b). Cpd1 was metabolized more 226 slowly by human liver microsomes; it was reduced to 20% at 60 min. 227 The duration of the effect of Cpd1 in mice was also evaluated using measurements of 228 in vivo muscle strength, since we showed that the drug inhibited twitch and tetanic contractions 229 in isolated skeletal muscle (see Fig. 2). Grip strength tests demonstrated that muscle strength 230 of WT mice was reduced by 15% 10 min after administration of 10 mg/kg Cpd1, and that this 231 deficit was almost completely recovered at 60 min, suggesting short duration of the medicinal 232 effect in vivo (Fig. 6c). No sex nor genotype differences were observed in the effect of Cpd1 233 on in vivo muscle contraction strength (Supplementary Fig. 6a-6c). 234 The above results indicate that transient effects of Cpd1 in the isoflurane challenge 235 and heat challenge experiments are due to rapid metabolism of the drug in mice. In fact, second 236 and third administrations of the 3 mg/kg dose when rectal temperature reached 39°C 237 successfully decreased the rectal temperature (Supplementary Fig. 7). We therefore tested 238 whether repeated administrations of Cpd1 could prevent mice from the crisis. Eight 239 administrations of 1 mg/kg Cpd1 at 10 min intervals successfully prevented rise in the rectal 240 temperature by exposure either to isoflurane (Fig. 6d) or to heat stress (Fig. 6e) and all the mice 241 survived the full 90 min after initiating either challenge (Fig. 6f). Pharmacokinetic analysis of 242 Cpd1 after repeated administrations revealed that Cpd1 rapidly declined from mouse blood as 243 was case of single administration (Fig. 6g). Interestingly, t1/2 was slightly longer in female 244 (14.8±2.1 min, n=3) than in male (9.6±2.7 min, n=3), whereas Cmax was significantly higher in 245 males (0.67±0.11 µg/mL, n=3) than in females (0.15±0.11 µg/mL, n=3). Muscle weakness was 246 transiently observed after the repeated administrations in both male and female, but it returned 247 to normal by 60 min (Fig. 6h) smaller. When mice were exposed to 1.5% (v/v) isoflurane, [Ca 2+ ]i rapidly increased in 258 untreated R163C muscles (1226 ± 154 nM, n = 19), indicating a MH episode (Fig. 7b, 7c). 259 Administration of isoflurane had no effect on [Ca 2+ ]i in WT muscles (121 ± 4 nM, n = 18) (Fig.  260 7c). Pretreatment with Cpd1 suppressed increase in [Ca 2+ ]i in R163C muscle in a dose-261 dependent manner (Fig. 7b, 7c) and effectively prevented the MH episode and death at doses 262 of 5 and 10 mg/kg (Fig. 7d). When Cpd1 at 10 mg/kg was administered to R163C mice 15 min 263 after induction of isoflurane anesthesia, it was able to abort the MH crisis and rapidly decreased 264 [Ca 2+ ]i to the levels measured in WT muscle (Fig. 7e, 7f). We also tested the effect of Cpd1 on a third MH model using homozygous RYR1-p.G2435R 273 mice 30 . The G2435R mice exhibit severe heat stroke by environmental heat stress. When 274 unanesthetized restrained G2435R mice were exposed to an ambient temperature of 38 C, 275 their rectal temperature rapidly increased and reached 40°C within 20 min (the experiment was 276 terminated at this point) (Fig. 8a). Pretreatment of G2435R mice with Cpd1 (30 mg/kg) greatly 277 slowed the rise in rectal temperature (Fig. 8b).
compared to WT which is thought to be in response to chronic reduced SR Ca 2+ load 30 . To test 280 the effect of Cpd1 on sarcolemmal cation influx, we measured resting Mn 2+ quench of fura-2 281 fluorescence in isolated G2435R and WT skeletal muscle fibers. The rate of Mn 2+ quench in 282 G2435R fibers was twice that in WT fibers (Fig. 8c, 8d). Cpd1 reduced Mn 2+ quench in G2435R 283 fibers in a dose-dependent manner and the Mn 2+ quench rate at the 10 µM dose was the same 284 as untreated WT fibers. 285

286
Discussion 287 Here we examined the therapeutic effects of Cpd1, a novel RyR1-selective inhibitor 288 with high potency 26 , on the volatile anesthetic and increased environmental temperature-289 induced MH crisis. Cpd1 showed good solubility in normal saline (Table 1) These results provide crucial evidence that Cpd1 is capable of effectively preventing and 297 treating the MH crisis and heat stroke by inhibiting the RyR1 channel. 298 Several strains of MH mouse models carrying mutations in the RYR1 gene have 299 been reported to date 29, 30, 33, 34 . We created a novel mouse model carrying p.R2509C mutation a HEK293 expression system, the p.R2508C mutant exhibited the highest CICR activity 302 among mutants in the central region tested 28 .Whereas homozygous mice were embryonic 303 lethal, heterozygous mice grew normally and were as fertile as WT (Fig. 1). R2509C mice 304 showed a halothane-and isoflurane-induced increase in [Ca 2+ ]i (Fig. 2) and caffeine-and 305 heat-induced contracture in skeletal muscle (Fig. 3). In addition, they exhibited a MH crisis 306 when exposed to isoflurane anesthesia ( Fig. 1) and heat stroke when exposed to 307 environmental heat stress (Fig. 5, Supplementary Fig. 3). These properties are common to 308 those seen in the other model mice carrying severe MH mutations in the RYR1 gene, e.g., 309 p.R163C 29 and p.Y524S 33 . Thus, R2509C mice are a useful model for MH research. 310 Interestingly, time to death by isoflurane anesthesia of R2509C mice (64±30 min, Fig. 1) was 311 much longer than that of R163C mice (<20 min, Fig. 7). Correspondingly, increase in resting 312 [Ca 2+ ]i appeared smaller in R2509C muscle (Fig. 2) than in R163C muscle (Fig. 7, 313 Supplementary Fig. 8). These findings suggest that R2509C mice show milder phenotype 314 than R163C mice. 315 Although dantrolene is the only compound that is available and used to treat and 316 prevent MH, a major problem of dantrolene in clinical use is its poor aqueous solubility 22 . 317 First, dantrolene must be solubilized with sterile water and injected or infused by the 318 intravenous route, since it is virtually insoluble in normal saline. Second, dantrolene is 319 prepared at a diluted concentration (0.33 mg/mL) due to its low water solubility, which makes 320 rapid preparation difficult in emergency situations. Whereas the second problem has recently 321 been solved by nanocrystalline suspension of dantrolene (Ryanodex ® ) which can be injected 322 at a concentration of 50 mg/mL 43 , the first problem remains unsolved to date. We found that of the drug by infusion using the intravenous route and expands usage of the drug before and 326 after surgery for prevention of MH and postoperative MH 44 . 327 Another drawback of dantrolene in clinical use is its long plasma half-life (10-12 h 328 in humans after intravenous injection) 45 . Therefore, it is difficult to control the appropriate 329 plasma concentration and reduce side effects such as muscle weakness 22 . Pharmacokinetic 330 analysis revealed that Cpd1 is rapidly metabolized in mice with plasma half-life of ~8 min, 331 probably by metabolism by the liver (Fig. 6). Muscle weakness was completely recovered 332 within 60 min (Fig. 6). Rapid metabolism of Cpd1 was also true even after repeated 333 administration of 1 mg/kg Cpd1 at 10 min intervals, which successfully prevented rise in the 334 rectal temperature by exposure either to isoflurane or heat stress (Fig. 6). Thus, Cpd1 may be 335 beneficial for a treatment of MH episodes without the concern of residual weakness. 336 Heat stroke is a life-threatening condition clinically diagnosed as a severe elevation 337 in body temperature with central nervous system dysfunction 12, 13 . It has been reported that 338 MH mutations in the RYR1 gene is implicated in some heat stroke 14, 15, 16 . Dantrolene has 339 therapeutic effects on some patients with severe exertional heat stroke 46 . Cpd1 effectively 340 prevents and treats fulminant heat stroke caused by environmental heat stress in both R2509C 341 (Figs. 5, 6) and G2435R (Fig. 8) model mice. Since Cpd1 is soluble in normal saline, it has 342 the potential to be administered by continuous infusion which could be necessary for 343 treatment of severe heat stroke without the concern for prolonged muscle weakness. 344 Dantrolene is also used for the treatment of neuroleptic malignant syndrome, a life-345 threatening neurologic emergency associated with the use of antipsychotic agents 47 as well as 346 overdose of 2,4-dinitrophenol (a prohibited weight loss agent that interrupts ATP synthesis 347 and causes hyperthermia) 48 . Since Cpd1 has similar effects in preventing and treating MH 348 and heat stroke, it might also be a potential candidate for treatment of these emergency 349 situations. was chemically synthesized for homologous recombination (Fig. 1) with the sCMOS camera (Zyla, Andor, Belfast, Northern Ireland). Cal520 was excited at 421 480±15 nm, and fluorescence was measured at wavelengths of 525±25 nm. Fura-2 was 422 excited at alternating lights of 340±6 nm and 380±6 nm, fluorescent light was detected at 423 510±40 nm, and the ratio of fluorescence excited at 340 nm to that at 380 nm was determined. 424 Imaging experiments were carried out at 26°C by superfusing a HEPES-Krebs solution with or without halothane or isoflurane. Cpd1 was administered 5 min before measurements. For   were generated by CRISPR/Cas9 system using single-stranded donor oligonucleotide carrying