Characterizing SERCA function in murine skeletal muscles after 35-37 days of spaceflight

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Though the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage and thus may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content (sarcolipin, phospholamban, and neuronatin), and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35-37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA) we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA. As the soleus is severely affected by spaceflight, future studies should determine whether improving SERCA function in this muscle can mitigate muscle atrophy and weakness.


Introduction 54
It is well established that microgravity exposure during spaceflight comes with a great 55 deal of physiological and psychosocial challenges that can compromise astronaut health [1][2][3]. 56 Loss of muscle mass and strength is an important factor that can impede the astronaut's ability 57 to perform mission-related duties during space travel and upon return to Earth or partial gravity 58 (i.e., Moon or Mars). Mammals (i.e., humans and rodents) have evolved with the never-ending 59 downward pull of gravity on Earth, and therefore postural muscles like the soleus are known to 60 be most affected with spaceflight. Similar to unloading models on Earth, spaceflight and 61 microgravity exposure in rodents unloads the postural soleus causing extensive muscle atrophy 62 and a fiber type shift from slow-oxidative to fast-glycolytic [4][5][6][7][8][9]. Similar changes have also been 63 observed in human soleus muscles after 17 days of spaceflight [10]. Not surprisingly, the 64 reduction in muscle mass has also been associated with muscle weakness [10]. However, a 65 recent study highlights the fact that skeletal muscle unloading causes disproportionate losses in 66 muscle mass and strength, with the decline in muscle strength occurring at a faster rate than 67 muscle mass [11]. While this suggests that the muscle weakness caused by unloading is not 68 merely due to a reduction in muscle size, the underlying cellular mechanisms behind this 69 disproportionate loss in muscle force are still poorly understood, and some have suggested a 70 role for Ca 2+ dysregulation and increased reactive oxygen/nitrogen species (RONS) production 71 [11,12]. 72 The sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) pump is responsible for 73 lowering cytosolic Ca 2+ by actively bringing it into the sarco(endo)plasmic reticulum (SR) [13, 74 14]. There are two main SERCA isoforms in skeletal muscle: SERCA1a and SERCA2a (the fast 75 and slow isoforms, respectively) [15]. Partly consistent with the slow-to-fast fiber type transition, 76 recent proteomic analyses have shown that ~30 days of spaceflight significantly increased 77 SERCA1a mRNA in the murine soleus, without altering SERCA2a mRNA [9]. This is similar to 78 Results 104 SERCA function in soleus muscles from male and female spaceflown mice 105 We first characterized SERCA function in murine soleus muscles obtained from NASA's 106 Rodent Research 9 (RR9) mission. These mice were aboard the International Space Station 107 (ISS) for 33 days and exposed to microgravity for 35 days. Absolute soleus weights were 108 smaller in the flight group compared with Vivarium (VIV) and Ground Controls (GC) ( Table 1). 109 Assessment of SERCA function in the RR9 soleus demonstrates that spaceflight caused 110 significant impairments in the amount of ATP-dependent Ca 2+ uptake with significantly elevated 111 area under the curve (AUC) when compared to VIV and GC ( Figure 1A, B). To investigate 112 whether the reductions in Ca 2+ uptake were due to changes in SERCA protein content, Western 113 blotting was employed. Interestingly, we found significant increases in both SERCA2a and 114 SERCA1a protein content in the flight group compared to VIV and GC ( Figure 1C, D). We also 115 found significant reductions in PLN and NNAT, but significant increases in SLN in the flight 116 group compared to GC and VIV ( Figure 1C, D). Due to limitations in the amount of sample 117 available, we could not measure directly the amount of S-nitrosylation and T-nitration on the 118 SERCA isoforms specifically. However, measuring total protein levels revealed that the flight 119 group had significant elevations in both total protein S-nitrosylation and T-nitration compared to 120 GC and VIV ( Figure 1C, D). 121 In consideration of a potential effect of biological sex, we also obtained murine soleus 122 samples from female mice from NASA's RR1 mission. These mice were aboard the ISS for 33 123 days and exposed to microgravity for 37 days. Consistent with soleus muscles from male mice, 124 soleus muscles from female mice were smaller in the flight group vs GC and VIV (Table 1). 125 However, since we were only able to obtain a limited number of samples (n = 4 per group) we 126 combined GC and VIV groups for statistical comparisons as there were no differences revealed 127 between these groups. Similar to the data obtained from male mice from the RR9 mission 128 ( Figure 1), we found that SERCA function was impaired with a significant reduction in Ca 2+ 129 uptake compared with GC/VIV (Figure2A and B). While there were no differences observed in 130 SERCA2a, we did find a significant increase in SERCA1a content as well as a significant 131 decrease in PLN in the flight group vs GC/VIV ( Figure 2C, D). We did not find any changes in 132 SLN or NNAT ( Figure 2C, D). Similar to male mice from the RR9 mission, protein S-nitrosylation 133 and T-nitration were higher in the flight group from the RR1 mission, albeit non-significantly 134 ( Figure 2C, D). 135 136 SERCA function in TA muscles from male spaceflown mice

137
We next examined SERCA function in the fast-glycolytic TA muscle. Like the soleus 138 muscle, TA muscles were smaller in the flight group compared with GC and VIV (Table 1). 139 However, and in contrast with the slow-twitch soleus, SERCA assessment showed a significant 140 increase in Ca 2+ uptake in the flight group with a significant reduction in AUC compared to VIV 141 and GC (Figure 2A, B). While fiber type transformations in the unloaded soleus have been well 142 characterized, less is known regarding the potential changes in fiber type composition in the TA 143 in response to spaceflight. Here, we found that the TA muscles from the flight group had no 144 changes in myosin heavy chain (MHC) IIa or IIx content, however, there was a significant 145 increase in MHC IIb compared with both VIV and GC ( Figure 2C). When examining SERCA 146 isoform content, we found no changes in SERCA2a, but there was a significant increase in 147 SERCA1a content in the flight group vs GC and VIV ( Figure 2D). SERCA regulatory proteins, 148 PLN, SLN and NNAT, were not detected in the TA with up to a 40 µg of total protein loaded 149 (data not shown). Furthermore, we did not find any changes in total protein S-nitrosylation and 150 T-nitration ( Figure 2D To our knowledge, ours is the first study to investigate SERCA function in soleus and TA 155 muscles from mice exposed to microgravity via spaceflight. We observed that, in the postural 156 soleus, Ca 2+ uptake was significantly reduced in the flight group in both male and female mice. 157 Associated with these findings were elevations in total protein T-nitration and S-nitrosylation, 158 which could implicate elevated RONS production in the impairment of SERCA function in the 159 postural soleus muscle during spaceflight. Contrarily, in the TA, we saw a significant 160 enhancement in Ca 2+ uptake in the flight group compared with GC and VIV, which was 161 associated with a fast fiber type transition and no change in total protein T-nitration or S-162 nitrosylation. Thus, our findings reveal important differences on the effects of spaceflight on 163 SERCA function between slow and fast muscle types. 164 Previous work with ground based and spaceflight models in both rodents and humans 165 have demonstrated that the postural soleus undergoes a fiber type shift towards a faster 166 phenotype and that the muscle displays significant atrophy and weakness [3, 5, 7, 10, 12, 17, 167 30-34]. While some previous work has suggested a potential role for Ca 2+ dysregulation [11,12], 168 we are the first to demonstrate impaired SERCA Ca 2+ uptake in the space flown soleus of both 169 male and female mice. Consistent with such work, we did observe a significant increase in 170 SERCA1a protein content, the isoform predominantly associated with fast skeletal muscle [15]. 171 It is important to note that irrespective of isoform, the primary determinant of Ca 2+ uptake is 172 SERCA density [35]. However, the increase in SERCA1a and SERCA2a could not overcome 173 the stress of spaceflight in the postural soleus. While an increase in SERCA2a protein content 174 was also observed, we speculate that this may be a compensatory response attempting to (but 175 failing to) increase Ca 2+ uptake in the face of impaired Ca 2+ homeostasis. 176 Accompanying the changes in SERCA protein content, three SERCA regulators: PLN, 177 SLN, and NNAT were also investigated. PLN and NNAT are both highly conserved proteins 178 primarily found in oxidative skeletal muscle [22,36,37]. PLN allosterically binds to SERCA and 179 reduces its affinity for calcium [22,36], while NNAT has been shown to inhibit Ca 2+ uptake and 180 promote SERCA uncoupling in vitro [37]. In response to spaceflight, significant reductions in 181 both PLN and NNAT protein content were seen, and thus, we believe may not contribute to the 182 impairments in SERCA function observed in the postural soleus. Further, since both PLN and 183 SLN are found primarily in oxidative skeletal muscle [22,36,37], the reduction in both of their 184 protein contents is not entirely surprising given the fast fiber type shift. Conversely, we found 185 that SLN was significantly upregulated in the soleus in response to spaceflight, which again 186 was, to some extent, expected. SLN is a well-established SERCA uncoupler [24,38] that is 187 upregulated in many muscle wasting conditions, including muscular dystrophy [39], sarcopenia 188 [40], and soleus unloading [33,41]. SLN has been shown to promote calcineurin signaling via 189 SERCA inhibition, thereby promoting the oxidative phenotype and muscle mass [42,43]. Its 190 deletion has also been shown to cause more severe muscle atrophy and a more pronounced 191 fast fiber type transition in the tenotomized soleus [33]. Thus, the increase in SLN presumably 192 contributes to the impairment in SERCA function observed with spaceflight, but whether it 193 contributes to muscle atrophy and weakness requires further investigation. 194 SERCA is highly susceptible to RONS mediated post-translational modifications such as 195 T-nitration and S-nitrosylation that can ultimately impair its ability to regulate cytosolic Ca 2+ 196 levels [25, 27, 28, 44]. Despite not having enough sample to conduct SERCA-specific analysis 197 of RONS modifications, our total protein assessment provides novel insight. Specifically, we 198 found dramatic increases in total protein T-nitration and S-nitrosylation in the soleus muscles 199 from the flight group vs VIV and GC in the RR9 mission. We found similar effects in the soleus 200 muscles from female mice in the RR1 mission; albeit non-significant presumably due to low 201 sample size. Nevertheless, our results could suggest that elevations in RONS production 202 contribute to the SERCA impairments observed in the soleus muscle after spaceflight. It is 203 known that chronically elevated [Ca 2+ ]I can increase RONS production by activating cytosolic 204 NADPH oxidase enzymes and by causing mitochondrial dysfunction via increased mitochondrial 205 Ca 2+ uptake [45][46][47]. On this note, a recent study has found that improving SERCA function 206 through pharmacological activation reduces mitochondrial RONS production in both aged mice 207 [48] and in a mouse model of elevated oxidative stress [19]. Taken together, our results may 208 reveal a negative cyclical relationship where in response to spaceflight, elevated RONS 209 production in the soleus may damage SERCA, contributing to less Ca 2+ uptake which only adds 210 further to RONS production. This highlights the importance of determining whether improving 211 SERCA function can alleviate soleus muscle weakness and atrophy observed during 212 spaceflight. 213 We also attempted to examine a potential effect of biological sex, measuring SERCA 214 function in soleus muscles from both male and female mice. Though we observed significant 215 reductions in Ca 2+ uptake in soleus muscles from space flown male and female mice, there 216 were some subtle differences in the changes in SERCA isoform, SERCA regulatory proteins 217 and total protein T-nitration and S-nitrosylation assessed via Western blotting. However, direct 218 comparisons between male and female mice were prevented due to differences in sample size, 219 sample preparation and mission. Nonetheless, our study could point towards a potential effect 220 of biological sex and highlights the importance of including both male and female mice in the 221 same study. 222 In addition to characterizing SERCA function in the postural soleus, we also investigated 223 the effects of spaceflight in the fast-glycolytic TA muscle. TA muscles were smaller in the flight 224 group and had significantly elevated MHC IIb protein levels. Thus, similar to the soleus muscle, 225 it appears that spaceflight causes a reduction in TA muscle size and a fast fiber type shift. 226 However, unlike the soleus muscle, we found that SERCA function was enhanced in the TA 227 muscle in response to spaceflight. We attribute this effect to the slow-to-fast fibre type shift that 228 came with a significant increase in SERCA1a protein content, and perhaps more importantly, no 229 change in total protein T-nitration or S-nitrosylation. That is, unlike the soleus muscle, TA 230 muscles displayed no signs of elevated RONS production. In turn, the reduction in muscle size 231 in the soleus was accompanied by an impairment in SERCA function and increased RONS 232 production, whereas the reduction in TA muscle size was accompanied by an improvement in 233 SERCA function and no alterations in RONS. The exact reasons explaining this difference 234 between slow and fast muscles are unknown, but we speculate that it may be due to differences 235 in duty. The postural soleus functions as a chronically active and loaded muscle, whereas the 236 TA muscle is more phasic in its activation and load, being used for more explosive type 237 movements. For this reason, the impact of muscle unloading would be more prominent in the 238 postural soleus. It is also important to note that the reduction in TA muscle size may be simply 239 due to the reduction in body mass observed in the RR9 flight group vs the GC and VIV 240 (Supplemental Figure 2) and not actually representative of atrophy per se. Though normalizing 241 soleus to body mass removed any significant differences between groups, we also noticed a 242 trending increase in TA:body mass ratio in the flight vs VIV groups. However, these results are 243 limited as a more accurate measure of muscle size would be myofiber cross-sectional area 244 (CSA). In this respect, it has been recently shown that spaceflight significantly reduces muscle 245 fibre cross-sectional area (CSA) in the murine soleus both in absolute and relative to body mass 246 measures [7]. In contrast, the fast glycolytic extensor digitorum longus (EDL) muscle showed no 247 reductions in absolute or relative CSA after 91 days of spaceflight. Therefore, we speculate that 248 like the EDL, the TA muscle from 35 days of spaceflight would not display any reductions in 249 CSA suggesting that there is no actual atrophy occurring in this muscle type. Future studies 250 should investigate this further in addition to examining TA muscle contractility to determine 251 whether the improvement in SERCA function observed in this muscle would lead to 252 improvements in force production. 253

Conclusions 254
We investigated SERCA function in the soleus and TA muscles of space flown mice. We 255 saw reductions in Ca 2+ uptake and increases in RONS in the soleus. In contrast, we found a 256 significant enhancement in Ca 2+ uptake, a fast fiber type shift with increased MHC IIb and 257 Archive Institutional Scientific Collection Biospecimen Sharing Program. We were specifically 266 provided with soleus and TA samples from male C57BL/6J mice from the RR9 mission (n = 10 267 per group) and soleus samples from female C57BL/6J mice from the RR1 mission (n = 4 per 268 group). The male mice from the RR9 mission were 10 weeks of age at launch and the female 269 mice from the RR1 mission were 16 weeks of age. All mice originated from Jackson 270 Laboratories. Mice in the flight group and ground control group were housed in NASA's Rodent 271 Flight Hardware and were provided ad libitum access to food (food bars) and water. Mice in the 272 VIV group were housed in standard Laboratory cages. For RR9, we received TA muscles that 273 were snap frozen in liquid nitrogen and stored at -80°C, and soleus muscles that were stored in 274 RNALater (ThermoFisher Scientific) at -80°C. For RR1, we received soleus muscles that were 275 snap frozen in liquid nitrogen and stored at -80°C. For all samples, we homogenized the 276 muscles in homogenizing buffer (250 mM Sucrose, 5 mM HEPES, 0.2 mM PMSF, 0.2% NaN3, 277 pH 7.5) prior to storing them at -80°C. In addition to the flight, GC and VIV groups, we also 278 received two cohort control groups from the RR9 mission. Due to Hurricane Irma (September 279 2017), the original RR9 GC and VIV experiments were prematurely terminated. The GC and VIV 280 experiments were then repeated in May 2018 using the same strain of mice that were used for 281 the flight experiment. Along with the new GC and VIV groups, an additional set of mice were 282 used as cohort controls to normalize the variation due to differences in cohorts. That is, the mice 283 originally dedicated to serve as the VIV group in 2017 were labeled as cohort 1, and another set 284 of similarly matched (both in age, sex and treatment) mice were run as cohort 2 in 2018. 285 Importantly, we found no differences in Ca 2+ uptake in either the soleus or TA muscles from 286 Cohort Control 1 (CC1) and Cohort Control 2 (CC2) (Supplemental Figure 1A and B), and 287 therefore did not need to normalize to any variation due to cohorts. 288 289 Calcium Uptake

290
Rates of Ca 2+ uptake in the muscle homogenates were measured using the Indo-1 Ca 2+ 291 fluorophore as previously described [38,[49][50][51], but fitted onto a 96-well plate [37]. Briefly, 292 muscle homogenate was added to reaction buffer (200 mM KCl, 20 mM HEPES, 10 mM NaN3, 293 5 µM TPEN, 15 mM MgCl2, pH 7.0) containing Indo-1 (4 µM final concentration; 57180, Sigma-294 Aldrich). Samples were then plated in duplicate, to which ATP (10 mM final concentration) was 295 added to initiate Ca 2+ uptake. The ratio of Ca 2+ -bound to Ca 2+ -free Indo-1 (405/485nm emission) 296 was measured using a Molecular Devices M2 plate reader upon excitation at 355nm at 37°C. 297 The amount of Ca 2+ uptake was then calculated as the change in the ratio of Ca 2+ -bound to 298 Ca 2+ -free Indo-1 and measuring the area under the curve, with the smaller the area under the 299 curve being indicative of more Ca 2+ uptake over time. 300 301 Western Blotting

302
To assess SERCA2a, SERCA1a, PLN, SLN, and NNAT protein content as well as the 303 amount of post-translational modifications -nitrotyrosine and nitrocysteine -present in the 304 soleus, Western blotting was used [23,28,37]. Using a BCA assay to assess protein 305 concentration, a total protein load of 2.5µg, 10µg, 20µg, 25µg, and 15µg was used for the 306 aforementioned proteins, respectively, and 20µg for nitrotyrosine and nitrocysteine in the soleus 307 muscle. For the TA, 10µg of protein was loaded for SERCA2a and 2.5µg for SERCA1a with 308 20µg similarly loaded for post-translational modifications. To investigate the MHC protein 309 content, 8µg of protein was loaded. Muscle homogenates were solubilized in Laemmli buffer 310 (#161-0747, BioRad) before being separated by SDS-PAGE using TGX BioRad PreCast 4-15% 311 gradient gels (#4568086, BioRad) and transferred to a polyvinylidene difluoride (PVDF) 312 membrane using the BioRad Transblot Turbo for all except for SLN which was separated using 313 tricine based SDS-PAGE and transferred to a nitrocellulose membrane using a wet-transfer. All 314 membranes were blocked using Everyblot (#12010020, BioRad) for 5min at room temperature 315 before the addition of primary antibodies. SERCA1a Tween 20 (TBST) prior to 1hr, room temperature, incubation with the corresponding anti-mouse 323 (SERCA2a, SERCA1a, PLN, nitrotyrosine, nitrocysteine) or anti-rabbit (SLN, NNAT) secondary 324 antibodies diluted in 5% (w/v) milk in TBST. Following another 3 washes in TBST, membranes 325 were imaged using Immobilon ® ECL Ultra Western HRP Substrate (MilliporeSigma) and a 326 BioRad ChemiDoc Imager. Ponceau stains were used to quantify total protein loads and all 327 images were analyzed using ImageLab software (BioRad). 328 329

330
All data are presented as means + standard error of the mean (SEM). A one-way 331 ANOVA with a Tukey's post-hoc test was used to compare the VIV, GC, and flight groups with 332 respect to Ca 2+ uptake via area under the curve analyses as well as protein contents. A 333 student's t test was used to compare flight vs combined GC and VIV for the RR1 soleus as well 334 as for comparisons between the two cohort control groups. Statistical significance was set at p ≤ 335 0.05 and outliers were detected using the ROUT method (Q = 2%) and were removed prior to 336 analyses. All statistical tests were employed using GraphPad Prism 8. Karhanek 8.2 ± 1.2*** 7.9 ± 0.9** 6.1 ± 0.7 RR1 Soleus 7.0 ± 0.5** 7.0 ± 0.4** 5.0 ± 0.8 All values are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.005 using a one-way ANOVA with a 530 Tukey's post-hoc test (n = 4 per group, RR1; n = 9-10 per group, RR9). Western blot data are presented relative to VIV control. *p < 0.05; **p < 0.01; ***p < 0.005; ****p 544 < 0.0001 using a one-way ANOVA and Tukey's post-hoc test (n = 9-10 per group). Representative Western blot images and analyses of SERCA isoform content and total protein T-559 nitration and S-nitrosylation. All values are means ± SEM and Western blot data are presented 560 relative to VIV control. *p < 0.05; **p < 0.01; ****p < 0.0001; values above bars indicate p values 561 using a one-way ANOVA with a Tukey's post-hoc test (n = 9-10 per group). 562