These changes in Ca2+ release amount can reveal the mechanism underlying how the interaction between Ca2+ release and Ca2+ uptake induces Ca2+ alternans. This study of alternans by calculating the values of Ca2+ release properties with impaired SERCA has not been explored before. Here, we induced Ca2+ alternans by using an impaired SERCA pump under ischemic conditions. The results showed that the recruitment and refractoriness of the Ca2+ release increased as Ca2+ alternans occurred. This indicates triggering Ca waves. As the propagation of Ca waves is linked to the occurrence of Ca2+ alternans, the “threshold” for Ca waves reflects the key factor in Ca2+ alternans development, and it is still controversial nowadays. We proposed the ratio between the diastolic network SR (NSR) Ca content (Cansr) and the cytoplasmic Ca content (Cai) (Cansr/Cai) as the “threshold” of Ca waves and Ca2+ alternans. Diastolic Cansr, Cai, and their ratio were recorded at the onset of Ca2+ alternans. Compared with certain Cansr and Cai, the “threshold” of the ratio can better explain the comprehensive effects of the Ca2+ release and the Ca2+ uptake on Ca2+ alternans onset. In addition, these ratios are related with the function of SERCA pumps, which vary with different ischemic conditions. Thus, values of these ratios could be used to differentiate Ca2+ alternans from different ischemic cases. This agrees with some experimental results. Therefore, the certain value of diastolic Cansr/Cai can be the better “threshold” for Ca waves and Ca2+ alternans.Impaired sarcoplasmic reticulum (SR) calcium transport ATPase (SERCA) gives rise to Ca Meanwhi([ADP]i)  and pH i([ADP]i) . Accordi2+ alternans can occur or not in different ischemic conditions. We attempted to find the ranges of CL in which Ca2+ alternans could arise in these three cases. In our simulations, the starting CL is 250 ms and the increasing step is 10 ms. Finally, we determined the ranges of CL in ischemic cases 1, 2, and 3, which are from 250 ms to 280 ms, from 250 ms to 380 ms, and from 250 ms to 300 ms, respectively.The CL also affects whether Ca2+ ) comprised the amount of diastolic NSR Ca2+ and the junctional SR (JSR) Ca2+. The amount of Ca2+ release (Cak)release( (mmol)) was expressed as the integral of the Ca2+ release flux (Jk)rel( (mmol/L/ms)) on the kth beat (equation (k)release( to Cak−1)sr_total( to represent the fraction of SR Ca2+ release:2+ included free and buffered Ca2+ (Cajsr_free (mmol/L) and Cajsr_buff (mmol/L)); vnsr and vjsr represented the volume of NSR and JSR; and Cak)sr( (mmol/L) and Cak)nsr( (mmol/L), respectively, referred to the diastolic SR and NSR Ca2+ content on the kth beat.In our simulations, the total amount of diastolic SR Caequation ). Then, 2+ is released through CRUs. One CRU is set to have six neighbors in the 3D-distribution cell simulation [N0 represents the total number of CRUs and NK is the number of that activated on the kth beat [α represents the probability of a Ca spark being activated spontaneously or by the L-type Ca2+ current (ICaL); β is the probability of a Ca spark triggered on the kth beat being unavailable during the (k + 1)th beat; γ indicates the probability of a Ca spark recruiting one of its neighboring; and f represents the percentage of secondary Ca sparks in the remaining available CRUs [k + 1)th beat is given as follows [2+ depletion of each CRU and Casr is the average Ca2+ content of each CRU before release [b> refers to the average Ca2+ content of these Nk CRUs after they sparked [k)releaserelease( and Cak−1)sr_totalNK+1= release . <Cab> r sparked . We put equation . The lefquations ), were lNK and N0 in equation (k)releasesr_totalrelease( and Cak−1)sr_total was from zero to the maximum Casr_total/(vnsr + vjsr). Then, these values were input to solve equation amplitude more prominent, and leads to a slightly deeper notch of the AP. Subsequently, the voltage-dependent repolarization currents cause different repolarization phases.According to equation , increasinset of . APs alsinset of , due to 2+ alternans can be observed in all three ischemic cases when CL = 250 ms. However, when the CL increases to 300 ms, it can only be observed in cases 2 and 3. In case 1, the maximum Casr with CL of 300 ms does not reach the value of Casr at which bifurcations occur with CL of 250 ms. In addition, the slopes of curves change slightly before alternans onset  affects the ratios more effectively. Furthermore, how diastolic Casr, Cai, and their ratio change with sequent heartbeats is analyzed under transient Ca2+ alternans . This means the maximum diastolic Cansr/Cai can be affected by ischemic cases. In addition, the onset of alternans is induced by ischemia, and thus its “threshold” is taken to be different with ischemic degrees. In nsr/Cai differs with ischemic conditions. As a contrast, neither Cai nor Cansr is able to distinguish different ischemic cases. In 2+ alternans, Casr and Cai increase. Correspondingly, Iup goes on retaking the increasing released Ca2+ and diastolic Cansr/Cai keeps rising. However, to what extent Iup and diastolic Cansr/Cai can increase is limited by the thermodynamic constrains. Ca2+ alternans and Ca waves form when the unbalance between the Ca2+ uptake and Ca2+ release occurs. Subsequently, as diastolic Cai and Cansr keep on increasing, the Ca2+ uptake rate increases enough to uptake all released Ca2+ and Ca2+ alternans disappears. This final constant value of diastolic Cansr/Cai indicates the new balance between the Ca2+ release and Ca2+ uptake. On the other hand, if diastolic Cai or Cansr is the “threshold” for Ca waves and Ca2+ alternans, these two increasing values will initiate larger Ca waves and Ca2+ alternans will not disappear.SERCA pumps contribute to maintain the Caequation , the max2+ efflux cooperates with the influx to affect the “threshold” for alternans. An intermediate SR Ca2+ uptake rate and a larger SR Ca2+ release work synergistically to produce alternans at longer CLs [Iup contributes to unbalanced Iup and Irel and promotes abnormal intracellular Ca2+ handling. In addition, other studies demonstrated that the properties of RyRs affect the “threshold” of Casr for alternans [sr [2+ uptake rate becomes larger accordingly. As the physiological conditions  are identical, the maximum Ca2+ uptake rate is kept the same. Thus, maximum Iup occurs at a lower Casr, resulting in a reduced “threshold” of Casr for alternans (Xie et al.  demonstrnger CLs . Accordilternans , 41, 42.Irel during the formation of Ca2+ alternans. Our new “theory” shows that the unbalance between Iup and Irel begins at the “threshold” of diastolic Cansr/Cai. These two currents interact with each other and result in Ca2+ alternans. We should also note that Ca2+ alternans of case 3 is transient (Iup and Irel get balanced, Ca2+ alternans is suppressed. Therefore, this “threshold” theory may disclose a novel therapeutic strategy for Ca2+ alternans. In theory, keeping the ratio below the “threshold” stops Ca2+ alternans occurrence. In clinical, the treatment aiming at the interaction between these two currents may have a promising effect. Although direct therapeutic tools modulating the SR release channels have not been fully developed [This “threshold” theory provides a new idea for changes of ransient , which s2+ alternans. That is, these components of Ca cycling interact with each other to affect Ca2+ alternans development. In addition, compared with isolated changes of diastolic Casr and Cai, the value of diastolic Cansr/Cai is more appropriate to function as the “threshold” for alternans. By defining this new “threshold,” we can better explain how the interplay between the Iup and Irel causes alternans. Furthermore, this proposed “threshold” theory may help find therapeutic targets for suppressing Ca2+ alternans.The integrated cell model can be used to simulate the SERCA pump function in specific ischemic conditions, whereas the original ORd model cannot be used. The simulated results indicate that Ca waves can be induced by impaired SERCA pump and thus give rise to Cai chosen in our simulation can also determine the results by influencing the inputs. We also need to take use of other cell models to calculate values of these properties during Ca2+ alternans occurrence and identify our “threshold” theory. In addition, although we conclude that Iup and Irel interact to produce Ca2+ alternans, we just identify how the Ca release is affected by impaired SERCA pump, and the effect of Irel on Iup should also be identified in detail in the future.This model is just used to simulate the impaired SERCA pump function during ischemia, whereas L-type calcium and other currents are also affected under ischemia, and these changes are not included in our model. We need to further improve this integrated model to simulate more accurate ischemic conditions. It is noted that the values of the three properties can be different by using different groups of inputs. The range of Ca


Mutations in genes encoding RyRs cause various muscle and arrhythmogenic heart diseases. Although RyR channels are activated by Ca2+, the actual mechanism of Ca2+ binding remains largely unknown. Here, we report the molecular basis of Ca2+ binding to RyRs for channel activation and discuss its implications in disease states. RyR1 and RyR2 carrying mutations in putative Ca2+ and caffeine-binding sites were functionally analysed. The results were interpreted with respect to recent near-atomic resolution RyR1 structures in various ligand states. We demonstrate that a tryptophan residue in the caffeine-binding site controls the structure of the Ca2+-binding site to regulate the Ca2+ sensitivity. Our results reveal the initial step of RyR channel activation by Ca2+ and explain the molecular mechanism of Ca2+ sensitization by caffeine and disease-causing mutations.Ryanodine receptors (RyRs) are Ca Takashi Murayama et al. report the molecular basis of calcium binding to ryanodine receptors, a process essential for muscle contraction. They find that a tryptophan residue in the caffeine binding site controls the structure of the calcium binding site, affecting calcium sensitivity. RyRs are huge (~2 MDa) homotetrameric protein complexes with a large cytoplasmic structure and a cation channel domain of six transmembrane segments at the carboxyl (C)-terminus4. Type 1 RyR (RyR1) is a skeletal muscle isoform and mutations in RYR1 cause several muscle diseases, including malignant hyperthermia and central core disease6. Type 2 RyR (RyR2) is expressed mainly in the heart, and mutations in RYR2 are associated with various arrhythmogenic heart diseases, including catecholaminergic polymorphic ventricular tachycardia (CPVT)8. To date, a total of nearly 800 disease-related mutations have been identified in RYR1 and RYR2 genes.Ryanodine receptors (RyRs) are Ca2+-induced Ca2+ release (CICR), in which the channel is directly activated by Ca2+9. The binding of Ca2+ to the Ca2+-binding site is an initial step for channel activation9; therefore, regulation of the Ca2+-binding is important for channel activity and is implicated in disease states. Indeed, CICR activity is enhanced by many RYR1 mutations found in malignant hyperthermia and central core disease12 and by RYR2 mutations identified in CPVT15. The molecular details of the Ca2+ binding site and its regulation, however, remain largely unknown.The RyR channel mediates Ca20. Furthermore, des Georges et al.20 reported putative binding sites for three major activating ligands, Ca2+, ATP, and caffeine, on a near-atomic resolution structure of RyR1. They proposed that the Ca2+-binding site is located in the core domain just above the transmembrane domain  of a tryptophan in the putative caffeine-binding site of human RyR2 has been reported to cause CPVT23, indicating the physiological significance of the caffeine-binding site.Recent cryo-electron microscopy structures of RyR1 and RyR2 have revealed a complex architecture, involving a superhelical scaffold in the cytoplasmic domain and a channel domain of the voltage-gated ion channel superfamilyain Fig.  and consain Fig. . Caffeinins Fig. . The clo2+ binding for channel activation and to assess the role of this mechanism in disease states, we conducted functional analysis of RyR1 and RyR2 channels carrying mutations in these sites using a heterologous expression system in HEK293 cells. Based on high similarity in ligand-binding sites between RyR1 and RyR2, the results were interpreted with respect to recently reported near-atomic resolution structures of RyR1. We identified these as the actual binding sites and demonstrate that a tryptophan residue in the caffeine-binding site controls the structure of the Ca2+-binding site to regulate the Ca2+ sensitivity. Our results reveal the initial step of RyR channel activation by Ca2+ and explain the molecular mechanism of Ca2+ sensitization by caffeine and disease-causing mutations.To elucidate the molecular basis of Ca2+ binding to RyRs and its regulation via the caffeine-binding site, we analysed a CPVT mutation in the putative caffeine-binding site of human RyR2 (W4645R)23. The corresponding mutation  and ER lumen ([Ca2+]ER), respectively15 /(Fmax− Fmin), in which Fmin and Fmax were determined in the presence of ionomycin with 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) plus cyclopiazonic acid and CaCl2, respectively  was greater in the mutants than in WT RyR2    , which is brought about by a ~2 Å upward shift of the CTD . Thus, the Ca2+-binding site in the presence of caffeine appears more favorable for the binding of Ca2+ compared with the site in the absence of caffeine. This conformational change reasonably explains enhancement of Ca2+ sensitivity by caffeine.The putative binding sites for Casee Fig. ; therefoAP) Fig. , in the B0) Fig. , and in 15) Fig. . Compari15) Fig. . In addiure Fig. . Moreove2+-binding pocket larger and less favorable for the binding of Ca2+. The binding of caffeine will alter the orientation of the tryptophan indole group, which breaks the interaction causing movement of the CTD upward to make the Ca2+-binding pocket smaller and more favorable for Ca2+ binding. This reasonably explains why the CPVT mutant (W4644R) enhances the Ca2+ sensitivity; the mutated residues may weaken the interaction to move the CTD upward.By looking at the binding site in more detail, we found that isoleucine (I4996 in RyR1 and I4925 in RyR2) in the CTD is located close to the tryptophan in the EGTA state Fig. . Based o2+- and caffeine-binding sites are well conserved in three RyR isoforms  using available PDB data19  the Ca2+-binding pocket was considerably smaller in the presence of Ca2+ compared with EGTA, , (2) the CTD moved upward in the presence of Ca2+ (~3 Å), (3) the CSol moved to the right in the presence of Ca2+ (~3 Å), and (4) hydrophobic residues responsible for caffeine action  are all conserved in RyR2.The amino acid residues around the putative Ca2+-binding site, in which Ca2+ is proposed to be coordinated directly by E3893, E3967, and the carbonyl oxygen of T5001, and also indirectly by Q397020 in RyR1  and no Mg2+, which removes inactivation by low-affinity Ca2+/Mg2+-binding sites22. Under these conditions, WT RyR2 exhibited monophasic Ca2+ dependence with pCa50 of ~5 . I4925A exhibited greatly enhanced Ca2+ sensitivity comparable to that of W4644A   Fig. . These r2+-sensitizing effect of caffeine by interaction between tryptophan and isoleucine. However, we found that caffeine further sensitized I4925A to Ca2+  gets to phenylalanine (F3753 in RyR1 and F3713 in RyR2) within ~2 Å in the presence of Caf + ATP  than WT (pCa50 = 4.79 ± 0.03)  should be unresponsive to caffeine . Loss of phenylalanine side chain is expected to weaken the interaction, which causes a reduced Caine Fig. . In the 03) Fig. . Caffein WT Fig. . We nextine Fig. . The Ca2all Fig. . These r2+ release, indicating a typical gain-of-function phenotype12. F3573A was much less responsive to caffeine in releasing Ca2+ compared with WT. The fact that 4-CmC caused large Ca2+ release in F3753A suggests similar level of ER Ca2+ as in WT. A caffeine-sensitive gain-of-function of I4996A and a reduced caffeine sensitivity of F3753A are consistent with the phenotype of I4925A and F3713A in RyR2, respectively.To address whether isoleucine and phenylalanine residues are also important for RyR1, functional analysis was done with HEK293 cells that express RyR1 carrying I4966A or F3753A mutation  the caffeine-binding site negatively regulates the Ca2+ sensitivity through an interaction between tryptophan in S2S3 and isoleucine in CTD, (2) caffeine sensitizes RyR to Ca2+ in two ways: by breaking the interaction between the tryptophan and the isoleucine and by forming another interaction between the tryptophan and a phenylalanine in Csol, and (3) CPVT mutations weaken the tryptophan-isoleucine interaction to enhance the Ca2+ sensitivity. Thus, the tryptophan residue serves as a switch to regulate the Ca2+ sensitivity. Our findings revealed the molecular basis for Ca2+ binding to RyR and provide an explanation for disease states.Ca2+ ([Ca2+]i and [Ca2+]ER) measurements and [3H]ryanodine binding. These assays measure the activity of the RyR channels22. Thus, our results by functional analysis provide an indirect evidence for binding of Ca2+ or caffeine to their binding sites. However, highly quantitative nature of [3H]ryanodine binding enabled us to address the Ca2+ sensitivity and the effect of caffeine in the mutant RyRs. We interpreted the functional results of mouse RyR2 with rabbit RyR1 structure, since there is no structural information about the Ca2+- and caffeine-binding sites in the reported RyR2 structure19. One might concern about misinterpretation by different isoforms. However, we are confident of our strategy for the following reasons. First, the effect of caffeine is common between RyR1 and RyR222. Second, the residues responsible for caffeine binding are highly conserved within the three mammalian RyR isoforms , due to no other available structures with caffeine. This might raise a concern that some structure changes in the Ca2+-binding site with Caf + ATP (5TAP) is overlapped with that with Ca2+ (5T15)   Fig. . One posaf/ATP 5TV  greatly sensitizes the RyR2 channel to Ca2+  produced in plants32. Although methylxanthines are not present in animals, including humans, xanthine is a metabolite of purine nucleotides in humans. It has been reported that 9-methyl-7-bromoeudistomin D (MBED), an artificial β-carboline analogue, has a potent Ca2+ releasing activity by binding to the caffeine-binding site33. Some β-carbolines may be formed naturally in humans34. Thus, it is quite possible that some derivatives of xanthine or β-carbolines act as the endogenous regulators of RyRs through the caffeine-binding site to regulate physiological Ca2+ sensitivity in vivo.What we propose in this study is a very simple and clear-cut mechanism of Ca12 and RyR215 were generated using the Flp-In T-REx system . Each mutation was introduced into rabbit RyR1 (GenBank accession number X15209.1) or mouse RyR2 (NM_023868.2) cDNA by inverse polymerase chain reaction using primers listed in Supplementary Table −1 hygromycin. The selective medium was changed every three days until the hygromycin-resistant foci were identified. Several hygromycin-resistant foci were tested for the inducible expression of RyRs and clones with suitable expression were selected and used for experiments.HEK293 cells, stably and inducibly expressing WT and mutant forms of RyR12+ imaging was carried out in HEK293 cells expressing WT or mutant RyR1 and RyR235. In Ca2+ measurements with RyR2, the Ca2+ signals from cytoplasm ([Ca2+]i) and ER lumen ([Ca2+]ER) were monitored using G-GECO1.1 36 and R-CEPIA1er 37, respectively. Cells were transfected with cDNAs for these Ca2+ indicators 28–32 h before measurement. Expression of RyR2 was induced by doxycycline (2 µg/ml) 4–6 h before measurement depending on the mutant. Experiments were carried out with HEPES-buffered Krebs solution . In experiments with RyR1, cells were loaded with fluo-4 AM 24 h after induction and fluorescence signals in response to caffeine (0.03–10 mM) were determined. At the end of each experiment, Fmin and Fmax were obtained with 0Ca Krebs solution containing 20 µM ionomycin, 5 mM BAPTA, and 20 µM cyclopiazonic acid and 20Ca Krebs solution containing 20 µM ionomycin and 20 mM CaCl2, respectively15. Averaged Fmin was 7.655% of Fmax for R-CEPIA1er and 0% for G-GECO1.1 and fluo-4. The fluorescence signal (F − Fmin) was normalized to the maximal fluorescence intensity (Fmax− Fmin). Measurements were carried out at 26°C.Single-cell CaHEK293 cells expressing RyR2 mutants (5 × 150 mm dishes) were collected and rinsed twice with phosphate-buffered saline. The cell pellets were resuspended with 5 ml of 0.3 M sucrose, 20 mM 3-morpholinopropanesulfonic acid (MOPS), pH 7.4 with a protease inhibitor cocktail and processed for nitrogen cavitation for 15 min at 1000 psi. The homogenate was centrifuged at 1000 × g for 5 min and the supernatant was ultracentrifuged for 30 min at 100,000 × g. The microsomal pellet was resuspended with 5 ml of the above buffer and ultracentrifuged again. The pellet was resuspended with 1 ml of the above buffer, quickly frozen with liquid nitrogen, and stored at −80 °C until used.3H]ryanodine in a medium containing 0.17 M NaCl, 20 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO) at pH 7.0, 2 mM dithiothreitol, 1 mM AMP, 1 mM MgCl2 and various concentrations of free Ca2+ buffered with 10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA)35. Free Ca2+ concentrations were calculated using WEBMAXC STANDARD (http://web.stanford.edu/~cpatton/webmaxcS.htm)38. The [3H]ryanodine binding data (B) were normalized to the maximum number of functional channels (Bmax), which was separately determined by Scatchard plot analysis using varied concentrations (3–20 nM) of [3H]ryanodine in a high-salt medium. The resultant B/Bmax represents the averaged activity of each mutant. For mutants regarding the Ca2+-binding sites, medium containing 1 M NaCl and no Mg2+ was used to remove inactivation through the low-affinity Ca2+/Mg2+ site.Microsomes isolated from HEK293 cells expressing WT and mutant RyR2 were incubated for 1 h at 25°C with 5 nM ) are expressed as:KA and KI are dissociation constants, and nA and nI are Hill coefficients for Ca2+ of A- and I-sites, respectively. The Hill coefficients were set at 2.0 and 1.0 for nA and nI, respectively, for WT and all the mutant channels, which maximize the sum of R2 values for curve fitting. Curve fitting was performed using the Prism 6 software . The curves for fA, 1 – fI, and A are shown in Supplementary Figure 2+]i, A at pCa = 7 was calculated by Eqs. .To determine the parameters of Ca40 (1: 5000 dilution) and calnexin  (1: 5000 dilution).Microsomal proteins (10 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with a 3–12% gel and transferred onto a polyvinylidene fluoride membrane. Western blotting was performed using antibodies for pan RyR28 was diagnosed with CPVT and introduced to Shiga University of Medical Science for genetic analysis. The patient and her family provided written informed consent in accordance with the guidelines approved by our institutional review boards (23-128-3). Genomic DNA was extracted from peripheral blood lymphocytes by use of the DNA Isolation Kit . Genetic screening for 50 genes related to inherited arrhythmias including RYR2, CASQ2, KCNJ2 and TRDN were performed by using a bench-top next generation sequencer (NGS) . Detected variants were confirmed by Sanger methods.The patient carrying C4193W mutation in RyR2t-test and one-way ANOVA followed by Dunnett’s test, were performed to compare two groups and three or more groups, respectively. Structural figures were prepared with PyMOL .Statistical analysis was performed using Prism 6 . Unpaired two-tailed Student’s The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. The PDB accession numbers we used in this paper are 5TAP, 5TB0, 5T15, 5T9V, 5GO9, 5GOA.Supplementary Information


MSTN) gene can cause the ‘double-muscling’ phenotype. Although there have been many studies performed on MSTN-mutant animals, there have been few studies that have investigated the cystine-knot motif in exon 3 of MSTN in rabbits. In the current study, CRISPR/Cas9 sgRNA anchored exon 3 of a rabbit’s MSTN was used to disrupt the cystine-knot motif to change the MSTN construction and cause a loss of its function. Eleven MSTN-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational MSTN amino acid sequences of the 11 founder rabbits were modeled to the tertiary structure using the SWISS-MODEL, and the results showed that the structure of the cystine-knot motif of each protein in the founder rabbits differed from the wild-type (WT). The MSTN-KO rabbits displayed an obvious ‘double-muscling’ phenomena, with a 20−30% increase in body weight compared with WT rabbits. In the MSTN-KO rabbits, all of the −/−MSTN rabbits showed teeth dislocation and tongue enlargement, and the percentage of rabbits having pelvic tilt was 0% in MSTN+/+, 0% in MSTN+/−, 77.78% in female MSTN−/− rabbits, and 37.50% in male MSTN−/− rabbits. The biomechanical mechanism of pelvic tilt and teeth dislocation in the MSTN-KO rabbits requires further investigation.Gene mutations at different gene sites will produce totally different phenotypes or biological functions in gene-edited animals. An allelic series of mutations in the myostatin (MSTN-KO rabbits will serve as an important animal model, not only for studying skeletal muscle development, but also for biomedical studies in pelvic tilt correction and craniofacial research.These newly generated  Also, in Piedmontese cattle [myostatin gene results in a truncation of the bioactive carboxyterminal domain of the protein [myostatin gene consists of three exons and two introns. The third exon of the rabbit myostatin gene codes for mature processed myostatin, which is the active region of the molecule and shares extensive homology with other TGF-β members [myostatin: a precursor protein consisting of an N-terminal signal sequence; a propeptide domain; and a growth factor domain that contains the characteristic cystine-knot motif . C339 forms the intermolecular dimerization disulfide bond [in vivo, with its absence leading to a double-muscled phenotype (‘double-muscling’) [Since its discovery in mice , MSTN hae cattle . An 11-be cattle , a G to  protein ,10. In a protein . These tide bond ,13. The scling’) . Hence, MSTN naturally mutant cattle generally show a 20−40% increase in muscle weights due to muscle hypertrophy or hyperplasia relative to wild-type (WT) cattle [MSTN-KO mice (MSTN−/− mice) produced by the homozygous deletion of the C-terminal region of the myostatin gene in embryonic stem cells were double or triple in mass compared with those of their heterozygous and WT littermates [−/−MSTN mice were over 50% larger in mass than WT controls due to larger cross-sections of the muscle fibers and increased numbers of muscle cells [−/−MSTN mice were used to explore the effects of increased muscle mass on sagittal suture complexity. The mice were also used to indicate that a consistent amount of force needs to be applied over a much greater area of the craniofacial skeleton in the myostatin knockout, and this should, in turn, lead to altered biomechanical stress and bony morphology [−/−MSTN mouse has significantly larger temporalis, masseter, and medial and lateral pterygoid muscles than WT controls [−/−MSTN mice , provide further evidence of an altered craniofacial loading environment due to MSTN deficiency [MSTN-KO mice have been used as a model for studying muscle–bone interactions, such as to investigate the role of increased mandibular elevators and bite forces on temporomandibular joint morphology. In the past 5 years, many MSTN-KO animals  with ‘double-muscling’ have been generated due to the emergence of the CRISPR/Cas9 gene editing technique as a newly versatile genome engineering tool [MSTN-KO animals have rarely been investigated.Previous studies have reported that ) cattle . This isle cells . Thereforphology ,16. An Mcontrols . These cficiency ,16–20. Ting tool . HoweverMSTN-KO rabbits using the CRISPR/Cas9 system anchored to the cystine-knot motif by microinjecting Cas9 mRNA and sgRNA into fertilized rabbit eggs. The MSTN-KO rabbits can be used as a basis to create new breeds of rabbit to improve muscle yield, with the ultimate goal of approval for human consumption. In addition, an important animal model is proposed for use in further studies that investigate muscle–bone interactions.The aim of the present study is to generate MSTN rabbit gene were selected as target genes  deleted C372 and C374, and four rabbits (M8−M11) deleted C339 and C340 in two alleles, C281–C340. M7♀ and M10♀ were homozygous, and both of them lost C372 and C374 in the cystine-knot motif. In addition, M10♀ also deleted C339 and C340. As shown in MSTN monomers. The typical phenotype of double-muscling was observed in F0 MSTN-KO rabbits at four months of age, and they were compared with their WT counterparts, as shown in −/−MSTN and +/−MSTN rabbits compared with +/+MSTN counterparts.Genomic DNA was extracted from the ear tissue of the newborn pups. The 12 individuals used for genotyping were numbered M1−M12. The T-cloning and PCR-sequence results showed that the MSTN-KO rabbits and their WT counterparts were recorded from 9 to 14 weeks. It was found that the average body weights of male MSTN-KO rabbits were significantly higher than the male WT rabbits from 9 to 14 weeks of age  from 10 to 14 weeks after birth were found between the MSTN-KO male rabbits and the WT male rabbits. Significant differences (*P<0.05) were found between the MSTN-KO female rabbits and the WT female rabbits from 13 to 14 weeks after birth. At 14 weeks, the average body weights of the female (2318.40 ± 183.15 g) and male (2579.33 ± 67.99 g) MSTN-KO rabbits increased by 17.91 and 26.74%, respectively, compared with the female  and male (2063.33 ± 186.47 g) WT rabbits, respectively.Rabbits experience an exuberant growth period from 2 to 3 months, so the body weights of the s of age C. The avs of age D. SignifMSTN-KO rabbits. Then the same genotype of the F1 male and female rabbits was mated for homozygous mutants. The breeding results are shown in Supplementary Table S1.The male founders  were mated with the female WT rabbits, and the female founders  were mated with the male WT rabbits. Genomic DNA was extracted from the ear tissues of the F1 rabbits. PCR amplification and TA-cloning sequencing analysis showed that these F1 rabbits were monoallelic A total of ten potential off-target (OT) sites  were successfully amplified using PCR and subjected to Sanger sequencing. No overlapping peaks were detected near the OTs.MSTN-KO rabbits exhibited the double-muscled phenotype (−/−MSTN rabbits (3512.20 ± 439.17 μm2) was substantially larger than that of the +/+MSTN rabbits . The average size of the gluteus maximus myofibers in the +/−MSTN rabbits (2610.37 ± 604.44 μm2) increased in comparison with the +/+MSTN rabbits . Similarly, the average size of tongue myofibers in the −/−MSTN rabbits (2109.22 ± 206.75 μm2) was substantially larger than that of the +/+MSTNrabbits . The average size of tongue myofibers in the +/−MSTN rabbits (1677.24 ± 247.50 μm2) increased in comparison with the +/+MSTN rabbits . The distribution of different sizes of gluteus maximus and tongue myofibers indicates that the percentage of smaller fiber cells in the MSTN-KO rabbits was lower than the percentage in the WT rabbits . The levels of CREA (325.97 ± 18.92 μmol/l) and CK (599.67 ± 13.79 U/l) were increased in the MSTN−/− rabbits compared with the MSTN+/− rabbits . The levels of CK  were increased in the MSTN+/− rabbits compared with the MSTN+/+ rabbits . CREA is a product of muscle tissue metabolism in the body. Serum CK is related to the total amount of muscle. This means that the greater the muscle capacity, the higher the serum CK activity. Increased serum CREA and CK levels in the MSTN-KO rabbits should correlate with increased muscle mass. The serum CREA content reported for MSTN-KO Meishan pigs was also higher than that in WT pigs [MSTN−/− rabbits than that in the MSTN+/+ rabbits at 6 months of age. Further experiments are required to investigate this phenomenon in MSTN−/− rabbits. There were no differences in the hematology characteristics and other biochemical parameters. The results are listed in Supplementary Tables S2 and S3.The hematological and biochemical parameters from 6-month-old MSTN mutation on the rabbits, the body structure and physiological activity of the MSTN-KO rabbits were closely observed. It was found that the MSTN−/− rabbits showed a greater mass of the masseter muscles compared with the WT and MSTN+/− rabbits  was significantly higher than that in male −/−MSTN rabbits (37.50%). The conclusion drawn was that this may be due to the difference in the pelvic structures of males and females. In mammals, the pelvis is significantly larger in females than in males to allow babies to pass through pelvis when they are born. However, it was still unclear if these craniofacial and pelvic tilt abnormalities were related to the MSTN knockout condition during the pre- or neonatal period or were simply a result of postnatal compensatory changes. The effect of the molecular mechanism of the loss of myostatin function on the craniofacial and pelvic tilt abnormalities in rabbits needs further investigation.In the present study, all of the homozygous mutant rabbits showed pelvic tilt, teeth dislocation, and tongue enlargement. This OT phenomenon was not found in the  rabbits ,31 and M-KO pigs . Previou-KO pigs . Howevere tissue . It has e tissue . Studiese tissue . Byron ee tissue  and Vecce tissue  employedn et al.  further mutation . To our -KO mice . In thisficiency . In thesMSTN-KO mouse has typically been used as a new animal model for the study of the relationship among craniofacial morphology, hypermuscularity, and increased bite force since its muscles have been demonstrated to be significantly larger [MSTN-KO rabbits will be a better model for pelvic tilt correction and craniofacial research due to their observable pelvis and skulls, appropriate body, longer life cycles, and easy management due to low feeding costs.The y larger ,38. In ay larger . It can MSTN-KO rabbits were generated with remarkably high efficiency by microinjecting Cas9 mRNA and sgRNA into pronuclear-stage embryos. The results of the present study suggest that Cas9 mRNA microinjection may provide a fast and efficient approach for gene knockout rabbit models. Although muscle overgrowth caused by homozygous mutations in myostatin was impressive, bone abnormalities caused by excessively growing muscles may be a health hazard for livestock. In addition, based on the abnormality results found in MSTN-KO rabbits, it is suggested that these rabbits would make an important animal model for craniofacial and pelvic research.In conclusion, The New Zealand rabbits used in the present study were kept at the Animal Genetic Engineering Laboratory of Yangzhou University. They were fed twice a day and offered water limitlessly. Animal experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China) and approved by the Animal Care and Use Committee of Yangzhou University, Yangzhou, China (license number: SYXK(Su)2017-0044).MSTN (sgRNA1 sited E3: g.4881-4903 and sgRNA2 sited E3: g.4796-4818) were performed, as shown in We constructed two CRISPR/Cas9 expression plasmids that were targeted to the cystine-knot motif. To ensure successful targeting, two sgRNAs independently targeting exon 3 of in vitro transcription, a pair of complementary DNA oligos, shown in Supplementary Table S4, were annealed to be double-stranded. They were then subcloned into the linearized PYSY-T7-Cas9-T7-MSTN-gRNA cloning vector, which was provided by the Nanjing YSY Biotech Com., Ltd., Nanjing, China. The PCR product for the in vitro transcription of Cas9 mRNA was amplified using T7 primers (T7-1:5′-TGTAAAACGACGGCCAGT-3′ and T7-2: 5′-TGGCACCGAGTCGGTGCTTT -3′). The PCR products for the in vitro transcription of sgRNA were amplified using T7 primers (T7-3: 5′-ATTAACCCTCACTAAAGGGA-3′ and T7-4: 5′-AAAAAAAGCACCGACTCGGTGCCAC-3′). Cas9 mRNA and sgRNAs were transcribed in vitro using the ScriptMAX Thermo T7 Transcription Kit  and purified using a lithium chloride precipitation solution  according to the manufacturer’s instructions. The concentration and quality of the synthesized mRNA were determined using the spectrophotometer  and agarose gel electrophoresis, respectively. Cas9 mRNA and sgRNAs were diluted into RNase-free water and stored at −80°C for future use.To construct the recombinant vector for the preparation of sgRNA by Healthy donors with regular estrus cycles were selected for zygote collection. Zygotes were collected using surgical oviduct flushing from donors after superovulation treatment and natural mating. In brief, donors were injected with follicle stimulating hormone (FSH) for estrus synchronization. A total of 60 IU FSH were administered by intramuscular injection in six dosages at 12-h intervals (the first dose was 15 IU and other doses decreased progressively to 5 IU). The animals were then injected with 100 IU of HCG 12 h after the FSH was administered. The females were then subsequently mated with a male rabbit.in vitro transcribed sgRNA (10 ng/μl) and Cas9 mRNA (40 ng/μl) was injected into the cytoplasm of pronuclear stage embryos. The injected embryos were transferred to M16 medium  with a 1% penicillin–streptomycin solution and 10% FBS for a 30–60 min culture at 38.5°C, 5% carbon dioxide, and humidity conditions. Then approximately 15−20 injected embryos were transferred into the oviducts of the recipient mother.Rabbit embryos at the pronuclear stage (approximately 18–20 h post-mating) were transferred into M2 medium  with a 1% penicillin–streptomycin solution  and 10% fetal bovine serum . A mixture of https://www.swissmodel.expasy.org/).The genomic DNA of the sample was extracted from a small piece of ear biopsy using the phenol–chloroform extraction method. The target sites were PCR amplified using the primers (CAS9SG12-1 and CAS9SG12-2). The sequences of primers are shown in Supplementary Table S5. The PCR products were subjected to 1% agarose gel electrophoresis, and the map is shown in MSTN+/+, MSTN+/−, and MSTN−/− rabbits using radio-immunoprecipitation assay (RIPA) lysis buffer  with protease inhibitors according to the manufacturer’s instructions. The protein concentrations were determined using the bicinchoninic assay (BCA) method . A 40-μg protein sample was subjected to a 12% polyacrylamide Tris-glycine gel for 1 h. Separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were then blocked for 2 h in a Tris buffered saline with Tween 20 (TBST) buffer containing 5% milk at room temperature. The membranes were subsequently incubated for 1.5 h at 37°C with primary monoclonal antibodies against MSTN  diluted to 1:100 with TBST buffer. The membranes were subsequently washed three times for five minutes with the TBST buffer and incubated with anti-mouse secondary antibodies  for 1.5 h at 37°C. The GAPDH antibody  was used as a reference control. The proteins were visualized with the ECL substrate solution  using an imaging system .Total protein was extracted from gluteus maximus tissue in MSTN+/+, MSTN+/−, and MSTN−/− rabbits were collected and fixed with 4% paraformaldehyde, followed by embedding in paraffin according to routine protocol. Deparaffinized tissues were sectioned at 4-μm thicknesses and blocked in 5% bovine serum albumin (BSA). Sections were stained with a primary monoclonal antibody against MSTN , and diluted to 1:50 with a blocking buffer by incubating at 4°C overnight. After washing, sections were incubated with secondary antibodies  for 1 h at 37°C and then for an additional 5 min with 4,6-diamidino-2-phenylindole . Images were taken using a microscope .Gluteus maximus tissue from MSTN-KO founder rabbits and WT counterpart controls were bred and housed under the same conditions. Their feed was adjusted with respect to growth stages after weaning at 30 days of age. Their body weights were recorded daily from 9 to 14 weeks of age.In the preent study, the in vivo, all possible sites of the entire rabbit genome with homology to the 23-bp sequence (sgRNA + PAM) were predicted using the CRISPR design tool (http://tools.genomeengineering.org). The mismatch parameter for the target sequence was set as 5′–NGG and was chosen as the protospacer adjacent motifs (PAM). The sites with 12-bp conserved proximal to PAM and total mismatches <4 and sites with total mismatches <5 were chosen as potential OT sites for subsequent testing. The selected potential OT sites were initially PCR-amplified using genomic DNA from MSTN-KO rabbits. The PCR products were then used for sequencing. The information of the OT sites and primer pairs used is listed in Supplementary Tables S6 and S7.To determine the site-specific cleavage of the CRISPR-Cas9 system MSTN−/− and a WT rabbit of the same age and anatomical position were collected and fixed in 4% paraformaldehyde. These tissues were dehydrated in a graded alcohol series and then embedded in paraffin wax and slide sectioned at 3–5 μm. The slide sections were then stained with Hematoxylin and Eosin (HE) and viewed using a microscope . For each sample, five fields of view (areas) were randomly selected in the HE-stained sections using a 200× objective and then analyzed using Image-Pro Plus 6.0 software . For each sample area, 100–150 myofibers were measured, and the relative size of the myofibers and the distribution of different sizes of the myofibers were determined.Tissues from the gluteus maximus muscles and tongues of an Blood samples were obtained from rabbits that had been fasted for 12 h, and ethylenediaminetetraacetic acid (EDTA)-plasma and plasma samples were collected after centrifugation at 4°C for 20 min.An automatic biochemical analyzer  was used to test the concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatine kinase (CK), creatinine (CREA), glucose (GLU), total cholesterol (TC), triglycerides (TG), calcium (CA), phosphorus (P), and LIP with commercial kits . SPSS13.0 software was used to analyze the measured results. Hematology parameters, like RBC, HGB, HCT, MCV, MCH, MCHC, WBC, RDW-CV, PLT, and MPV, were tested using an auto-hematology analyzer .MSTN-KO and WT rabbits were performed using a Student’s t test, and all data were expressed as a mean ± S.E.M. Statistical analyses were conducted using GraphPad Prism software, and P<0.05 was considered as statistically significant .Statistical comparisons of body weight, myofiber size, and blood parameters among the The data supporting the results of this manuscript are included in the body of the manuscript and as Supplementary data.


Bone substitutes are commonly used when bone grafts are necessary in human craniofacial surgery. To study the properties and biologic behaviour of those substitutes, they can be implanted in experimental animals. A frequently used model involves the creation of critical-sized defects  in the rabbits calvaria (the top part of the skull). The procedure was initially described in 1989 and the authors considered and reported that post-operative analgesia was not necessary. In our experience, this procedure is invasive and can result in severe postoperative pain. The anaesthetic management of rabbits undergoing this procedure is challenging. Most of the recent publications using this model fail to provide sufficient information on perioperative animal management. With this report we have aimed to document a possible practical and simple anaesthetic and postoperative management of rabbits undergoing this procedure. Particular emphasis has been placed on postoperative pain assessment, duration and treatment.®) provided clinically suitable anaesthesia. Supplemental oxygen was administered throughout recovery. Monitoring was clinical and instrumental . Lidocaine was infiltrated at the surgical site and meloxicam was injected subcutaneously as perioperative analgesia. After surgery, pain was assessed five times daily . Postoperative analgesia included SC meloxicam once daily for four days and buprenorphine every 8 h for three days (unless both pain scores were at the lowest possible levels). Rescue analgesia (buprenorphine) was administered in case of the score > 3/8 in the composite pain scale, >4/10 on the grimace scale or if determined necessary by the caregivers. Airway management with a V-gel® was possible but resulted in respiratory obstruction during the surgery in two cases. Hypoventilation was observed in all rabbits. All rabbits experienced pain after the procedure. Monitoring, pain assessments and administration of postoperative analgesia were recommended for 48 h.Calvarial bone surgery on rabbits is frequently performed. This report aims to document a simple and practical anaesthetic and perioperative management for this procedure. Fourteen male New Zealand white rabbits were included in the study. Subcutaneous (SC) dexmedetomidine, ketamine and buprenorphine ± isoflurane vaporized in oxygen administered through a supraglottic airway device (V-gel In Vivo Experiments) guidelines .,31.30,31Particular attention was given to the overall behaviour of the rabbits and water and food intakes, presence of urine and faeces in the litter . Abdomens were palpated each day. Animals were weighed 48 h after surgery. Weight loss of >10% was considered as an end-point.−1  and xylazine 4 mg kg−1 , pentobarbital sodium 150 mg kg−1  was administered intravenously.Between one and six months after the surgery, the rabbits were euthanised. After sedation with SC ketamine 65 mg kgGraphPad Prism 7 for Mac OS X  was used for the graphical representation of simple descriptive statistics. No statistical test was applied to this data set.−1 IV). Those rabbits were the two rabbits who scored lower sedation scores (Rabbits 1 and 11) and were the first rabbits to be anaesthetized on the first and third day of procedures. A R4 V-gel® was inserted into all but one rabbit (Number 11). That rabbit had a smaller head compared to the others and the R4 V-gel® was too large to be advanced smoothly so a R3 V-Gel® was placed. Despite pre-oxygenation, desaturation happened in several cases during airway management. In two cases, obvious respiratory obstruction developed during the early stages of surgery . The V-gels were promptly removed and oxygen +/− isoflurane were administered via a tight facemask connected to the Mapleson F breathing system for the rest of the procedure.The mean ± SD weight before surgery was 3.3 ± 0.4 kg. The median (range) sedation score was 13 (10–13). Individual sedation scores are presented in E’CO2 ranges are reported in 2 of 59 to 79 mmHg) developed in all but the first rabbit. Arterial oxygen tension was between 99 and 407 mmHg in all but two animals: rabbit 3 before insertion of the V-Gel® (68 mmHg) and rabbit 7 which had a facemask at the time of blood sampling (77 mmHg). Base excess was between 0 and 21 and blood glucose concentration was elevated (between 10 and 21 mmol L−1). The individual duration of anaesthesia (55 to 80 min), surgery (30 to 45 min) and return of righting reflex (20 to 55 min) are reported in Individual isoflurane requirements are reported in The evolution of the composite pain score and grimace scale in the three days following surgery are presented in The dura in the right bone defect of rabbit 14 was partially damaged during surgery resulting in moderate bleeding. Recovery from anaesthesia was slower than in the other animals. Mentation was reduced in the first hours after surgery and the animal developed head tilting and walking on the circle. After 48 h of intensive observation and appropriate analgesia administration, this condition did not improve. Weight loss was around 15%, therefore the rabbit was euthanized.® in this surgical procedure had the potential to lead to respiratory obstruction. Second, hypoventilation and ventilation/perfusion inequalities were documented in all animals and may have been unobserved without arterial blood gas analysis. Third, despite early multimodal analgesia, rabbits undergoing this surgical procedure did show signs of pain in at least the first 48 h after surgery. This is particularly 3R relevant as post-operative analgesia of rabbits undergoing this surgical model is inconsistently reported.The description of the presented anaesthetic and perioperative management raises several clinically relevant aspects. First, the use of the V-gel® placement was difficult in the two less sedated animals. Although V-Gel® insertion was easy and successful in the other rabbits, respiratory obstruction developed in the early surgical stages of two rabbits. Endotracheal intubation can be challenging in rabbits [® is specifically designed for rabbits [®, however, has the potential to cause laryngeal compression without an obvious clinical sign [The V-gel rabbits  although rabbits . The V-G rabbits  and has  rabbits  potentia rabbits . The V-Gcal sign . The res® was not completely sealing the airway. This finding would have been unobserved had blood gases not been analysed as SpO2 was > 95% throughout the procedure in all rabbits. This underlines the fact that oxygen administration in the perianaesthetic context is reasonable in rabbits.Arterial blood gas analysis documented ventilation–perfusion inequalities and lower arterial oxygen tension than what could be expected in a small animal that is administered pure oxygen. The rabbits enrolled in this experiment were obtained from a colony free of respiratory pathogens and appeared clinically healthy. Hypoxaemia with a similar anaesthesia protocol without buprenorphine has been documented in rabbits . The lowE’CO2 and arterial PaCO2 are not surprising. Superiority of arterial blood gas analysis over PE’CO2 monitoring was already previously established in pet rabbits [−1 of buprenorphine in rabbits one hour before the administration of a similar anaesthesia protocol (SC medetomidine 0.25 mg kg−1 and ketamine 15 mg kg−1) [2 measured in the last four rabbits at the beginning of anaesthesia (approximately 15–20 min after injection) was lower than PaCO2 during the surgery but higher than what was reported by Murphy et al. [A sidestream capnograph and relatively high fresh gas flows were used so differences between P rabbits . In all  rabbits ,40,41,42Anaesthetic risk is higher in rabbits  than in other domestic species . PerianaDespite early multimodal analgesia  every animal enrolled in this experiment experienced pain. This was a major finding and contrasts the reports on the early application of this technique . This maThis study has several limitations. First, it is well established that the pain response is sex-dependent . The finThe clinical relevance of the documented hypercapnia is unknown. Although outcomes were mostly good from an anaesthesia point of view, peri-anaesthetic management can still be improved. Future studies will look at investigating the impact of opioids and other factors on hypoventilation during anaesthesia and will aim to further refine the post-operative analgesia protocol.® was possible, but the continuous intraoperative monitoring is important due to a potential respiratory obstruction. Rabbits will experience pain after this surgical procedure and we recommend maximum postoperative monitoring and regular administration of postoperative analgesia for 48 h after surgery. This is particularly 3R relevant as post-operative analgesia of rabbits undergoing this surgical model is inconsistently reported.Within its limitations, the present report describes a simple and practical anaesthetic management for rabbits undergoing calvarial bone surgery. Subcutaneous administration of dexmedetomidine, ketamine and buprenorphine ± addition of low-dose isoflurane provided clinically suitable anaesthesia of sufficient duration. Nevertheless, it resulted in marked hypoventilation of unknown clinical importance. Airway management with a V-gel


The interplay between the endoplasmic reticulum (ER), a major store of intracellular Ca2+, and mitochondria, an important source of adenosine triphosphate (ATP), has been the subject of much research, as their dysfunction has been linked with metabolic diseases. Interestingly, throughout the cell’s cytosolic domain, these two organelles share common microdomains called mitochondria-associated ER membranes (MAMs), where their membranes are in close apposition. The role of MAMs is critical for intracellular Ca2+ dynamics as they provide hubs for direct Ca2+ exchange between the organelles. A recent experimental study reported correlation between obesity and MAM formation in mouse liver cells, and obesity-related cellular changes that are closely associated with the regulation of Ca2+ dynamics. We constructed a mathematical model to study the effects of MAM Ca2+ dynamics on global Ca2+ activities. Through a series of model simulations, we investigated cellular mechanisms underlying the altered Ca2+ dynamics in the cells under obesity. We predict that, as the dosage of stimulus gradually increases, liver cells from obese mice will reach the state of saturated cytosolic Ca2+ concentration at a lower stimulus concentration, compared to cells from healthy mice.Multiple cellular organelles tightly orchestrate intracellular calcium (Ca 2+ oscillations carry encoded signals in their amplitude and frequency to regulate various cellular processes, and accumulating evidence supports the importance of the interplay between the ER and mitochondria in cellular Ca2+ homeostasis. Miscommunications between the organelles may be involved in the development of metabolic diseases. Based on a recent experimental study that spotlighted a correlation between obesity and physical interactions of the ER and mitochondria in mouse hepatic cells, we constructed a mathematical model as a tool to probe the effects of the cellular changes linked with obesity on global cellular Ca2+ dynamics. Our model successfully reproduced the experimental study that observed a positive correlation between an increase in ER-mitochondrial junctions and the magnitude of mitochondrial Ca2+ responses. We postulate that hepatic cells from lean animals exhibit Ca2+ oscillations that are more robust under higher concentrations of stimulus, compared to cells from obese animals.It is well known that intracellular Ca For better comparison, we normalized the model outputs in the same notion, using the mitochondrial Ca2+ dynamics from the control model as a reference. As shown in Cmito transient, and the result is consistent with the experimental observation reported by Arruda et al. Given that the MCU has a low Ca2+ affinity, i.e., it requires high concentrations of Ca2+ for channel activation, increasing the mitochondrial surface portion that faces MAMs, and thus exposing more MCUs to higher concentrations of Ca2+, would lead to augmented MCU Ca2+ flux, and consequently, induce larger amplitudes of mitochondrial Ca2+ activities.To determine whether the model can reproduce the experimental observations of Arruda et al. , we perfo et al.  estimatea et al.  quantifia et al. . Fig 2 s2+ transients in the model show a faster reaction time to the perturbation, compared to the experimental data. There are several factors that may play a role in this discrepancy. For simplicity, we modeled the stimulation as a pulse of IP3, with a fast increase in the concentration of IP3 followed by a relatively slow decay. However, the time course of IP3 concentration was not measured in the experiment, and thus we cannot verify whether the model’s IP3 trajectory mimics the IP3 dynamics in the experiment. Furthermore, the model assumes spatially homogeneous mitochondrial Ca2+ concentration, whereas in reality, the Ca2+ concentration first rises near domains with a dense population of MCUs, then subsequently spreads to other parts of the domain. Although the model accurately reproduced the mitochondrial Ca2+ peak increase associated with the upregulation of MAMs, it certainly lacks finesse for simulating the precise timescale of the mitochondrial Ca2+ spikes.The Ca2+ concentration and mitochondrial metabolic products, such as ATP and NADH, to investigate the coordination of calcium signals and the target responses. Recorded by Gaspers et al. [2+. The study reported a fast single cytosolic Ca2+ spike accompanied by an increase in the mitochondrial NADH concentration and then a prolonged phase of NADH oxidation. Moreover, continuous Ca2+ oscillations were paired with sustained mitochondrial NADH metabolic cycles at a raised basal concentration. In follow-up studies, Gaspers et al. [2+ concentration and mitochondrial membrane potential, which was the opposite of what the authors had expected. In a separate study, histamine-dependent mitochondrial Ca2+ signals were accompanied by relatively slower ATP responses in both cytosol and mitochondria [A number of studies traced cytosolic Cas et al. , the dyns et al. , 36 obsechondria .3, M = 5, t0 = 100, and Δ = 0.2. Then the second phase of IP3 dynamics was generated from the following condition:H is the Heaviside function. The corresponding IP3 timeseries and the resulting Ccyt are shown by the inset graph of N rapidly increased with the increase in Ccyt and slowly decreased to the baseline level, in contrast to the fast downstroke of Ccyt. With a raised baseline level, the evolution of N during Ca2+ oscillations was analogous to the experimental observation described above. A similar pattern was observed in other mitochondrial variables. At the onset of the cytosolic Ca2+ spikes, the model generated rapid increases in Vm, ATPc, and ATPm, followed by an extended period of decrease at a considerably slower rate than Ccyt. Similar to the evolution of N, Vm, ATPc, and ATPm showed relatively slower dynamics than Ccyt  mice, for mouse models of obesity. The ob/ob mouse cells showed higher expression levels of IPR and MCU, as well as a higher degree of MAM formation.Arruda et al. also compared hepatic cellular characteristics between different groups of mice. They had a group of lean mice as their control, and two groups of mice, one that had been under high fat diet (HFD) and the other genetically obese , which is determined by Ca2+ fluxes across the plasma membrane. Though we have not introduced any changes to these fluxes for model simulations of the obesity condition, there is experimental evidence that suggests otherwise. Arruda et al. [2+ depletion, facilitates store-operated Ca2+ entry (SOCE). Such protein-protein interactions form another type of Ca2+ microdomain, and they are not explicitly included in the model as they are not the main subject of this paper. However, a follow-up theoretical study of these ER-plasma membrane junctions in a whole cell context, similar to our ER-mitochondria contact model, would be relevant and potentially useful in gaining a complete understanding.Our model is an open-cell type that exhibits dynamic total Caa et al.  showed d2+ oscillations (periodic solutions). Furthermore, as in many previous mathematical models of Ca2+ dynamics [2+ profile throughout the bulk cytosol. These underlying assumptions are physiologically inaccurate, as many experimental studies show the stochastic nature of intracellular Ca2+ activities and clustered spatial distributions of Ca2+ channels that regulate such activities. Nevertheless, Cao et al. [2+ signals that occur due to the intrinsically stochastic nature of the IPR activities, and the cell level global Ca2+ spikes that are more likely to arise from a deterministic mechanism. The authors showed that the oscillation periods generated from a deterministic version of their model lie within the interspike interval distributions simulated from a stochastic version of the model. Thus, we find it reasonable to utilize a deterministic model, such as ours, to simulate and probe the attributes of Ca2+ activities, at least for those at the cell level.The model is deterministic, which assumes synchronized behaviors of the activated IPRs, within each compartment. This allows relatively fast simulations of Cadynamics , 26, 28,o et al.  demonstro et al.  presente2+ dynamics on mitochondrial metabolism. For the obesity model simulations, we only modified the parameters that are directly associated with Ca2+ dynamics. This is not to diminish the criticality of mitochondrial metabolic mechanisms nor to suggest that there is no effect of obesity on the mechanisms. There certainly is a plethora of experimental evidence that suggests correlations between obesity and mitochondrial dysfunction that range from structural functions such as fusion and fission to biochemical functions. What we do not fully comprehend yet is, which parts of the pathways are linked with obesity, and how they communicate with each other. Furthermore, the model is not comprehensive enough to describe mitochondrial metabolic pathways under different cell conditions. For instance, the process of glycolysis is represented by a single parameter, kgly, which is an over simplification of the process that is highly correlated with the nutrient availability, i.e., the cell’s metabolic state. Admittedly, the current state of the model and assumptions is not suitable for investigating possible mechanisms underlying the adverse effects of obesity on mitochondrial metabolic dynamics.The model is not applicable for studying the effects of obesity and obesity-related changes in Ca2+ dynamics would be a system of stochastic partial differential equations with spatial and temporal discretization. However, there is a trade-off between sufficient accuracy, computational efficiency, and data availability.The best mathematical model for describing cellular CaS1 File(ODE)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.