IP3 receptors contribute to muscle atrophy and maintain ribosomal RNA content during 3-day hind limb suspension in rats

Abstract

Aim: Skeletal muscle unloading leads to the upregulation of proteolytic gene expression, downregulation of protein synthesis markers, and the development of muscle atrophy. These changes are accompanied by alterations in calcium signaling. We investigated the role of Inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) in regulating calcium signaling and controlling gene expression in skeletal muscles during unloading.

Methods: Male Wistar rats were randomly assigned to 4 groups (n = 8 per group). C - vivarium control; C+2APB - vivarium control with daily intraperitoneal injections of the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB, 10 mg/ kg b.w.); HU - 3-day hind limb unloading; HU+2APB - 3-day hind limb unloading with daily 2-APB injections. After the intervention, soleus muscles were analyzed for markers of calcium metabolism and proteostasis.

Results: Three days of unloading resulted in a significant increase in nuclear phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMK II) content and calcineurin (CaN) expression compared to the C group (P < 0.05); this effect was prevented in the HU+2APB group. In HU+2APB rats, the decline in soleus muscle weight-to-body weight ratio was partially prevented, and the downregulation of protein synthesis markers (as seen in the HU group) was also prevented. However, proteolytic signaling markers were equally upregulated in the HU+2APB and HU groups compared to C.

Conclusion: IP3 receptor inhibition during 3-day hind limb suspension in rats partially prevented the decline in m. soleus weight index and protein synthesis markers. This effect may be attributed to alterations in the regulation of nuclear calcium signaling.

Keywords

IP3 receptors / atrophy / m. soleus / rRNA / unloading

References

[1] ShenkmanBS.Calcium-dependent signaling mechanisms and soleus fiber remodeling under gravitational unloading.J Muscle Res Cell Motil2008;29:221-30

[2] IngallsCP,ArmstrongRB.Intracellular Ca²⁺ transients in mouse soleus muscle after hindlimb unloading and reloading.J Appl Physiol1999;87:386-90

[3] YangH,PanF.New findings: hindlimb unloading causes nucleocytoplasmic Ca²⁺ overload and DNA damage in skeletal muscle.Cells2023;12:1077 PMCID:PMC10093444

[4] BelovaSP,ShenkmanBS.The blockade of dihydropyridine channels prevents an increase in μ-calpain level under m. soleus unloading.Dokl Biochem Biophys2015;460:1-3

[5] SharloKA,TyganovSA.The effect of SERCA activation on functional characteristics and signaling of rat soleus muscle upon 7 days of unloading.Biomolecules2023;13:1354 PMCID:PMC10526198

[6] CárdenasC,MolgóJ,MigneryGA.Nuclear inositol 1,4,5-trisphosphate receptors regulate local Ca²⁺ transients and modulate cAMP response element binding protein phosphorylation.J Cell Sci2005;118:3131-40

[7] ChinER.Intracellular Ca²⁺ signaling in skeletal muscle: decoding a complex message.Exerc Sport Sci Rev2010;38:76-85

[8] ChinER.The role of calcium and calcium/calmodulin-dependent kinases in skeletal muscle plasticity and mitochondrial biogenesis.Proc Nutr Soc2004;63:279-86

[9] CrabtreeGR.Generic signals and specific outcomes: signaling through Ca²⁺, calcineurin, and NF-AT.Cell1999;96:611-4

[10] ChibalinAV,ZakyrjanovaGF,KrivoiII.Early endplate remodeling and skeletal muscle signaling events following rat hindlimb suspension.J Cell Physiol2018;233:6329-36

[11] KravtsovaVV,VilchinskayaNA.Chronic ouabain prevents Na,K-ATPase dysfunction and targets AMPK and IL-6 in disused rat soleus muscle.Int J Mol Sci2021;22:3920 PMCID:PMC8069997

[12] KravtsovaVV,MatchkovVV.Skeletal muscle Na,K-ATPase as a target for circulating ouabain.Int J Mol Sci2020;21:2875 PMCID:PMC7215781

[13] ArayaR,CárdenasJC.Dihydropyridine receptors as voltage sensors for a depolarization-evoked, IP₃R-mediated, slow calcium signal in skeletal muscle cells.J Gen Physiol2003;121:3-16 PMCID:PMC2217318

[14] Arias-CalderónM,Díaz-VegasA.Characterization of a multiprotein complex involved in excitation-transcription coupling of skeletal muscle.Skelet Muscle2016;6:15 PMCID:PMC4827232

[15] CasasM,JaimovichE.ATP signaling in skeletal muscle: from fiber plasticity to regulation of metabolism.Exerc Sport Sci Rev2014;42:110-6

[16] Atakpa-AdajiP.IP₃R at ER-mitochondrial contact sites: beyond the IP₃R-GRP75-VDAC1 Ca²⁺ funnel.Contact2023;6:25152564231181020 PMCID:PMC10328019

[17] DubininMV,IgoshkinaAD,MikinaNV.Effect of 2-aminoethoxydiphenyl borate on the functions of mouse skeletal muscle mitochondria.Biochem Biophys Res Commun2024;712-713:149944

[18] RossiAM.IP₃ receptors - lessons from analyses ex cellula.J Cell Sci2018;132:jcs222463

[19] Foskett J, Daniel Mak D. Regulation of IP₃R channel gating by Ca²⁺ and Ca²⁺ binding proteins. structure and function of calcium release channels.Curr Top Membr2010;66:235-72

[20] MirzoevT,VilchinskayaN,ShenkmanB.Key markers of mTORC1-dependent and mTORC1-independent signaling pathways regulating protein synthesis in rat soleus muscle during early stages of hindlimb unloading.Cell Physiol Biochem2016;39:1011-20

[21] BelovaSP,KostrominovaTY,NemirovskayaTL.P38α-MAPK signaling inhibition attenuates soleus atrophy during early stages of muscle unloading.Int J Mol Sci2020;21:2756 PMCID:PMC7215762

[22] KilkennyC,CuthillIC,AltmanDG.Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research.PLoS Biol2010;8:e1000412 PMCID:PMC2893951

[23] Morey-holtonE,KaplanskyA.The Hindlimb unloading rat model: literature overview, technique update and comparison with space flight data. experimentation with animal models in space.Adv Space Biol Med2005;10:7-40

[24] LinL,YanW.Influence of orai1 intervention on mouse airway epithelium reactions in vivo and in vitro.Ann Allergy Asthma Immunol2012;108:103-12

[25] WangG,XuC,GaoY.Inhibition of SOCs attenuates acute lung injury induced by severe acute pancreatitis in rats and PMVECs injury induced by lipopolysaccharide.Inflammation2016;39:1049-58

[26] BelovaSP,SharloK,ShenkmanBS.Metformin attenuates an increase of calcium-dependent and ubiquitin-proteasome markers in unloaded muscle.J Appl Physiol2022;133:1149-63

[27] PfafflMW.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res2001;29:e45 PMCID:PMC55695

[28] GigerJM,ZengM,HaddadF.Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin.J Appl Physiol2009;107:1204-12 PMCID:PMC2763827

[29] ThomasonDB.Atrophy of the soleus muscle by hindlimb unweighting.J Appl Physiol1990;68:1-12

[30] ZaripovaKA,SharloKA,NemirovskayaTL.Blocking IP3 receptors with 2-APB alters cellular signaling during 7-day soleus unloading in rats.J Evol Biochem Phys2024;60:1795-806

[31] SaleemH,MolinskiTF.Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor.Br J Pharmacol2014;171:3298-312 PMCID:PMC4080982

[32] RostasJAP.Calcium/calmodulin-stimulated protein kinase II (CaMKII): different functional outcomes from activation, depending on the cellular microenvironment.Cells2023;12:401 PMCID:PMC9913510

[33] HudsonMB.Calcineurin: a poorly understood regulator of muscle mass.Int J Biochem Cell Biol2013;45:2173-8 PMCID:PMC3947871

[34] FajardoVA,ChambersPJ.Effects of sarcolipin deletion on skeletal muscle adaptive responses to functional overload and unload.Am J Physiol Cell Physiol2017;313:C154-61

[35] BaiH,YanQ.TRPV2-induced Ca²⁺-calcineurin-NFAT signaling regulates differentiation of osteoclast in multiple myeloma.Cell Commun Signal2018;16:68 PMCID:PMC6191893

[36] ZaripovaKA,SharloKA.SERCA activation prevents Ca²⁺ and ATP upregulation during 3-day soleus muscle unloading in rats.Am J Physiol Regul Integr Comp Physiol2025;329:R108-22

[37] SharloKA,VilchinskayaNA.Molecular signaling effects of electrical stimulation of the soleus muscle during 6 days of dry immersion.Sports Med Health Sci2025;

[38] MoinardC.Direct or indirect regulation of muscle protein synthesis by energy status?.Clin Nutr2021;40:1893-6

[39] MillwardDJ,JamesWP,RyattJS.Relationship between protein synthesis and RNA content in skeletal muscle.Nature1973;241:204-5

[40] TyganovSA,BelovaSP.Effects of plantar mechanical stimulation on anabolic and catabolic signaling in rat postural muscle under short-term simulated gravitational unloading.Front Physiol2019;10:1252 PMCID:PMC6776874

[41] RozhkovSV,MirzoevTM.Temporal changes in the markers of ribosome biogenesis in rat soleus muscle under simulated microgravity.Acta Astronautica2021;186:252-8

[42] Jiménez-VidalM,PutneyLK.Nuclear-localized calcineurin homologous protein CHP1 interacts with upstream binding factor and inhibits ribosomal RNA synthesis.J Biol Chem2010;285:36260-6 PMCID:PMC2978553

[43] RobersonPA,WellesJE.A time course for markers of protein synthesis and degradation with hindlimb unloading and the accompanying anabolic resistance to refeeding.J Appl Physiol2020;129:36-46 PMCID:PMC7469230

[44] FigueiredoVC.Regulation of ribosome biogenesis in skeletal muscle hypertrophy.Physiology2019;34:30-42

[45] KotaniT,KouzakiK,IsemuraM.Percutaneous electrical stimulation-induced muscle contraction prevents the decrease in ribosome RNA and ribosome protein during pelvic hindlimb suspension.J Appl Physiol2022;133:822-33

[46] RozhkovSV,ShenkmanBS.Inhibition of mTORC1 differentially affects ribosome biogenesis in rat soleus muscle at the early and later stages of hindlimb unloading.Arch Biochem Biophys2022;730:109411

[47] RoseAJ,JensenTE.A Ca²⁺-calmodulin-eEF2K-eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions.J Physiol2009;587:1547-63

[48] IngallsCP,ArmstrongRB.Time course changes in [Ca2+]i, force, and protein content in hindlimb-suspended mouse soleus muscles. Aviat Space Environ Med 2001;72:471-6.

[49] HizliAA,SwangerJ.Phosphorylation of eukaryotic elongation factor 2 (eEF2) by cyclin a-cyclin-dependent kinase 2 regulates its inhibition by eEF2 kinase.Mol Cell Biol2013;33:596-604

[50] BodineSC.Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1.Am J Physiol Endocrinol Metab2014;307:E469-84 PMCID:PMC4166716

[51] HansonAM,YoungMH,FergusonVL.Longitudinal characterization of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb suspension.Muscle Nerve2013;48:393-402

[52] LeermakersPA,ScholsAMWJ.Skeletal muscle unloading results in increased mitophagy and decreased mitochondrial biogenesis regulation.Muscle Nerve2019;60:769-78 PMCID:PMC6900132