International Journal of Molecular Sciences Review Calcium Mechanisms in Limb-Girdle Muscular Dystrophy with CAPN3 Mutations Jaione Lasa-Elgarresta 1,2, Laura Mosqueira-Martín 1,2, Neia Naldaiz-Gastesi 1,2, Amets Sáenz 1,2, Adolfo López de Munain 1,2,3,4,* and Ainara Vallejo-Illarramendi 1,2,5,* 1 Biodonostia, Neurosciences Area, Group of Neuromuscular Diseases, 20014 San Sebastian, Spain; [email protected] (J.L.-E.); [email protected] (L.M.-M.); [email protected] (N.N.-G.); [email protected] (A.S.) 2 CIBERNED, Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, 28031 Madrid, Spain 3 Departmento de Neurosciencias, Universidad del País Vasco UPV/EHU, 20014 San Sebastian, Spain 4 Osakidetza Basque Health Service, Donostialdea Integrated Health Organisation, Neurology Department, 20014 San Sebastian, Spain 5 Grupo Neurociencias, Departmento de Pediatría, Hospital Universitario Donostia, UPV/EHU, 20014 San Sebastian, Spain * Correspondence: [email protected] (A.L.d.M.); [email protected] (A.V.-I.); Tel.: +34-943-006294 (A.L.d.M.); +34-943-006128 (A.V.-I.) Received: 4 August 2019; Accepted: 11 September 2019; Published: 13 September 2019 Abstract: Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a rare disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness of shoulder, pelvic, and proximal limb muscles that usually appears in children and young adults and results in loss of ambulation within 20 years after disease onset in most patients. The pathophysiological mechanisms involved in LGMDR1 remain mostly unknown, and to date, there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of Ca2+ homeostasis in the skeletal muscle is a significant underlying event in this muscular dystrophy. We also review and discuss specific clinical features of LGMDR1, CAPN3 functions, novel putative targets for therapeutic strategies, and current approaches aiming to treat LGMDR1. These novel approaches may be clinically relevant not only for LGMDR1 but also for other muscular dystrophies with secondary calpainopathy or with abnormal Ca2+ homeostasis, such as LGMD2B/LGMDR2 or sporadic inclusion body myositis. Keywords: calpain 3; calcium; LGMD2A; LGMDR1; muscular dystrophies; calpainopathy 1. Overview of Calcium Homeostasis in the Skeletal Muscle Ca2+ plays a vital role in a wide range of cellular processes such as gene transcription, membrane resealing, secretion, neurotransmission, as well as cell differentiation, proliferation, or survival [1,2]. In skeletal muscle fibers, Ca2+ is crucial for both electric activation along the motor endplate and skeletal muscle contraction. In addition, Ca2+ is involved in many other functions such as protein synthesis, protein degradation, fiber type shifting, Ca2+-regulated proteolysis, transcription factor modulation, mitochondrial adaptation, cell plasticity, and respiration [3]. Therefore, tight regulation of Ca2+ levels is essential for the proper function of skeletal muscle (Figure1). Int. J. Mol. Sci. 2019, 20, 4548; doi:10.3390/ijms20184548 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 4548 2 of 22 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 22 FigureFigure 1. Representation1. Representation of of Ca Ca2+2+ fluxesfluxes in the the muscle muscle fiber. fiber. Upon Upon sarcolemmal sarcolemmal depolarization depolarization reaching reaching T-tubulesT-tubules (1), (1), DHPRs DHPRs undergo undergo a a conformational conformational change that that activates activates RyR1 RyR1 channels channels and and results results in in Ca2Ca+ release2+ release from from the the SR SR (2). (2). CaCa22++ diffusesdiffuses to to the the sarcomere sarcomere where where it itinitiates initiates muscle muscle contraction contraction (3). (3). 2+ MuscleMuscle relaxation relaxation takes takes place place when when Ca Ca2+ isis sequesteredsequestered into into the the SR SR by by SERCAs SERCAs (4) (4) or orpumped pumped out out of of 2+ thethe fiber fiber by membraneby membrane channels channels (NCX, (NCX, PMCA) PMCA) (5). (5). Cytosolic Cytosolic Ca Ca2+ also also binds binds CaM, CaM, which which activates activatesthe 2+ 2+ Ca2the+-dependent Ca -dependent signaling signaling pathways pathways resulting resulting in muscle in muscle gene regulationgene regulation (6). Cytosolic (6). Cytosolic Ca2+ alsoCa reachesalso reaches mitochondria (7), where it stimulates metabolism and ATP synthesis required for muscle mitochondria (7), where it stimulates metabolism and ATP synthesis required for muscle contraction contraction and relaxation. and relaxation. 1.1.1.1. Ca 2Ca+ 2+in in Excitation-Contraction Excitation-Contraction CouplingCoupling MuscleMuscle contraction contraction initiates initiates by by depolarizationdepolarization of the the sarcolemma sarcolemma in in response response to toacetylcholine acetylcholine releaserelease from from motoneurons. motoneurons. TheThe actionaction potentialpotential propagates into into the the triads, triads, which which are are anatomical anatomical structures formed by the association of sarcolemmal transverse tubules (T-tubules) and sarcoplasmic structures formed by the association of sarcolemmal transverse tubules (T-tubules) and sarcoplasmic reticulum (SR) terminal cisternae [4]. Triads play an essential role in excitation-contraction coupling reticulum (SR) terminal cisternae [4]. Triads play an essential role in excitation-contraction coupling (ECC) since they allow close contact and synchronization between crucial receptors in the (ECC) since they allow close contact and synchronization between crucial receptors in the sarcolemma sarcolemma and the SR. See [3,5] for review. Membrane depolarization activates dihydropyridine andreceptors the SR. (DHPRs) See [3,5 ]in for the review.T-tubules Membrane [6], and this depolarizationresults in activation activates of the closely dihydropyridine apposed ryanodine receptors (DHPRs)receptors in the(RyRs), T-tubules the main [6], Ca and2+ release this results channels in activation in the SR [7]. of theRyR1, closely the predominant apposed ryanodine RyR isoform receptors in 2+ (RyRs),skeletal the muscle, main Ca releasesrelease Ca channels2+ from the in theSR into SR [ 7the]. RyR1, cytosol the either predominant after activation RyR isoform by DHPRs in skeletal or 2+ muscle,increased releases cytosolic Ca Cafrom2+ levels the [8]. SR RyR1 into thefunction cytosol is modulated either after by activation post-translational by DHPRs modifications, or increased cytosolicsuch as Ca S-nitrosylation,2+ levels [8]. S-glutathionylation, RyR1 function is and modulated phosphorylation by post-translational by both Protein modifications, Kinase A (PKA) such as S-nitrosylation,and Ca2+/calmodulin-dependent S-glutathionylation, protein and kinase phosphorylation II (CaMKII) [9]. by RyRs both operate Protein in Kinase coordination A (PKA) with and Ca2other+/calmodulin-dependent proteins in order to maintain protein the kinase balance II (CaMKII) between Ca [9].2+ RyRsrelease, operate Ca2+ storage, in coordination and Ca2+ reuptake with other proteins[10]. Indeed, in order a variety to maintain of proteins the balance and small between molecules, Ca2+ bothrelease, in the Ca SR2+ storage,lumen and and cytosol Ca2+ arereuptake needed [10 ]. Indeed,for this a varietytight coordination of proteins [9]. and On smallthe cytosolic molecules, side, calmodulin both in the (CaM), SR lumen a Ca and2+ sensor, cytosol has area dual needed effect for thison tight RyR1, coordination functioning [as9]. an On activator the cytosolic at low side,cytosolic calmodulin Ca2+ levels (CaM), and as a an Ca inhibitor2+ sensor, at hashigh a cytosolic dual eff ect 2+ onCa RyR1, levels functioning [11]. On the as anSR activatorluminal side, at low calsequestrin cytosolic Ca(CSQ)2+ levels forms and a complex as an inhibitor with RyRs, at junctin, high cytosolic and 2+ Ca2triadin.+ levels RyR [11 ].function On the is SRinhibited luminal by side,the binding calsequestrin of CSQ, (CSQ)which formsessentially a complex depends with on luminal RyRs, junctin,Ca concentration [12]. CSQ is the main Ca2+-binding protein in the SR lumen, and it functions as an and triadin. RyR function is inhibited by the binding of CSQ, which essentially depends on luminal endogenous regulator of Ca2+ fluxes and as a Ca2+ reservoir with a moderate affinity but high capacity Ca2+ concentration [12]. CSQ is the main Ca2+-binding protein in the SR lumen, and it functions as an endogenous regulator of Ca2+ fluxes and as a Ca2+ reservoir with a moderate affinity but high capacity Int. J. Mol. Sci. 2019, 20, 4548 3 of 22 to bind Ca2+ [13]. After Ca2+ release into the cytosol through RyR1, Ca2+ binds to various cytosolic Ca2+ buffers, such as ATP and CaM, and it can also be sequestered by mitochondria. At the sarcomere, the contractile unit of the skeletal muscle, troponin C undergoes a Ca2+-dependent conformational change that ultimately results in myosin and actin cross-bridge cycling and muscle contraction [14]. Upon muscle excitation, cytosolic Ca2+ levels raise from ~100 nM (resting levels) to ~10µM in slow fibers, and ~18 µM in fast fibers [15]. Muscle relaxation initiates by lowering of cytosolic Ca2+ back to resting levels, which mainly relies on sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps [16], and sarcolemmal Ca2+ transporters such as