Jin Seok Woo

S165F mutation of junctophilin 2 affects Ca2+ signalling in skeletal muscle

JPs (junctophilins) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the t-tubule (transverse-tubule) and SR (sarcoplasmic reticulum) membranes. In humans with HCM (hypertrophic cardiomyopathy), mutations in JP2 are linked to altered Ca2+ signalling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. In the present study, we investigated the role of the dominant-negative JP2-S165F mutation (which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, overexpression of JP2-S165F in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Single myotube Ca2+ imaging experiments showed reductions in both the excitation-contraction coupling gain and RyR (ryanodine receptor) 1-mediated Ca2+ release from the SR. Immunoprecipitation assays revealed defects in the PKC (protein kinase C)-mediated phosphorylation of the JP2-S165F mutant protein at Ser165 and in binding of JP2-S165F to the Ca2+ channel TRPC3 (transient receptor potential cation canonical-type channel 3) on the t-tubule membrane. Therefore both the hypertrophy and altered intracellular Ca2+ signalling in the JP2-S165F-expressing skeletal myotubes can be linked to altered phosphorylation of JP2 and/or altered cross-talk among Ca2+ channels on the t-tubule and SR membranes.

Hypertrophy in Skeletal Myotubes Induced by Junctophilin-2 Mutant, Y141H, Involves an Increase in Store-operated Ca2+ Entry via Orai1

Background: Junctophilin-2 (JP2) contributes to the formation of junctional membrane complexes (JMC) in striated muscle. Results: Different from the S165F mutant of JP2, Y141H induces hypertrophy in skeletal myotubes involving abnormal JMC and altered Ca2+ signaling due to the increased store-operated Ca2+ entry (SOCE) via Orai1. Conclusion: JP2 is linked to muscle hypertrophy via various Ca2+ signaling pathways. Significance: SOCE is a novel factor in understanding muscle hypertrophy. Junctophilins (JPs) play an important role in the formation of junctional membrane complexes (JMC) in striated muscle by physically linking the transverse-tubule and sarcoplasmic reticulum (SR) membranes. Researchers have found five JP2 mutants in humans with hypertrophic cardiomyopathy. Among these, Y141H and S165F are associated with severely altered Ca2+ signaling in cardiomyocytes. We previously reported that S165F also induced both hypertrophy and altered intracellular Ca2+ signaling in mouse skeletal myotubes. In the present study, we attempted to identify the dominant-negative role(s) of Y141H in primary mouse skeletal myotubes. Consistent with S165F, Y141H led to hypertrophy and altered Ca2+ signaling (a decrease in the gain of excitation-contraction coupling and an increase in the resting level of myoplasmic Ca2+). However, unlike S165F, neither ryanodine receptor 1-mediated Ca2+ release from the SR nor the phosphorylation of the mutated JP2 by protein kinase C was related to the altered Ca2+ signaling by Y141H. Instead, abnormal JMC and increased SOCE via Orai1 were found, suggesting that the hypertrophy caused by Y141H progressed differently from S165F. Therefore JP2 can be linked to skeletal muscle hypertrophy via various Ca2+ signaling pathways, and SOCE could be one of the causes of altered Ca2+ signaling observed in muscle hypertrophy.

TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts.

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without α1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in α1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or α1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.

Glutamate at position 227 of junctophilin-2 is involved in binding to TRPC3

Canonical-type transient receptor potential cation channel type 3 (TRPC3) allows the entry of extracellular Ca2+ and Na+ into various cells. In mouse skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor type 1/Ca2+-release channel on sarcoplasmic reticulum membrane) regulates the gain of excitation–contraction coupling. Junctophilin-2 (JP2) is a TRPC3-interacting protein in mouse skeletal myotubes. Based on these knowledge from bona-fide TRPC3-expressing cells, to identify critical binding region(s) of JP2 that participate in binding to TRPC3, various JP2 portions were subjected to co-immunoprecipitation assay with intact TRPC3 from rabbit skeletal muscle. A region covering 143 to 234 amino acids of JP2 (F1-2) was the most efficient portion binding to TRPC3. Through mutational studies, we found that the binding ability of JP2 to TRPC3 was mainly due to glutamate in the F1-2 region (E227). This substantial binding between JP2 and TRPC3 suggests that JP2 can be a regulatory protein of TRPC3 and/or TRPC3-mediated Ca2+ homeostasis in skeletal muscle.

Stromal interaction molecule 1 (STIM1) regulates sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Stromal interaction molecule 1 (STIM1) mediates Ca2+ movements from the extracellular space to the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca2+ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca2+ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca2+ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca2+ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca2+ movements between the SR and the cytosol.

Heteromeric TRPC3 with TRPC1 formed via its ankyrin repeats regulates the resting cytosolic Ca2+ levels in skeletal muscle

The main tasks of skeletal muscle are muscle contraction and relaxation, which are mediated by changes in cytosolic Ca(2+) levels. Canonical-type transient receptor potential 3 (TRPC3) contains an ankyrin repeat (AR) region at the N-terminus (38-188 amino acids) and forms extracellular Ca(2+)-entry channels by homo or heteromerization with other TRP subtypes in various cells including skeletal myotubes. However, previous research has not determined which region(s) of TRPC3 is responsible for the heteromerization, whether the AR region participates in the heteromerizations, or what is the role of heteromeric TRPC3s in skeletal muscle. In the present study, the heteromerization of TRPC3 with TRPC1 was first examined by GST pull-down assays of TRPC3 portions with TRPC1. The portion containing the AR region of TRPC3 was bound to the TRPC1, but the binding was inhibited by the very end sub-region of the TRPC3 (1-37 amino acids). In-silico studies have suggested that the very end sub-region possibly induces a structural change in the AR region. Second, the very end sub-region of TRPC3 was expressed in mouse primary skeletal myotubes, resulting in a dominant-negative inhibition of heteromeric TRPC3/1 formation. In addition, the skeletal myotubes expressing the very end sub-region showed a decrease in resting cytosolic Ca(2+) levels. These results suggest that the AR region of TRPC3 could mediate the heteromeric TRPC3/1 formation, and the heteromeric TRPC3/1 could participate in regulating the resting cytosolic Ca(2+) levels in skeletal muscle.

Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca(2+)-uptake through SERCA1a (more than 35%) at micromolar Ca(2+) but not at nanomolar Ca(2+), suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.

Angiopoietin 1 enhances the proliferation and differentiation of skeletal myoblasts.

Angiopoietin 1 (Ang1) plays an important role in various endothelial functions, such as vascular integrity and angiogenesis; however, less is known about its function outside of the endothelium. In this study, we examined whether Ang1 has direct effects on skeletal muscle cells. We found that Ang1 exhibited myogenic potential, as it promoted the proliferation, migration, and differentiation of mouse primary skeletal myoblasts. The positive effect of Ang1 on myoblast proliferation could have been mediated by the α7 and β1 integrins. We also found that Ang1 potentiated cellular Ca2+ movements in differentiated myotubes in response to stimuli, possibly through the increased expression of two Ca2+‐related proteins, namely, Orai1 and calmodulin. Ang1 also increased Orai1 and calmodulin expression in mouse hearts in vivo. These results provide an insight into the molecular mechanisms by which Ang1 directly affects the myogenesis of striated muscle. J. Cell. Physiol.

Interaction between mitsugumin 29 and TRPC3 participates in regulating Ca2+ transients in skeletal muscle

Mitsugumin 29 (MG29) is related to the fatigue and aging processes of skeletal muscle. To examine the roles of MG29 in conjunction with its binding protein, the canonical-type transient receptor potential cation channel 3 (TRPC3), in skeletal muscle, the binding region of MG29 to TRPC3 was studied along with the functional relevance of the binding in mouse primary skeletal myotubes using co-immunoprecipitation assays and Ca(2+) imaging experiments. The N-terminus and the I-II loop of MG29 constitute the binding region for TRPC3. The myotubes that expressed the MG29 mutant missing the entire TRPC3-binding region showed a disrupted binding between endogenous MG29 and TRPC3 and a reduction in Ca(2+) transients in response to membrane depolarization without affecting ryanodine receptor 1 activity, the resting cytosolic Ca(2+) level, and the amount of releasable Ca(2+) from the sarcoplasmic reticulum. Among the proteins mediating Ca(2+) movements in skeletal muscle, TRPC4 expression was significantly decreased by the MG29 mutant. Therefore, MG29 could be a new factor for regulating Ca(2+) transients during skeletal muscle contraction possibly via a correlation with TRPC3 and TRPC4.

A focus on extracellular Ca 2+ entry into skeletal muscle

The main task of skeletal muscle is contraction and relaxation for body movement and posture maintenance. During contraction and relaxation, Ca2+ in the cytosol has a critical role in activating and deactivating a series of contractile proteins. In skeletal muscle, the cytosolic Ca2+ level is mainly determined by Ca2+ movements between the cytosol and the sarcoplasmic reticulum. The importance of Ca2+ entry from extracellular spaces to the cytosol has gained significant attention over the past decade. Store-operated Ca2+ entry with a low amplitude and relatively slow kinetics is a main extracellular Ca2+ entryway into skeletal muscle. Herein, recent studies on extracellular Ca2+ entry into skeletal muscle are reviewed along with descriptions of the proteins that are related to extracellular Ca2+ entry and their influences on skeletal muscle function and disease.