The reduced contraction capacity of palatopharyngeal muscle in OSAHS is related to the decreased intra-cellular [Ca2+] mediated by low RyR1 and DHPRα1s expression.

PURPOSE: To aim of the study was to explore the possible mechanisms for the decreased contraction capacity of the palatopharyngeal muscle in cases with obstructive sleep apnea hypopnea syndrome (OSAHS). METHODS: Palatopharyngeal muscle specimens from patients with OSAHS were taken as the case group. Palatopharyngeal muscle tissue by surgical removal of oropharyngeal malignant tumors was used as a control cohort. The palatopharyngeal muscle contraction capacity was measured by assessing diaphragm peak-twitching force / cross-sectional area (Pt/CSA), fatigue index (FI) twitch tension, and force per cross-sectional area (Force/CSA). Myofibril and sarcoplasmic reticulum (SR) ultra-structures were observed by electron microscopy. The intra-cellular calcium concentration was measured by fluorescence spectrophotometry. DHPRα1s and RyR1 expression profiles were probed through RT-qPCR and Western blot, and the colocalization of them was determined by immunofluorescence. RESULTS: In comparison with the control cohort, the OSAHS cohort demonstrated decreased Pt/CSA (P < 0.01), FI twitch tension (P < 0.01), together with contraction capacity (P < 0.01). This cohort also had lower intra-cellular [Ca2+] of palatopharyngeal muscle cells with abnormal ultrastructure of sarcoplasmic reticulum (SR) (P < 0.01). In addition, transcriptomic (P < 0.01) and proteomic expression (P < 0.01) for RyR1 and DHPRα1s were markedly reduced within OSAHS cohort, although the degree of colocalization of them was not altered. CONCLUSION: RyR1 and DHPRα1s downregulation may disrupt intra-cellular [Ca2+] homeostasis and subsequently decrease the palatopharyngeal muscle contraction capacity in patients with OSAHS, thus providing a novel insight into the pathogenesis of OSAHS.

Dysferlin-deficient myotubes show tethering of different membrane compartments characterized by TMEM16E and DHPRα.

TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.

Transport of the alpha subunit of the voltage gated L-type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles.

Contraction of skeletal muscle is initiated by excitation-contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca2+ release. EC coupling requires L-type voltage gated Ca2+ channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule-independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRß, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons.

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Structural and biophysical analyses of the skeletal dihydropyridine receptor ß subunit ß1a reveal critical roles of domain interactions for stability.

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and ß subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR ß subunit ß1a Removal of the intrinsically disordered N and C termini and the hook region of ß1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined ß isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the ß1a and ß2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both ß isoforms by ∼6-8 °C. Similar to other ß isoforms, ß1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of ß1a while also providing a conduit for allosteric signaling events.

STAC3 incorporation into skeletal muscle triads occurs independent of the dihydropyridine receptor.

Excitation-contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine-rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.

Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the ß1a Subunit.

The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the ß1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that ß1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of ß1a, which are essential to support EC coupling. Direct FRET measurements between α1S and ß1a confirmed that both wild type and truncated ß1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the ß1a subunit in supporting skeletal muscle excitation-contraction coupling.

Elucidation of the complex metabolic profile of cerebrospinal fluid using an untargeted biochemical profiling assay.

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy.

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

INTRODUCTION: Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. METHODS: We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. RESULTS: Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca2+ ] were significantly decreased in the rat sepsis model compared with controls. DISCUSSION: Decreased intracellular [Ca2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017.

Structure-Function Relationship of the Voltage-Gated Calcium Channel Cav1.1 Complex.

Voltage-gated calcium (Cav) channels are miniature membrane transistors that convert membrane electrical signals to intracellular Ca2+ transients that trigger many physiological events. In mammals, there are ten subtypes of Cav channel, among which Cav1.1 is the first Cavα1 to be cloned. Cav1.1 is specified for the excitation-contraction coupling of skeletal muscles, and has been a prototype in the structural investigations of Cav channels. This article summarized the recent advances in the structural elucidation of Cav1.1 and the mechanistic insights derived from the 3.6 Å structure obtained using single-particle, electron cryomicroscopy. The structure of the Cav1.1 complex established the framework for mechanistic understanding of excitation-contraction coupling and provides the template for molecular interpretations of the functions and disease mechanisms of Cav and Nav channels.

Bridging the myoplasmic gap II: more recent advances in skeletal muscle excitation-contraction coupling.

In skeletal muscle, excitation-contraction (EC) coupling relies on the transmission of an intermolecular signal from the voltage-sensing regions of the L-type Ca(2+) channel (Ca(V)1.1) in the plasma membrane to the channel pore of the type 1 ryanodine receptor (RyR1) nearly 10 nm away in the membrane of the sarcoplasmic reticulum (SR). Even though the roles of Ca(V)1.1 and RyR1 as voltage sensor and SR Ca(2+) release channel, respectively, have been established for nearly 25 years, the mechanism underlying communication between these two channels remains undefined. In the course of this article, I will review current viewpoints on this topic with particular emphasis on recent studies.

Cardiac-specific disruption of Bin1 in mice enables a model of stress- and age-associated dilated cardiomyopathy.

Non-compensated dilated cardiomyopathy (DCM) leading to death from heart failure is rising rapidly in developed countries due to aging demographics, and there is a need for informative preclinical models to guide the development of effective therapeutic strategies to prevent or delay disease onset. In this study, we describe a novel model of heart failure based on cardiac-specific deletion of the prototypical mammalian BAR adapter-encoding gene Bin1, a modifier of age-associated disease. Bin1 deletion during embryonic development causes hypertrophic cardiomyopathy and neonatal lethality, but there is little information on how Bin1 affects cardiac function in adult animals. Here we report that cardiomyocyte-specific loss of Bin1 causes age-associated dilated cardiomyopathy (DCM) beginning by 8-10 months of age. Echocardiographic analysis showed that Bin1 loss caused a 45% reduction in ejection fraction during aging. Younger animals rapidly developed DCM if cardiac pressure overload was created by transverse aortic constriction. Heterozygotes exhibited an intermediate phenotype indicating Bin1 is haplo-insufficient to sustain normal heart function. Bin1 loss increased left ventricle (LV) volume and diameter during aging, but it did not alter LV volume or diameter in hearts from heterozygous mice nor did it affect LV mass. Bin1 loss increased interstitial fibrosis and mislocalization of the voltage-dependent calcium channel Cav 1.2, and the lipid raft scaffold protein caveolin-3, which normally complexes with Bin1 and Cav 1.2 in cardiomyocyte membranes. Our findings show how cardiac deficiency in Bin1 function causes age- and stress-associated heart failure, and they establish a new preclinical model of this terminal cardiac disease.

L-Dopa Might Be Insufficient to Suppress Development of Prolactinomas in Dihydropteridine Reductase-Deficiency Patients.

Dihydropteridine reductase (DHPR) deficiency is a disorder that prevents regeneration of tetrahydrobiopterin (BH4), causing hyperphenylalaninemia (HPA) and low levels of neurotransmitters, including dopamine. Due to low levels of dopamine, patients present with hyperprolactinemia. Treatment consists of a phenylalanine (Phe)-restricted diet, hydroxytryptophan and levodopa (L-Dopa) supplementation, leading to a rapid normalization of prolactin (PRL) levels. We report a case of a patient with DHPR deficiency presenting with new symptomatic hyperprolactinemia and amenorrhea in adolescence despite appropriate management. The prolactinoma was confirmed with pituitary magnetic resonance imaging. The patient was started on cabergoline with rapid normalization of PRL levels and resolution of symptoms, in keeping with previous reports. Cabergoline has a stronger affinity for the D2R receptor and longer half-life than L-Dopa, leading to lactotroph apoptosis, tumor shrinkage, and rapid and maintained normalization of PRL levels, with a better side-effect profile. Patients with DHPR deficiency need to be actively monitored for symptomatic hyperprolactinemia, as L-Dopa monotherapy is insufficient to suppress PRL secretion, leading to lactotroph hypertrophy and proliferation over time and development of prolactinomas in later life.

Case report: Dihydropyridine receptor (CACNA1S) congenital myopathy, a novel phenotype with early onset periodic paralysis.

Introduction: CACNA1S related congenital myopathy is an emerging recently described entity. In this report we describe 2 sisters with mutations in the CACNA1S gene and the novel phenotype of congenital myopathy and infantile onset episodic weakness. Clinical description: Both sisters had neonatal onset hypotonia, muscle weakness, and delayed walking. Episodic weakness started in infancy and continued thereafter, provoked mostly by cold exposure. Muscle imaging revealed fat replacement of gluteus maximus muscles. Next generation sequencing found the missense p.Cys944Tyr variant and the novel splicing variant c.3526-2A>G in CACNA1S. Minigene assay revealed the splicing variant caused skipping of exon 28 from the transcript, potentially affecting protein folding and/or voltage dependent activation. Conclusion: This novel phenotype supports the notion that there are age related differences in the clinical expression of CACNA1S gene mutations. This expands our understanding of mutations located in regions of the CACNA1S outside the highly conserved S4 segment, where most mutations thus far have been identified.

Reduced DHPRα1S and RyR1 expression levels are associated with diaphragm contractile dysfunction during sepsis.

INTRODUCTION: Sepsis often causes diaphragm contractile dysfunction. Dihydropyridine receptors (DHPRα1s and DHPRα1c) and ryanodine receptors (RyR1, RyR2, and RyR3) are essential for excitation-contraction coupling in striated muscles. However, their expression in diaphragm during sepsis have not been explored. METHODS: Eight rats received endotoxin, and 8 more rats received placebo. After 24 hours, 3) diaphragm isometric contractile force was measured. The mRNA and protein levels of DHPRs and RyRs in diaphragm muscles were determined. RESULTS: Sepsis weakened diaphragm contractile function. The expression levels of DHPRα1s and RyR1 were significantly lower in septic rats than in control rats. The expression levels of DHPRα1c and RyR3 were unaffected by sepsis. RyR2 was undetectable at both mRNA and protein levels in the control and sepsis groups. CONCLUSIONS: Weakened diaphragm contraction in the septic rats was associated with reduced mRNA and protein expression of DHPRα1s and RyR1, the isoforms of skeletal muscles.

Aberrant expression of circular RNA DHPR facilitates tumor growth and metastasis by regulating the RASGEF1B/RAS/MAPK axis in hepatocellular carcinoma.

BACKGROUND: Circular RNAs (circRNAs) are noncoding RNAs. Accumulating evidence suggests that circRNAs play a critical role in human biological processes, especially tumorigenesis, and development. However, the exact mechanisms of action of circRNAs in hepatocellular carcinoma (HCC) remain unclear. METHODS: Bioinformatic tools and RT-qPCR were used to identify the role of circDHPR, a circRNA derived from the dihydropteridine reductase (DHPR) locus, in HCC and para-carcinoma tissues. Kaplan-Meier analysis and the Cox proportional hazard model were used to analyze the correlation between circDHPR expression and patient prognosis. Lentiviral vectors were used to establish stable circDHPR-overexpressing cells. In vitro and in vivo studies have shown that tumor proliferation and metastasis are affected by circDHPR. Mechanistic assays, including Western blotting, immunohistochemistry, dual-luciferase reporter assays, fluorescence in situ hybridization, and RNA immunoprecipitation, have demonstrated the molecular mechanism underlying circDHPR. RESULTS: CircDHPR was downregulated in HCC, and low circDHPR expression was associated with poor overall survival and disease-free survival rates. CircDHPR overexpression inhibits tumor growth and metastasis in vitro and in vivo. Further systematic studies revealed that circDHPR binds to miR-3194-5p, an upstream regulator of RASGEF1B. This endogenous competition suppresses the silencing effect of miR-3194-5p. We confirmed that circDHPR overexpression inhibited HCC growth and metastasis by sponging miR-3194-5p to upregulate the expression of RASGEF1B, which is regarded as a suppressor of the Ras/MAPK signaling pathway. CONCLUSIONS: Aberrant circDHPR expression leads to uncontrolled cell proliferation, tumorigenesis, and metastasis. CircDHPR may serve as a biomarker and therapeutic target for HCC.

Advances in CaV1.1 gating: New insights into permeation and voltage-sensing mechanisms.

The CaV1.1 voltage-gated Ca2+ channel carries L-type Ca2+ current and is the voltage-sensor for excitation-contraction (EC) coupling in skeletal muscle. Significant breakthroughs in the EC coupling field have often been close on the heels of technological advancement. In particular, CaV1.1 was the first voltage-gated Ca2+ channel to be cloned, the first ion channel to have its gating current measured and the first ion channel to have an effectively null animal model. Though these innovations have provided invaluable information regarding how CaV1.1 detects changes in membrane potential and transmits intra- and inter-molecular signals which cause opening of the channel pore and support Ca2+ release from the sarcoplasmic reticulum remain elusive. Here, we review current perspectives on this topic including the recent application of functional site-directed fluorometry.

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation-contraction coupling.

Excitation-contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage-dependent L-type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine-rich domain-containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II-III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II-III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II-III loop. Therefore, this work demonstrates an isoform-specific interaction between STAC3 and the II-III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

Olfactory Stimulation Successfully Improves Swallowing Function of Aged Rats Through Activating Central Neuronal Networks and Downstream DHPR-RyR-mediated Neuromuscular Activities.

Presbyphagia is age-related changes in swallowing function, which imposes a high risk of aspiration in older adults. Considering olfactory stimulation (OS) can influence behavioral activities by modulating neuronal excitability, the present study aims to determine whether OS could improve the swallowing function of aged rats through activating the central neuronal networks and downstream muscular activities participated in the control of swallowing. Aged male Wistar rats received OS by inhaling a mixture of plant-based volatile molecules twice a day for 12 days were subjected to functional magnetic resonance imaging (fMRI) and c-fos, choline acetyltransferase (ChAT) immunostaining to detect the neuronal activities of the orbitofrontal cortex (OFC) and medullary nuclei engaged in swallowing control, respectively. The functional effects of OS on downstream pharyngeal muscle activity were examined by evaluating the dihydropyridine receptor-ryanodine receptor (DHPR-RyR)-mediated intramuscular Ca2+ expression, and analyzing the amplitude/frequency of muscle contraction, respectively. In untreated rats, only moderate signal of fMRI and mild c-fos/ChAT expression was detected in the OFC and medullary nuclei, respectively. However, following OS, intense signals of fMRI and immunostaining were clearly expressed in the orbitofronto-medullary networks. Functional data corresponded well with above findings in which OS significantly enhanced DHPR-RyR-mediated intramuscular Ca2+ expression, effectively facilitated a larger amplitude of pharyngeal muscle contraction, and exhibited better performance in consuming larger amounts of daily dietary. As OS successfully activates the neuromuscular activities participated in the control of swallowing, applying OS may serve as an effective, easy, and safe strategy to greatly improve the swallow function of aging populations.