UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

We report the case of a 22-year-old man with a diagnosis of dihydropteridine reductase (DHPR) deficiency who progressively developed movement disorders and epilepsy. Despite L-Dopa supplementation the patient continued to show high prolactin levels, with a discrepancy between the neurological clinical picture and the hormonal biochemical levels. For this reason, other potential causes were ruled out by performing a cerebral magnetic resonance imaging, which demonstrated a solid lesion in the pituitary gland strongly suggestive of a prolactinoma. As the association between metabolic disorders affecting biogenic amine synthesis and prolactinoma has not been previously reported in humans, this report suggests that a critical evaluation of the use of prolactin as a guide for therapy dosage should be made in patients with DHPR deficiency disorders.

BACKGROUND: Pulmonary hypertension occurs in approximately 1% of the global population, and the prognosis for such patients may be poor. However, the mechanisms underlying the development of this disease remain unclear. Thus, understanding the development of pulmonary hypertension and finding new therapeutic targets and approaches are important for improved clinical outcomes.
METHODS: Lung tissue specimens were collected from six patients with atrial septal defect and pulmonary hypertension (all women, with a mean age of 46.5 ± 4.7 years, and their condition could not be corrected with an internal medical occlusion device) and from nine control patients with lung cancer who underwent lobectomy (six men and three women, with a mean age of 56.7 ± 1.7 years). Isobaric tags for relative and absolute quantitation and liquid chromatography tandem mass spectrometry analyses were used to detect protein expression levels.
RESULTS: We found 74 significantly upregulated and 88 significantly downregulated differentially expressed proteins between control and pulmonary hypertensive lung tissue specimens. Gene ontology analyses identified the top 20 terms in all three categories, that is, biological process, cellular component, and molecular function. Kyoto Encyclopedia of Genes and Genomes and protein-protein interaction analyses determined the top 10 signaling pathways and found that the six hub proteins associated with the differentially expressed upregulated proteins (PRKAA1, DHPR, ACTB, desmin, ACTG1, and ITGA1) were all involved in hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and dilated cardiomyopathy.
CONCLUSIONS: Our results identified protein expression profile changes in lung tissue derived from patients with pulmonary hypertension, providing potential new biomarkers for clinical diagnosis and prognosis for patients with pulmonary hypertension and offering candidate protein targets for future therapeutic drug development.

Excitation-contraction coupling (ECC) is a physiological process that links excitation of muscles by the nervous system to their mechanical contraction. In skeletal muscle, ECC is initiated with an action potential, generated by the somatic nervous system, which causes a depolarisation of the muscle fibre membrane (sarcolemma). This leads to a rapid change in the transmembrane potential, which is detected by the voltage-gated Ca2+ channel dihydropyridine receptor (DHPR) embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca2+ channel, ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amount of Ca2+ ions from the SR that initiate muscle contraction. Despite the fundamental role of ECC in skeletal muscle function of all vertebrate species, the molecular mechanism underpinning the communication between the two key proteins involved in the process (DHPR and RyR1) is still largely unknown. The goal of this work is to review the recent progress in our understanding of ECC in skeletal muscle from the point of view of the structure and interactions of proteins involved in the process, and to highlight the unanswered questions in the field.

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.

Skeletal muscle is a highly organized tissue that has to be optimized for fast signalling events conveying electrical excitation to contractile response. The site of electro-chemico-mechanical coupling is the skeletal muscle triad where two membrane systems, the extracellular t-tubules and the intracellular sarcoplasmic reticulum, come into very close contact. Structure fits function here and the signalling proteins DHPR and RyR1 were the first to be discovered to bridge this gap in a conformational coupling arrangement. Since then, however, new proteins and more signalling cascades have been identified just in the last decade, adding more diversity and fine tuning to the regulation of excitation-contraction coupling (ECC) and control over Ca2+ store content. The concept of Ca2+ entry into working skeletal muscle has become attractive again with the experimental evidence summarized in this review. Store-operated Ca2+ entry (SOCE), excitation-coupled Ca2+ entry (ECCE), action-potential-activated Ca2+ current (APACC), and retrograde EC-coupling (ECC) are new concepts additional to the conventional orthograde ECC; they have provided fascinating new insights into muscle physiology. In this review, we discuss the discovery of these pathways, their potential roles, and the signalling proteins involved that show that the triad may become a crowded place in time.

During the complex series of events leading to muscle contraction, the initial electric signal coming from motor neurons is transformed into an increase in calcium concentration that triggers sliding of myofibrils. This process, referred to as excitation-contraction coupling, is reliant upon the calcium-release complex, which is restricted spatially to a sub-compartment of muscle cells (\"the triad\") and regulated precisely. Any dysfunction in the calcium-release complex leads to muscle impairment and myopathy. Various causes can lead to alterations in excitation-contraction coupling and to muscle diseases. The latter are reviewed and classified into four categories: (i) mutation in a protein of the calcium-release complex; (ii) alteration in triad structure; (iii) modification of regulation of channels; (iv) modification in calcium stores within the muscle. Current knowledge of the pathophysiologic mechanisms in each category is described and discussed.

BACKGROUND: Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low affinity calcium dye and high intracellular ethylene glycol-bis(2-aminoethylether)-n,n,n\',n\'-tetraacetic acid (EGTA) to measure Ca(2+) fluxes. Muscles were subjected to both current and voltage clamp conditions.
METHODS: Standard and confocal fluorescence microscopy was used to study Ca(2+) release in single fibers enzymatically isolated from hind limb muscles of wild-type and C3KO mice. Two microelectrode amplifier and experiments were performed under current or voltage clamp conditions. Calcium concentration changes were detected with an impermeant low affinity dye in the presence of high EGTA intracellular concentrations, and fluxes were calculated with a single compartment model. Standard Western blotting analysis was used to measure the concentration of RyR1 and the α subunit of the dihydropyridine (αDHPR) receptors. Data are presented as mean ± SEM and compared with the Student\'s test with significance set at p < 0.05.
RESULTS: We found that the peak value of Ca(2+) fluxes elicited by single action potentials was significantly reduced by 15-20 % in C3KO fibers, but the kinetics was unaltered. Ca(2+) release elicited by tetanic stimulation was also impaired in C3KO fibers. Confocal studies confirmed that Ca(2+) release was similarly reduced in all triads of C3KO mice. Voltage clamp experiments revealed a normal voltage dependence of Ca(2+) release in C3KO mice but reduced peak Ca(2+) fluxes as with action potential stimulation. These findings concur with biochemical observations of reduced RyR1 and αDHPR levels in C3KO muscles and reduced mechanical output. Confocal studies revealed a similar decrease in Ca(2+) release at all triads consistent with a homogenous reduction of functional voltage activated Ca(2+) release sites.
CONCLUSIONS: Overall, these results suggest that decreased Ca(2+) release is an early defect in calpainopathy and may contribute to the observed reduction of CaMKII activation in C3KO mice.

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.

In skeletal muscle, Ca(2+) release from sarcoplasmic reticulum (SR) following the action potential relies on the delicate architecture of the T-Tubule/SR junction. The T-Tubule/SR junction comprises two membrane systems: the integral proteins DHPR and RyR1 and the Ca(2+)-buffering apparatus within the SR lumen. The arrangement and interactions of the components have remained elusive due to technological limitations. Here, we determined whether electron tomography is effective fort the in situ determination of the relationships between RyR1 and DHPR, the SR membrane and other involved structures. First, we visually confirmed that RyR1 and DHPR are close neighbors that are mutually staggered with each other and directly interact with one of RyR1 subunits. Second, the Ca(2+) storage network within the SR lumen is directly correlated with RyR1. These results suggest that the excitation of the T-Tubule may induce Ca(2+) release through a direct interaction among DHPR, RyR1 and the Ca(2+) buffering apparatus. These results indicate that electron tomography has potential as an efficient method for the in situ characterization of the complex architecture and arrangement of two integral-integral-membrane proteins in the context of the surrounding phospholipid-bilayer and proteins.