RUOLO DELLE PROTEINE MINORITARIE DEL RETICOLO SARCOPLASMATICO NELL’ACCOPPIAMENTO ECCITAZIONE-CONTRAZIONE DEL MUSCOLO SCHELETRICO

Muscle strength is restored in JP45 and CASQ1 double knock-out mice. Skeletal muscle constitutes approximately 40% of human body mass, and alterations in muscle mass and in calcium release process are implicated in decrement of strength with age, in neuromuscular diseases, myopaties as well as obesity and diabetes. The action potential leads to muscle fiber contraction and generation of mechanical force in a process called Excitation-Contraction Coupling (ECC), that takes place at the triad, a structure made up of two membrane compartments: transverse tubules, invaginations of sarcolemma and the terminal cisternae of the Sarcoplasmic Reticulum (SR). The main components of ECC machinery are the 1,4 dihydropyridine receptor (DHPR) Cav 1.1 subunit on T tubules, Ryanodine receptor (RyR1) on the SR and Calsequestrin-1, which serve as channel and voltage sensor, Ca2+ release channel and main Ca storage protein of SR respectively. ECC is activated by a bidirectional signalling: depolarization of sarcolemma induces conformational changes of the DHPR that triggers the opening of RyR1 to release calcium ions via an orthograde signalling. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by RyR1. The myoplasmic calcium activates the contractile proteins and is subsequently pumped back into the SR by a Ca2+ ATPase (SERCA) pump leading to muscle relaxation. Yet to be identified minor protein components and other proteins aside the DHPR, RyR1 and calsequestrin are essential for the structure and regulation of the machinery involved in ECC. JP45 is a membrane protein interacting with Cav1.1 and the sarcoplasmic reticulum calcium storage protein calsequestrin (CASQ1). We hypothesized that JP45 and CASQ1 form a signalling pathway that modulates Cav1.1 channel activity. We tested this in flexor digitorum brevis (FDB) muscle fibres from JP45 and CASQ1 double knock-out mice (DKO). Our results show that calcium transient evoked by tetanic stimulation in DKO fibres, result from massive calcium influx due to enhanced Cav1.1 channel activity. This enhanced activity restores muscle strength both in vitro and in vivo. Downregulation of EC uncoupling in aged JP45 KO mice. The decline in muscular strength with age, termed sarcopenia, is caused largely by a loss of total muscle mass - but also a disproportionate loss of strength. The loss of muscle strength in old age is characterized in part by a deficit in Ca2+ release caused by activation of DHPR, a phenomenon known as excitation-contraction uncoupling (ECU). On the basis of our previous data showing that ablation of JP45 results in a significant loss of muscle strength in 3 months old mice, we hypothesized that ablation of JP45 expression will result in more marked muscle weakness in JP45KO than WT mice with aging. To our surprise however, we found that JP45KO exhibit sustained in vivo and in vitro skeletal muscle force with no further decline in ECC with aging. Calorie restriction induced by the absence of expression of JP45 in specific areas of the brain (nucleus of the solitary tract, area postrema and nucleus arcuate) associated with central regulation of food intake lead to a significant decrease of body weight in JP45KO compared with WT. The latter event inhibited downregulatation of the expression of DHPR which in turn prevented worsening of the EC uncoupling in aged JP45KO mice. Peroxisome proliferator-activated receptor-γ coactivator-1α (PCG-1α) affects calcium signals in skeletal muscle. Peroxisome proliferator-activated receptor-γ coactivator-1α (PCG-1α) is implicated in muscle plasticity promoting fibre switching towards oxidative activity. Since fibre type switch involves the activation of calcium dependent transcriptional factors, we analysed the effect of PGC-1α on calcium handling in muscles overexpressing the coactivator. We demonstrate that PGC-1α causes a reduction of maximal muscle force in vivo and ex vivo by diminishing the expression of calcium release molecules and altering calcium release and uptake process. Furthermore, we demonstrate that PGC-1α increases resistance to fatigue and drives fibre type switching partly through remodelling of calcium transients. Expression of retinaldehyde in skeletal muscle. We identified the primary structure and role of SRP35, a novel minor protein component of the sarcoplasmic reticulum. We show that SRP35 is a transmembrane component of the SR, is located near the SERCA Ca2+ATPase and is a short-chain dehydrogenase/reductase belonging to the DHRS7C subfamily. We demonstrated that retinol is the substrate of SRP35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. We show that transfection of C2C12 myotubes with the fusion protein encoding SRP35-EYFP, or adding retinoic acid to C2C12 cell culture, results in a decrease of calcium released by RyR1 and a significant reduction of RyR1 protein expression. The definition of the role of minor components which make up the ECC molecular machinery is important not only to understand how mutations in genes involved in calcium homeostasis cause myopathies, but also to define new therapeutic targets for innovative strategies aimed to treat neuromuscular disorders linked to defect of EC coupling

Alignment of protein structures in the presence of domain motions

Abstract Background Structural alignment is an important step in protein comparison. Well-established methods exist for solving this problem under the assumption that the structures under comparison are considered as rigid bodies. However, proteins are flexible entities often undergoing movements that alter the positions of domains or subdomains with respect to each other. Such movements can impede the identification of structural equivalences when rigid aligners are used. Results We introduce a new method called RAPIDO (Rapid Alignment of Proteins in terms of Domains) for the three-dimensional alignment of protein structures in the presence of conformational changes. The flexible aligner is coupled to a genetic algorithm for the identification of structurally conserved regions. RAPIDO is capable of aligning protein structures in the presence of large conformational changes. Structurally conserved regions are reliably detected even if they are discontinuous in sequence but continuous in space and can be used for superpositions revealing subtle differences. Conclusion RAPIDO is more sensitive than other flexible aligners when applied to cases of closely homologues proteins undergoing large conformational changes. When applied to a set of kinase structures it is able to detect similarities that are missed by other alignment algorithms. The algorithm is sufficiently fast to be applied to the comparison of large sets of protein structures.</p

Role of the JP45-Calsequestrin Complex on Calcium Entry in Slow Twitch Skeletal Muscles

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La3+- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca2+ concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca2+ concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²? released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism

Functional Characterization of Two Variants at the Intron 6-Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

Pathogenic variants in KCNQ2 encoding for Kv7.2 potassium channel subunits have been found in patients affected by widely diverging epileptic phenotypes, ranging from Self-Limiting Familial Neonatal Epilepsy (SLFNE) to severe Developmental and Epileptic Encephalopathy (DEE). Thus, understanding the pathogenic molecular mechanisms of KCNQ2 variants and their correlation with clinical phenotypes has a relevant impact on the clinical management of these patients. In the present study, the genetic, biochemical, and functional effects prompted by two variants, each found in a non-familial SLNE or a DEE patient but both affecting nucleotides at the KCNQ2 intron 6-exon 7 boundary, have been investigated to test whether and how they affected the splicing process and to clarify whether such mechanism might play a pathogenetic role in these patients. Analysis of KCNQ2 mRNA splicing in patient-derived lymphoblasts revealed that the SLNE-causing intronic variant (c.928-1G > C) impeded the use of the natural splice site, but lead to a 10-aa Kv7.2 in frame deletion (Kv7.2 p.G310Δ10); by contrast, the DEE-causing exonic variant (c.928G > A) only had subtle effects on the splicing process at this site, thus leading to the synthesis of a full-length subunit carrying the G310S missense variant (Kv7.2 p.G310S). Patch-clamp recordings in transiently-transfected CHO cells and primary neurons revealed that both variants fully impeded Kv7.2 channel function, and exerted strong dominant-negative effects when co-expressed with Kv7.2 and/or Kv7.3 subunits. Notably, Kv7.2 p.G310S, but not Kv7.2 p.G310Δ10, currents were recovered upon overexpression of the PIP2-synthesizing enzyme PIP5K, and/or CaM; moreover, currents from heteromeric Kv7.2/Kv7.3 channels incorporating either Kv7.2 mutant subunits were differentially regulated by changes in PIP2 availability, with Kv7.2/Kv7.2 G310S/Kv7.3 currents showing a greater sensitivity to PIP2 depletion when compared to those from Kv7.2/Kv7.2 G310Δ10/Kv7.3 channels. Altogether, these results suggest that the two variants investigated differentially affected the splicing process at the intron 6-exon 7 boundary, and led to the synthesis of Kv7.2 subunits showing a differential sensitivity to PIP2 and CaM regulation; more studies are needed to clarify how such different functional properties contribute to the widely-divergent clinical phenotypes

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorder

Gabapentin treatment in a patient with KCNQ2 developmental epileptic encephalopathy

De novo variants in KCNQ2 encoding for Kv7.2 voltage-dependent neuronal potassium (K+) channel subunits are associated with developmental epileptic encephalopathy (DEE). We herein describe a the clinical and electroencephalographic (EEG) features of a child with early-onset DEE caused by the novel KCNQ2 p.G310S variant. In vitro experiments demonstrated that the mutation induces loss-of-function effects on the currents produced by channels incorporating mutant subunits; these effects were counteracted by the selective Kv7 opener retigabine and by gabapentin, a recently described Kv7 activator. Given these data, the patient started treatment with gabapentin, showing a rapid and sustained clinical and EEG improvement over the following months. Overall, these results suggest that gabapentin can be regarded as a precision therapy for DEEs due to KCNQ2 loss-of-function mutations

The Geriatric G8 Score Is Associated with Survival Outcomes in Older Patients with Advanced Prostate Cancer in the ADHERE Prospective Study of the Meet-URO Network

Introduction: Androgen receptor pathway inhibitors (ARPIs) have been increasingly offered to older patients with prostate cancer (PC). However, prognostic factors relevant to their outcome with ARPIs are still little investigated. Methods and Materials: The Meet-URO network ADHERE was a prospective multicentre observational cohort study evaluating and monitoring adherence to ARPIs metastatic castrate-resistant PC (mCRPC) patients aged ≥70. Cox regression univariable and multivariable analyses for radiographic progression-free (rPFS) and overall survival (OS) were performed. Unsupervised median values and literature-based thresholds where available were used as cut-offs for quantitative variables. Results: Overall, 234 patients were enrolled with a median age of 78 years (73–82); 86 were treated with abiraterone (ABI) and 148 with enzalutamide (ENZ). With a median follow-up of 15.4 months (mo.), the median rPFS was 26.0 mo. (95% CI, 22.8–29.3) and OS 48.8 mo. (95% CI, 36.8–60.8). At the MVA, independent prognostic factors for both worse rPFS and OS were Geriatric G8 assessment ≤ 14 (p &lt; 0.001 and p = 0.004) and PSA decline ≥50% (p &lt; 0.001 for both); time to castration resistance ≥ 31 mo. and setting of treatment (i.e., post-ABI/ENZ) for rPFS only (p &lt; 0.001 and p = 0.01, respectively); age ≥78 years for OS only (p = 0.008). Conclusions: Baseline G8 screening is recommended for mCRPC patients aged ≥70 to optimise ARPIs in vulnerable individuals, including early introduction of palliative care