Characterization of fast-twitch and slow-twitch skeletal muscles of calsequestrin 2 (CASQ2)-knock out mice: unexpected adaptive changes of fast-twitch muscles only

This study investigates the functional role of calsequestrin 2 (CASQ2) in both fast-twitch and slow-twitch skeletal muscles by using CASQ2 12/ 12 mice; CASQ2 is expressed throughout life in slow-twitch muscles, but only in the developmental and neonatal stages in fast-twitch muscles. CASQ2 12/ 12 causes increase in calsequestrin 1 (CASQ1) expression, but without functional changes in both muscle types. CASQ2 12/ 12 mice have ultrastructural changes in fast-twitch muscles only, i.e., formation of pentads and stacks in the sarcoplasmic reticulum

Differential Analysis of Gly211Val and Gly286Val Mutations Affecting Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) in Congenital Pseudomyotonia Romagnola Cattle.

Congenital pseudomyotonia in cattle (PMT) is a rare skeletal muscle disorder, clinically characterized by stiffness and by delayed muscle relaxation after exercise. Muscle relaxation impairment is due to defective content of the Sarco(endo)plasmic Reticulum Ca2+ ATPase isoform 1 (SERCA1) protein, caused by missense mutations in the ATP2A1 gene. PMT represents the only mammalian model of human Brody myopathy. In the Romagnola breed, two missense variants occurring in the same allele were described, leading to Gly211Val and Gly286Val (G211V/G286V) substitutions. In this study, we analyzed the consequences of G211V and G286V mutations. Results support that the reduced amount of SERCA1 is a consequence of the G211V mutation, the G286V mutation almost being benign and the ubiquitin-proteasome system (UPS) being involved. After blocking the proteasome using a proteasome inhibitor, we found that the G211V mutant accumulates in cells at levels comparable to those of WT SERCA1. Our conclusion is that G211/286V mutations presumably originate in a folding-defective SERCA1 protein, recognized and diverted to degradation by UPS, although still catalytically functional, and that the main role is played by G211V mutation. Rescue of mutated SERCA1 to the sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca2+ concentration and prevent the appearance of pathological signs, paving the way for a possible therapeutic approach against Brody disease

Repairing folding-defective \u3b1-sarcoglycan mutants by CFTR correctors, a potential therapy for Limb Girdle Muscular Dystrophy 2D

Limb Girdle Muscular Dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for \u3b1-sarcoglycan. Nowadays more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of \u3b1-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of \u3b1-sarcoglycan partners, \u3b2-, \u3b3- and \u3b4-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy.Here, we show that the application of small molecules developed to rescue \u394F508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several \u3b1-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective \u3b1-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes

SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 \ub5M, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency

ITALIAN CANCER FIGURES - REPORT 2015: The burden of rare cancers in Italy = I TUMORI IN ITALIA - RAPPORTO 2015: I tumori rari in Italia

OBJECTIVES: This collaborative study, based on data collected by the network of Italian Cancer Registries (AIRTUM), describes the burden of rare cancers in Italy. Estimated number of new rare cancer cases yearly diagnosed (incidence), proportion of patients alive after diagnosis (survival), and estimated number of people still alive after a new cancer diagnosis (prevalence) are provided for about 200 different cancer entities. MATERIALS AND METHODS: Data herein presented were provided by AIRTUM population- based cancer registries (CRs), covering nowadays 52% of the Italian population. This monograph uses the AIRTUM database (January 2015), which includes all malignant cancer cases diagnosed between 1976 and 2010. All cases are coded according to the International Classification of Diseases for Oncology (ICD-O-3). Data underwent standard quality checks (described in the AIRTUM data management protocol) and were checked against rare-cancer specific quality indicators proposed and published by RARECARE and HAEMACARE (www.rarecarenet.eu; www.haemacare.eu). The definition and list of rare cancers proposed by the RARECAREnet "Information Network on Rare Cancers" project were adopted: rare cancers are entities (defined as a combination of topographical and morphological codes of the ICD-O-3) having an incidence rate of less than 6 per 100,000 per year in the European population. This monograph presents 198 rare cancers grouped in 14 major groups. Crude incidence rates were estimated as the number of all new cancers occurring in 2000-2010 divided by the overall population at risk, for males and females (also for gender-specific tumours).The proportion of rare cancers out of the total cancers (rare and common) by site was also calculated. Incidence rates by sex and age are reported. The expected number of new cases in 2015 in Italy was estimated assuming the incidence in Italy to be the same as in the AIRTUM area. One- and 5-year relative survival estimates of cases aged 0-99 years diagnosed between 2000 and 2008 in the AIRTUM database, and followed up to 31 December 2009, were calculated using complete cohort survival analysis. To estimate the observed prevalence in Italy, incidence and follow-up data from 11 CRs for the period 1992-2006 were used, with a prevalence index date of 1 January 2007. Observed prevalence in the general population was disentangled by time prior to the reference date (≤2 years, 2-5 years, ≤15 years). To calculate the complete prevalence proportion at 1 January 2007 in Italy, the 15-year observed prevalence was corrected by the completeness index, in order to account for those cancer survivors diagnosed before the cancer registry activity started. The completeness index by cancer and age was obtained by means of statistical regression models, using incidence and survival data available in the European RARECAREnet data. RESULTS: In total, 339,403 tumours were included in the incidence analysis. The annual incidence rate (IR) of all 198 rare cancers in the period 2000-2010 was 147 per 100,000 per year, corresponding to about 89,000 new diagnoses in Italy each year, accounting for 25% of all cancer. Five cancers, rare at European level, were not rare in Italy because their IR was higher than 6 per 100,000; these tumours were: diffuse large B-cell lymphoma and squamous cell carcinoma of larynx (whose IRs in Italy were 7 per 100,000), multiple myeloma (IR: 8 per 100,000), hepatocellular carcinoma (IR: 9 per 100,000) and carcinoma of thyroid gland (IR: 14 per 100,000). Among the remaining 193 rare cancers, more than two thirds (No. 139) had an annual IR <0.5 per 100,000, accounting for about 7,100 new cancers cases; for 25 cancer types, the IR ranged between 0.5 and 1 per 100,000, accounting for about 10,000 new diagnoses; while for 29 cancer types the IR was between 1 and 6 per 100,000, accounting for about 41,000 new cancer cases. Among all rare cancers diagnosed in Italy, 7% were rare haematological diseases (IR: 41 per 100,000), 18% were solid rare cancers. Among the latter, the rare epithelial tumours of the digestive system were the most common (23%, IR: 26 per 100,000), followed by epithelial tumours of head and neck (17%, IR: 19) and rare cancers of the female genital system (17%, IR: 17), endocrine tumours (13% including thyroid carcinomas and less than 1% with an IR of 0.4 excluding thyroid carcinomas), sarcomas (8%, IR: 9 per 100,000), central nervous system tumours and rare epithelial tumours of the thoracic cavity (5%with an IR equal to 6 and 5 per 100,000, respectively). The remaining (rare male genital tumours, IR: 4 per 100,000; tumours of eye, IR: 0.7 per 100,000; neuroendocrine tumours, IR: 4 per 100,000; embryonal tumours, IR: 0.4 per 100,000; rare skin tumours and malignant melanoma of mucosae, IR: 0.8 per 100,000) each constituted <4% of all solid rare cancers. Patients with rare cancers were on average younger than those with common cancers. Essentially, all childhood cancers were rare, while after age 40 years, the common cancers (breast, prostate, colon, rectum, and lung) became increasingly more frequent. For 254,821 rare cancers diagnosed in 2000-2008, 5-year RS was on average 55%, lower than the corresponding figures for patients with common cancers (68%). RS was lower for rare cancers than for common cancers at 1 year and continued to diverge up to 3 years, while the gap remained constant from 3 to 5 years after diagnosis. For rare and common cancers, survival decreased with increasing age. Five-year RS was similar and high for both rare and common cancers up to 54 years; it decreased with age, especially after 54 years, with the elderly (75+ years) having a 37% and 20% lower survival than those aged 55-64 years for rare and common cancers, respectively. We estimated that about 900,000 people were alive in Italy with a previous diagnosis of a rare cancer in 2010 (prevalence). The highest prevalence was observed for rare haematological diseases (278 per 100,000) and rare tumours of the female genital system (265 per 100,000). Very low prevalence (<10 prt 100,000) was observed for rare epithelial skin cancers, for rare epithelial tumours of the digestive system and rare epithelial tumours of the thoracic cavity. COMMENTS: One in four cancers cases diagnosed in Italy is a rare cancer, in agreement with estimates of 24% calculated in Europe overall. In Italy, the group of all rare cancers combined, include 5 cancer types with an IR>6 per 100,000 in Italy, in particular thyroid cancer (IR: 14 per 100,000).The exclusion of thyroid carcinoma from rare cancers reduces the proportion of them in Italy in 2010 to 22%. Differences in incidence across population can be due to the different distribution of risk factors (whether environmental, lifestyle, occupational, or genetic), heterogeneous diagnostic intensity activity, as well as different diagnostic capacity; moreover heterogeneity in accuracy of registration may determine some minor differences in the account of rare cancers. Rare cancers had worse prognosis than common cancers at 1, 3, and 5 years from diagnosis. Differences between rare and common cancers were small 1 year after diagnosis, but survival for rare cancers declined more markedly thereafter, consistent with the idea that treatments for rare cancers are less effective than those for common cancers. However, differences in stage at diagnosis could not be excluded, as 1- and 3-year RS for rare cancers was lower than the corresponding figures for common cancers. Moreover, rare cancers include many cancer entities with a bad prognosis (5-year RS <50%): cancer of head and neck, oesophagus, small intestine, ovary, brain, biliary tract, liver, pleura, multiple myeloma, acute myeloid and lymphatic leukaemia; in contrast, most common cancer cases are breast, prostate, and colorectal cancers, which have a good prognosis. The high prevalence observed for rare haematological diseases and rare tumours of the female genital system is due to their high incidence (the majority of haematological diseases are rare and gynaecological cancers added up to fairly high incidence rates) and relatively good prognosis. The low prevalence of rare epithelial tumours of the digestive system was due to the low survival rates of the majority of tumours included in this group (oesophagus, stomach, small intestine, pancreas, and liver), regardless of the high incidence rate of rare epithelial cancers of these sites. This AIRTUM study confirms that rare cancers are a major public health problem in Italy and provides quantitative estimations, for the first time in Italy, to a problem long known to exist. This monograph provides detailed epidemiologic indicators for almost 200 rare cancers, the majority of which (72%) are very rare (IR<0.5 per 100,000). These data are of major interest for different stakeholders. Health care planners can find useful information herein to properly plan and think of how to reorganise health care services. Researchers now have numbers to design clinical trials considering alternative study designs and statistical approaches. Population-based cancer registries with good quality data are the best source of information to describe the rare cancer burden in a population

Omeostasi del calcio nelle fibre muscolari scheletriche e nei neuroni del Purkinje

Dottorato di ricerca in patologia sperimentale. 7. ciclo. A.a. 1994-95. Coordinatore L. Montanaro. Tutore A. MargrethConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Glycogen- and PP1c-targeting subunit GM is phosphorylated at Ser48 by sarcoplasmic reticulum-bound CaMKII in rabbit fast-twitch skeletal muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i)

Structural study of skeletal muscle fibres in healthy and pseudomyotonia affected cattle

Cattle congenital pseudomyotonia (PMT), recognized as naturally occurring animal model of human Brody disease, is an inherited recessive autosomal muscular disorder due to missense mutations in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca2(+)-ATPase protein, isoform 1 (SERCA1). PMT has been described in the Chianina and Romagnola italian cattle breeds and as a single case in Dutch improved Red and White cross-breed. The genetic defect turned out to be heterogeneous in different cattle breeds, even though clinical symptoms were homogeneous. Skeletal muscles of affected animals are characterized by a selective deficiency of SERCA1 in sarcoplasmic reticulum (SR) membranes. Recently, we provided evidence that in Chianina breed, the ubiquitin proteasome system is responsible for SERCA1 mutant premature disposal, even when the mutation does not affect the catalytic properties of the pump. Results presented here show that all SERCA1 mutants described until now, although expressed at low level, are correctly targeted to SR membranes. Ultrastructural studies confirm that in pathological muscle fibres, structure, as well as triads, is well preserved. All together these results suggest that a possible therapeutical approach based on the rescue of the defective protein at SR membranes could be hypothesized. Only fully functionally active missense mutants, whem located at the SR membrane could restore the efficient control of Ca2+ homeostasis and prevent the appearance of the pathological signs. Moreover, these data demonstrate the increasing importance of domestic animals as genetic models of human pathologies

Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle

Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR