Calsequestrins in skeletal and cardiac muscle from adult Danio rerio

Calsequestrin (Casq) is a high capacity, low affinity Ca2+-binding protein, critical for Ca2+-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located

L'esercizio fisico e l'elettro-stimolazione, strategie per contrastare il declino muscolare negli anziani.

Aging is a multifactorial process characterized by a constant and relentless decline of the structure, functions and consequently of muscle performance. One of the most obvious effects of aging in humans is the reduction of muscle mass, known as sarcopenia, which its development changes from individual to individual and which leads to reduced functional capacity (strength and resistance). Factors that contribute to the progress of this state are the decrease in both the number and size of myofibers and the amount of motoneurons that innervate the muscle fibers. Histological examinations carried out on the muscles of the elderly have demonstrated that denervation can contribute to atrophy and in addition the immobility and a life of sedentary accelerates the process, while the physical exercise protects in part from the effects of aging. Several studies have shown that regular exercise can prolong life expectancy and slow the effects of aging. At the molecular level calcium, and consequently the sarcoplasmic reticulum, plays a vital role in metabolic processes and structural adaptation of muscle through the activation of specific signaling pathways, in response to the type of stimulus received. This thesis focuses precisely on the benefits of physical exercise and whether these can be compared to those obtained by using a training protocol induced by electrostimulation. This could be useful in rehabilitation as exercise, especially in the elderly, it is not always applicable due to pathological and psychological problems. To determine the effects of aging and to correlate them with different lifestyles were studied the vastus lateralis muscle of three distinct groups of people (a) healthy sedentary seniors (b) healthy seniors with at least 30 years of regular exercise (c) young athletes. In these subjects muscle was characterized in relation to age and type of exercise to determine the effects of the voluntary exercise on the progress of sarcopenia during aging. From the data obtained through a series of functional tests was showed that the physically active seniors have characteristics most similar to young compared to the sedentary group of the same age. This is evidence of the fact that physically active seniors are a very powerful and performing group and it's possible to compare them to master athletes of the same age. Morphological analysis and histological studies have shown that the sedentary seniors present more denervated fibers than the other two groups, and that the physically active seniors have a significantly higher percentage of slow fibers, which, however, does not depend by the type of exercise done. Also it was detected in all three groups fibers that coexpress both myosin fast and slow and specifically the group of the sedentary seniors presents an higher percentage of coexpressing fibers than the other two groups. However, the serial sections of sedentary seniors showed the presence of coexpressing fibers of small dimensions and angled (denervated); therefore, this fact suggests that these fibers are slow fibers that coexpress fast myosin isoforms through default myogenic programs. On the contrary, the coexpressing muscle fibers in the physically active seniors are similar in size to the innervated "pure" muscle fibers, only fast or only slow. Based on the results obtained high levels of exercise appear to have beneficial effects on reinnervation of muscle fibers, resulting in conservation of muscle function, size and structure, thus delaying the functional decline and loss of independence which are common in older people. However, some pathological conditions and also some psychological situations, typical of the elderly, limit the ability to perform physical activity. It is therefore necessary to find an alternative to physical exercise that can maintain the beneficial properties, and above all that can be safe; these features can all be found in electrostimulation (ES). To determine whether this type of therapy can be effective in the elderly was also developed a study to define the effects of ES on aged human muscle, compared with a group of seniors the performed a voluntary strength training program (Leg Press, LP), for a period of nine weeks. In both groups of seniors, after the two workouts, it was noted an improvement in functional parameters, but only those who received the ES treatment presented an increase in the maximum isometric strength of the muscles vastus lateralis. Electrostimulation, unlike the LP, has kept unchanged the overall average size of the fibers, while there was an increment of the diameter and the number of fast fibers and a decrement of both parameters in slow type fibers. Of note, no sign of fibrosis and/or infiltration of inflammatory cells was detected in the treated muscles for both types of training. In addition, analysis of ultrastructure not viewed any alteration of muscle structure before and after treatment. After both treatments of ES and LP was recorded an activation of both the pathway of calcineurin/NFAT and calcium calmodulin dependent kinase (CaMKII), both key points for muscle remodeling. The quantitative evaluation of all the proteins that constitute the sarcoplasmic reticulum, shows a significant increment in protein levels of SERCA2 and Sarcalumenin and a decrement CASQ1 after ES, while after LP there were no significant changes. Analysis of quantitative PCR (qPCR) were made in order to clarify whether the level of Sarcalumenin SERCA2 and CASQ RNA are regulated by electrostimulation. None of the genes is up-regulated or down-regulated in a statistically significant way by the ES training, suggesting a regulation at the post transcriptional level. The vastus lateralis muscle is a mixture of different types of muscle fibers, consequently, the increase in the average quantity of SERCA2 and Sarcalumenin in total homogenates can be attributed to all the fibers or to a specific subpopulation (ie, slow twitch fibers or intermediate oxidative fibers IIA). To see if the increase in SERCA2 is against fast fibers in muscles treated by electrostimulation, it was developed a protocol immunofluorescence to assess the co-expression of myosin fast and SERCA2. After the ES the number of mixed fiber SERCA2/fast myosin is significantly increased, but was unchanged after LP. From the analysis of the intensity of fluorescence of fast myosin it was found that certain fast-twitch fibers exhibit a lower signal of fast myosin respectively in sections post ES and post LP and about 50% of such fibers were also positive SERCA2. From these observations we conclude that the mixed fibers for fast myosin/SERCA2 are a heterogeneous population and a part of these fibers probably expresses both isoforms of myosin. In conclusion, in this thesis were developed experimental protocols that have allowed to identify some favorable changes induced by physical exercise in the seniors. Moreover, thanks to the stabilization of the nuclear translocation of NFATc1 that remains even after several days last training session, it can be assign to the role of biomarker of inactivity (NFATc1 cytoplasmic) and activation "muscle" (myonuclear translocation), whether induced from ES or LP . Also for the first time it was shown that the ES is able to neuro-modulate with beneficial effects for the quality of the muscle, the mobility and the functional performances of the seniors. Thus the ES may be a viable approach to counter the problems and the atrophy of the muscle caused by the aging process both for healthy seniors and subjects to chronic health conditions, having as ultimate goal to improve lifestyle

Long-term high-level exercise promotes muscle reinnervation with age.

The histologic features of aging muscle suggest that denervation contributes to atrophy, that immobility accelerates the process, and that routine exercise may protect against loss of motor units and muscle tissue. Here, we compared muscle biopsies from sedentary and physically active seniors and found that seniors with a long history of high-level recreational activity up to the time of muscle biopsy had 1) lower loss of muscle strength versus young men (32% loss in physically active vs 51% loss in sedentary seniors); 2) fewer small angulated (denervated) myofibers; 3) a higher percentage of fiber-type groups (reinnervated muscle fibers) that were almost exclusive of the slow type; and 4) sparse normal-size muscle fibers coexpressing fast and slow myosin heavy chains, which is not compatible with exercise-driven muscle-type transformation. The biopsies from the old physically active seniors varied from sparse fiber-type groupings to almost fully transformed muscle, suggesting that coexpressing fibers appear to fill gaps. Altogether, the data show that long-term physical activity promotes reinnervation of muscle fibers and suggest that decades of high-level exercise allow the body to adapt to age-related denervation by saving otherwise lost muscle fibers through selective recruitment to slow motor units. These effects on size and structure of myofibers may delay functional decline in late aging

Calsequestrins new calcium store markers of adult Zebrafish cerebellum and optic tectum

Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity

Electrical stimulation counteracts muscle atrophy associated with aging in humans

Functional and structural muscle decline is a major problem during aging. Our goal was to improve in old subjects quadriceps m. force and mobility functional performances (stair test, chair rise test, timed up and go test) with neuromuscular electrical stimulation (9 weeks, 2-3times/week, 20-30 minutes per session). Furthermore we performed histological and biological molecular analyses of vastus lateralis m. biopsies. Our findings demonstrate that electrical stimulation significantly improved mobility functional performancies and muscle histological characteristics and molecular markers

Forced Oscillatory Technique R5-19 values correlate with spirometry FEV1/FVC in severe eosinophilic asthma. An observational, prospective, cohort study

Introduction: Severe asthma affects 3-10% of asthmatic patients. Biologic therapies can act as disease modifying agents in severe asthma. The prevalence of small airways dysfunction (SAD) increases with asthma severity. Forced Oscillatory Technique (FOT) is a reliable method for studying small airways. The aim of the study was to analyze FOT parameters in a cohort of eosinophilic severe asthma patients naïve of biologic therapy, and to describe the presence of correlation with spirometry data. Variations of FOT parameters after 6 and 12 months from the start of biologic therapy were also prospectively recorded and analyzed. Methods: 47 severe eosinophilic asthma patients were consecutively enrolled. FOT, spirometry data, levels of asthma biomarkers, number of exacerbations, Asthma Control Test (ACT) were determined at baseline (T0: patients naïve of biologic treatment) and after 6 and 12 months. Results: at T0, a significant linear correlation was found between R5-19 and FEV1/FVC (Forced expiratory volume in 1 second/Forced Vital Capacity) values (p=0.0008). At T0, FOT R5-19 values were more elevated in obstructed patients. The cut-off value of R5-19 that best discriminates the presence of obstruction (FEV1/FVC <0.70) was determined at 0.81 cmH2O/(L/s) (sensitivity 0.58, specificity 0.76, ROC-AUC 0.67). A significant relationship was found between FEV1/FVC and FOT R5-19 also after 6 (p=0.007) and 12 months (p=0.027) of biologic therapy. No significant correlations were found between any other FOT parameter and blood eosinophils count, FeNO, number of exacerbations or ACT. Conclusions: FOT R5-19 values correlate with FEV1/FVC and are significantly higher in obstruction. This correlation could be explained by the higher resistances of small airways in obstructed patients

miR-24-3p secreted as extracellular vesicle cargo by cardiomyocytes inhibits fibrosis in human cardiac microtissues

Background and aims: Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix-producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models. Methods and results: Gain- and loss-of-function experiments were performed using human induced pluripotent stem cell-derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia-simulating conditions. hCM-derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo-cultured human cardiac slices.hCF transfection with miR-24-3p mimic prevented TGFβ1-mediated induction of FURIN, CCND1 and SMAD4-miR-24-3p target genes participating in TGFβ1-dependent fibrinogenesis -, regulating hCF-to-myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM-derived EVs modulated hCF activation. Ischaemia-simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant-derived agent with miR-24-3p-stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues. Conclusions: These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis