iMS-Bmal1-/- mice show evident signs of sarcopenia that are counteracted by exercise and melatonin therapies.

Sarcopenia is an age-related disease characterized by a reduction in muscle mass, strength, and function and, therefore, a deterioration in skeletal muscle health and frailty. Although the cause of sarcopenia is still unknown and, thus, there is no treatment, increasing evidence suggests that chronodisruption, particularly alterations in Bmal1 clock gene, can lead to those deficits culminating in sarcopenia. To gain insight into the cause and mechanism of sarcopenia and the protective effect of a therapeutic intervention with exercise and/or melatonin, the gastrocnemius muscles of male and female skeletal muscle-specific and inducible Bmal1 knockout mice (iMS-Bmal1-/- ) were examined by phenotypic tests and light and electron microscopy. Our results revealed a disruption of the normal activity/rest rhythm, a drop in skeletal muscle function and mass, and increased frailty in male and female iMS-Bmal1-/- animals compared to controls. A reduction in muscle fiber size and increased collagenous tissue were also detected, accompanied by reduced mitochondrial oxidative capacity and a compensatory shift towards a more oxidative fiber type. Electron microscopy further supports mitochondrial impairment in mutant mice. Melatonin and exercise ameliorated the damage caused by loss of Bmal1 in mutant mice, except for mitochondrial damage, which was worsened by the latter. Thus, iMS-Bmal1-/- mice let us to identify Bmal1 deficiency as the responsible for the appearance of sarcopenia in the gastrocnemius muscle. Moreover, the results support the exercise and melatonin as therapeutic tools to counteract sarcopenia, by a mechanism that does not require the presence of Bmal1.

Zebrafish as a Human Muscle Model for Studying Age-Dependent Sarcopenia and Frailty.

Currently, there is an increase in the aging of the population, which represents a risk factor for many diseases, including sarcopenia. Sarcopenia involves progressive loss of mass, strength, and function of the skeletal muscle. Some mechanisms include alterations in muscle structure, reduced regenerative capacity, oxidative stress, mitochondrial dysfunction, and inflammation. The zebrafish has emerged as a new model for studying skeletal muscle aging because of its numerous advantages, including histological and molecular similarity to human skeletal muscle. In this study, we used fish of 2, 10, 30, and 60 months of age. The older fish showed a higher frailty index with a value of 0.250 ± 0.000 because of reduced locomotor activity and alterations in biometric measurements. We observed changes in muscle structure with a decreased number of myocytes (0.031 myocytes/µm2 ± 0.004 at 60 months) and an increase in collagen with aging up to 15% ± 1.639 in the 60-month group, corresponding to alterations in the synthesis, degradation, and differentiation pathways. These changes were accompanied by mitochondrial alterations, such as a nearly 50% reduction in the number of intermyofibrillar mitochondria, 100% mitochondrial damage, and reduced mitochondrial dynamics. Overall, we demonstrated a similarity in the aging processes of muscle aging between zebrafish and mammals.

Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport.

The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed to elucidate the two seemingly opposing actions of melatonin regarding its relationship with free radicals. We analyzed the effects of melatonin on head and neck squamous cell carcinoma cell lines (Cal-27 and SCC-9), which were treated with 0.5 or 1 mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal-27 xenograft mice. Here we report that melatonin mediates apoptosis in head and neck cancer by driving mitochondrial reverse electron transport (RET) to induce ROS production. Melatonin-induced changes in tumoral metabolism led to increased mitochondrial activity, which, in turn, induced ROS-dependent mitochondrial uncoupling. Interestingly, mitochondrial complex inhibitors, including rotenone, abolished the ROS elevation indicating that melatonin increased ROS generation via RET. Melatonin also increased membrane potential and CoQ10 H2 /CoQ10 ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of cancer cells with alternative oxidase, which transfers electrons from QH2 to oxygen, inhibited melatonin-induced ROS generation, and apoptosis. RET restored the melatonin-induced oncostatic effect, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors in melatonin's effects in cancer cells and establish the dual effect of melatonin in protecting normal cells and inducing apoptosis in cancer cells.

The Zebrafish, an Outstanding Model for Biomedical Research in the Field of Melatonin and Human Diseases.

The zebrafish has become an excellent model for the study of human diseases because it offers many advantages over other vertebrate animal models. The pineal gland, as well as the biological clock and circadian rhythms, are highly conserved in zebrafish, and melatonin is produced in the pineal gland and in most organs and tissues of the body. Zebrafish have several copies of the clock genes and of aanat and asmt genes, the latter involved in melatonin synthesis. As in mammals, melatonin can act through its membrane receptors, as with zebrafish, and through mechanisms that are independent of receptors. Pineal melatonin regulates peripheral clocks and the circadian rhythms of the body, such as the sleep/wake rhythm, among others. Extrapineal melatonin functions include antioxidant activity, inducing the endogenous antioxidants enzymes, scavenging activity, removing free radicals, anti-inflammatory activity through the regulation of the NF-κB/NLRP3 inflammasome pathway, and a homeostatic role in mitochondria. In this review, we introduce the utility of zebrafish to analyze the mechanisms of action of melatonin. The data here presented showed that the zebrafish is a useful model to study human diseases and that melatonin exerts beneficial effects on many pathophysiological processes involved in these diseases.

Chronodisruption and Loss of Melatonin Rhythm, Associated with Alterations in Daily Motor Activity and Mitochondrial Dynamics in Parkinsonian Zebrafish, Are Corrected by Melatonin Treatment.

Beyond sleep/wake, clock genes regulate the daily rhythms of melatonin production, motor activity, innate immunity, and mitochondrial dynamics, among others. All these rhythms are affected in Parkinson's disease (PD), suggesting that chronodisruption may be an early stage of the disease. The aim of this study was to evaluate the connection between clock genes and these rhythms in PD, and whether melatonin administration reestablished the normal clock function. Parkinsonism was induced with 600 µM MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in 24-120 h post fertilization (hpf) zebrafish embryos and melatonin was administered at a dose of 1 µM. Day-night melatonin rhythm disappeared in MPTP-treated embryos, which showed an advance in the activity phase in parallel with changes in the rhythm of clock genes. An alteration in the fission-to-fusion mitochondrial dynamics was also detected in parkinsonian embryos, increasing the former and leading to apoptosis. Melatonin administration to MPTP-treated embryos fully restored the circadian system, including the rhythms of clock genes, motor activity, melatonin rhythm, and mitochondrial dynamics, and decreasing apoptosis. Because clock-controlled rhythms such as sleep/wake alterations are early events in PD, the data here reported may point to chronodisruption as one initial pathophysiological event of the disease.

From Chronodisruption to Sarcopenia: The Therapeutic Potential of Melatonin.

Sarcopenia is an age-related condition that involves a progressive decline in muscle mass and function, leading to increased risk of falls, frailty, and mortality. Although the exact mechanisms are not fully understood, aging-related processes like inflammation, oxidative stress, reduced mitochondrial capacity, and cell apoptosis contribute to this decline. Disruption of the circadian system with age may initiate these pathways in skeletal muscle, preceding the onset of sarcopenia. At present, there is no pharmacological treatment for sarcopenia, only resistance exercise and proper nutrition may delay its onset. Melatonin, derived from tryptophan, emerges as an exceptional candidate for treating sarcopenia due to its chronobiotic, antioxidant, and anti-inflammatory properties. Its impact on mitochondria and organelle, where it is synthesized and crucial in aging skeletal muscle, further highlights its potential. In this review, we discuss the influence of clock genes in muscular aging, with special reference to peripheral clock genes in the skeletal muscle, as well as their relationship with melatonin, which is proposed as a potential therapy against sarcopenia.

The Impact of Melatonin Supplementation and NLRP3 Inflammasome Deletion on Age-Accompanied Cardiac Damage.

To investigate the role of NLRP3 inflammasome in cardiac aging, we evaluate here morphological and ultrastructural age-related changes of cardiac muscles fibers in wild-type and NLRP3-knockout mice, as well as studying the beneficial effect of melatonin therapy. The results clarified the beginning of the cardiac sarcopenia at the age of 12 months, with hypertrophy of cardiac myocytes, increased expression of ß-MHC, appearance of small necrotic fibers, decline of cadiomyocyte number, destruction of mitochondrial cristae, appearance of small-sized residual bodies, and increased apoptotic nuclei ratio. These changes were progressed in the cardiac myocytes of 24 old mice, accompanied by excessive collagen deposition, higher expressions of IL-1α, IL-6, and TNFα, complete mitochondrial vacuolation and damage, myofibrils disorganization, multivesicular bodies formation, and nuclear fragmentation. Interestingly, cardiac myocytes of NLRP3-/- mice showed less detectable age-related changes compared with WT mice. Oral melatonin therapy preserved the normal cardiomyocytes structure, restored cardiomyocytes number, and reduced ß-MHC expression of cardiac hypertrophy. In addition, melatonin recovered mitochondrial architecture, reduced apoptosis and multivesicular bodies' formation, and decreased expressions of ß-MHC, IL-1α, and IL-6. Fewer cardiac sarcopenic changes and highly remarkable protective effects of melatonin treatment detected in aged cardiomyocytes of NLRP3-/- mice compared with aged WT animals, confirming implication of the NLRP3 inflammasome in cardiac aging. Thus, NLRP3 suppression and melatonin therapy may be therapeutic approaches for age-related cardiac sarcopenia.

Melatonin alleviates sepsis-induced heart injury through activating the Nrf2 pathway and inhibiting the NLRP3 inflammasome.

Melatonin improved the outcome of septic cardiomyopathy by inhibiting NLRP3 priming induced by reactive oxygen species. To get insights into these events, we studied the melatonin/Nrf2 antioxidant pathways during sepsis in the heart of NLRP3-deficient mice. Sepsis was induced by cecal ligation and puncture and melatonin was given at a dose of 30 mg/kg. Nuclear turnover of Nrf2 and p-Ser40 Nrf2 and expression of ho-1 were enhanced in nlrp3+/+ and nlrp3-/- mice during sepsis. Sepsis caused higher mitochondria impairment, apoptotic and autophagic events in nlrp3+/+ mice than in nlrp3-/- animals. These findings were accompanied by greater levels of Parkin and PINK-1, and lower Mfn2/Drp-1 ratio in nlrp3+/+ than in nlrp3-/- mice during sepsis, supporting less mitophagy in the latter. Ultrastructural analysis of myocardial tissue further confirmed these observations. The activation of NLRP3 inflammasome accounted for most of the deleterious effects of sepsis, whereas the Nrf2-dependent antioxidative response activation in response to sepsis was unable to neutralize these events. In turn, melatonin further enhanced the Nrf2 response in both mice strains and reduced the NLRP3 inflammasome activation in nlrp3+/+ mice, restoring myocardial homeostasis. The data support that the anti-inflammatory efficacy of melatonin against sepsis depends, at least in part, on Nrf2 activation.

The Impact of Melatonin and NLRP3 Inflammasome on the Expression of microRNAs in Aged Muscle.

Muscular aging is a complex process and underlying physiological mechanisms are not fully clear. In recent years, the participation of the NF-kB pathway and the NLRP3 inflammasome in the chronic inflammation process that accompanies the skeletal muscle's aging has been confirmed. microRNAs (miRs) form part of a gene regulatory machinery, and they control numerous biological processes including inflammatory pathways. In this work, we studied the expression of four miRs; three of them are considered as inflammatory-related miRs (miR-21, miR-146a, and miR-223), and miR-483, which is related to the regulation of melatonin synthesis, among other targets. To investigate the changes of miRs expression in muscle along aging, the impact of inflammation, and the role of melatonin in aged skeletal muscle, we used the gastrocnemius muscle of wild type (WT) and NLRP3-knockout (NLRP3-) mice of 3, 12, and 24 months-old, with and without melatonin supplementation. The expression of miRs and pro-caspase-1, caspase-3, pro-IL-1ß, bax, bcl-2, and p53, was investigated by qRT-PCR analysis. Histological examination of the gastrocnemius muscle was also done. The results showed that age increased the expression of miR-21 (p < 0.01), miR-146a, and miR-223 (p < 0.05, for both miRs) in WT mice, whereas the 24-months-old mutant mice revealed decline of miR-21 and miR-223 (p < 0.05), compared to WT age. The lack of NLRP3 inflammasome also improved the skeletal muscle fibers arrangement and reduced the collagen deposits compared with WT muscle during aging. For the first time, we showed that melatonin significantly reduced the expression of miR-21, miR-146a, and miR-223 (p < 0.05 for all ones, and p < 0.01 for miR-21 at 24 months old) in aged WT mice, increased miR-223 in NLRP3- mice (p < 0.05), and induced miR-483 expression in both mice strains, this increase being significant at 24 months of age.

Lack of retinoid acid receptor-related orphan receptor alpha accelerates and melatonin supplementation prevents testicular aging.

The role of retinoid acid receptor-related orphan receptor alpha (RORα) on male reproductive functions during aging is unclear. Here, we analyze the morphological changes in the testis of both young and aged RORα-deficient mice, with and without melatonin supplementation. Young mutants showed vacuolation, degeneration and pyknosis of spermatogenic epithelium and Sertoli cells. Aged mutants showed atrophy of the seminiferous tubules and absence of mitotic spermatogenic cells. Absence of sperms in many tubules, loss of acrosomal cap, vacuolation and hypertrophy of Sertoli cells were detected in aged mice, with a significant reduction in the number of seminiferous tubules and a significant increase in the number of Leydig cells and telocytes. Repair in seminiferous tubules and interstitial tissues with enhancement of spermatogenesis was observed in melatonin-treated aged mice. Young mutants overexpressed VEGF that was weaker in aged animals and observed only in the spermatocytes, while melatonin increased VEGF expression in spermatocytes and spermatids. Caspase 3 increased in both young and aged mutant mice in all seminiferous tubules and interstitium; caspase 3 immunostaining in seminiferous tubules, however, showed a normal pattern of apoptosis with melatonin supplementation. The present study reports that age-dependent testicular changes in RORα mutant mice were recovered by melatonin treatment.

Melatonin/Nrf2/NLRP3 Connection in Mouse Heart Mitochondria during Aging.

Aging is a major risk for cardiovascular diseases (CVD). Age-related disorders include oxidative stress, mitochondria dysfunction, and exacerbation of the NF-κB/NLRP3 innate immune response pathways. Some of the molecular mechanisms underlying these processes, however, remain unclear. This study tested the hypothesis that NLRP3 inflammasome plays a role in cardiac aging and melatonin is able to counteract its effects. With the aim of investigating the impact of NLRP3 inflammasome and the actions and target of melatonin in aged myocardium, we analyzed the expression of proteins implied in mitochondria dynamics, autophagy, apoptosis, Nrf2-dependent antioxidant response and mitochondria ultrastructure in heart of wild-type and NLRP3-knockout mice of 3, 12, and 24 months-old, with and without melatonin treatment. Our results showed that the absence of NLRP3 prevented age-related mitochondrial dynamic alterations in cardiac muscle with minimal effects in cardiac autophagy during aging. The deficiency of the inflammasome affected Bax/Bcl2 ratio, but not p53 or caspase 9. The Nrf2-antioxidant pathway was also unaffected by the absence of NLRP3. Furthermore, NLRP3-deficiency prevented the drop in autophagy and mice showed less mitochondrial damage than wild-type animals. Interestingly, melatonin treatment recovered mitochondrial dynamics altered by aging and had few effects on cardiac autophagy. Melatonin supplementation also had an anti-apoptotic action in addition to restoring Nrf2-antioxidant capacity and improving mitochondria ultrastructure altered by aging.

Lack of NLRP3 Inflammasome Activation Reduces Age-Dependent Sarcopenia and Mitochondrial Dysfunction, Favoring the Prophylactic Effect of Melatonin.

To investigate the role of NLRP3 inflammasome in muscular aging, we evaluated here the morphological and functional markers of sarcopenia in the NLRP3-knockout mice, as well as the beneficial effect of melatonin supplementation. The gastrocnemius muscles of young (3 months), early-aged (12 months), and old-aged (24 months) NLRP3-knockout female mice were examined. Moreover, locomotor activity and apoptosis were assessed. The results revealed early markers of sarcopenia at the age of 12 months, including reduction of lactate, ratio of muscle weight to body weight, muscle fibers number, and mitochondrial number. Increased interstitial tissues, apoptosis, and muscle fibers area, as well as mitochondrial damage were detected, with little muscular activity effects. In the old-aged, these alterations progressed with a reduction in locomotor activity, mitochondrial cristae destruction, nuclear fragmentation, tubular aggregates (TAs) formation, and increased frailty index. Oral melatonin supplementation preserved the normal muscular structure, muscle fibers number, and muscular activity in old age. Melatonin enhanced lactate production, recovered mitochondria, inhibited TAs formation, reduced apoptosis, and normalized frailty index. The fewer sarcopenic changes as well as the highly detectable prophylactic effects of melatonin treatment reported here in the muscle of NLRP3-knockout mice comparing with that previously detected in wild-type mice, confirming NLRP3 inflammasome implication in muscular aging and sarcopenia onset and progression.

Involvement of plasma miRNAs, muscle miRNAs and mitochondrial miRNAs in the pathophysiology of frailty.

Frailty is a geriatric syndrome that leads not only to the loss of physical functions, but also to a generalized decline of the organism and a high risk of disability and dependency. Frailty's detection and management represent important goals for current gerontology. The advance in its rapid diagnosis could play a relevant role in taking measures to reduce the negative consequences it exerts on the body and to take preventive measures. microRNAs are the one of multiple epigenetic biomarkers that reflect functional changes in aged subject. In this review we analyze microRNAs as molecules involved in the control of the pathways leading to the development of frailty. miRNAs can be present in different body fluids, including plasma/serum and saliva, can be associated with organelles like the mitochondria, and can be expressed in tissues. Based on the multifactorial physiopathology of frailty, we analyzed here the microRNAs linked to "inflammaging" (inflamma-miRs), to musculoskeletal health (myomiRs), and microRNAs that can directly or indirectly affect the mitochondria (mitomiRs). Subsequently, we analyze those microRNAs that can be modified by physical exercise. In this review we will analyze the latest experimental studies carried out in animals, cell cultures, and human samples, with the aim to identify gaps in the research and in order to try to dazzle the information about the pathways regulated by each miRNA. Multiple studies revised here suggest that several miRs can be considered as possible markers of frailty, including miR-1, miR-21, miR-34a, miR-146a, miR-185, and miR-206, miR-223, among others. Normalization of miRNAs data and standardization of the protocols used for their measurement to avoid confounding variables influencing the results, are important to use miRNAs as disease biomarkers.

Analysis of Plasma MicroRNAs as Predictors and Biomarkers of Aging and Frailty in Humans.

Although circulating microRNAs (miRNAs) can modulate gene expression and affect immune system response, little is known about their participation in age-associated frailty syndrome and sarcopenia. The aim of this study was to determine miRNAs as possible biomarkers of age and frailty and their correlation with oxidative and inflammatory state in human blood. Three inflammation-related miRNAs (miR-21, miR-146a, and miR-223) and one miRNA related with the control of melatonin synthesis (miR-483) were analyzed. Twenty-two healthy adults, 34 aged robust, and 40 aged fragile patients were selected for this study. The expression of plasma miRNAs was assessed by RT-qPCR; plasma cytokines (IL-6, IL-8, IL-10, and TNFα) were analyzed by commercial kits, and plasma advanced oxidation protein products (AOPP) and lipid oxidation (LPO) were spectrophotometrically measured. Fragile subjects had higher miR-21 levels than control subjects, whereas miR-223 and miR-483 levels increased at a similar extend in both aged groups. All cytokines measured increased in aged groups compared with controls, without differences between robust and fragile subjects. The fragile group had a TNFα/IL-10 ratio significantly higher than robust and control groups. Aged groups also had higher AOPP and LPO levels than controls. Women presented higher AOPP and LPO levels and increased expression of miR-483 compared with men. Positive correlations between miR-21 and AOPP and between miR-483 and IL-8 were detected. The expression of miR-21 and the TNFα/IL-10 ratio were correlated positively with the presence of frailty, which suggests that these markers can be considered as possible biomarkers for age-related frailty.