Cinnamaldehyde attenuates TNF-α induced skeletal muscle loss in C2C12 myotubes via regulation of protein synthesis, proteolysis, oxidative stress and inflammation.

Inflammation is the primary driver of skeletal muscle wasting, with oxidative stress serving as both a major consequence and a contributor to its deleterious effects. In this regard, regulation of both can efficiently prevent atrophy and thus will increase the rate of survival [1]. With this idea, we hypothesize that preincubation of Cinnamaldehyde (CNA), a known compound with anti-oxidative and anti-inflammatory properties, may be able to prevent skeletal muscle loss. To examine the same, C2C12 post-differentiated myotubes were treated with 25 ng/ml Tumor necrosis factor-alpha (TNF-α) in the presence or absence of 50 µM CNA. The data showed that TNF-α mediated myotube thinning and a lower fusion index were prevented by CNA supplementation 4 h before TNF-α treatment. Moreover, a lower level of ROS and thus maintained antioxidant defense system further underlines the antioxidative function of CNA in atrophic conditions. CNA preincubation also inhibited an increase in the level of inflammatory cytokines and thus led to a lower level of inflammation even in the presence of TNF-α. With decreased oxidative stress and inflammation by CNA, it was able to maintain the intracellular level of injury markers (CK, LDH) and SDH activity of mitochondria. In addition, CNA modulates all five proteolytic systems [cathepsin-L, UPS (atrogin-1), calpain, LC3, beclin] simultaneously with an upregulation of Akt/mTOR pathway, in turn, preserves the muscle-specific proteins (MHCf) from degradation by TNF-α. Altogether, our study exhibits attenuation of muscle loss and provides insight into the possible mechanism of action of CNA in curbing TNF-α induced muscle loss, specifically its effect on proteolysis and protein synthesis.

Evaluation of cinnamaldehyde derivatives as potential protective agents against oxidative-stress induced myotube atrophy using chemical, biological and computational analysis.

Skeletal muscle atrophy, associated with increased morbidity, mortality and poor quality of life, is a metabolic disorder with no FDA approved drug. Oxidative stress is one of the key mediators of atrophy that influences various cell signaling molecules. The goal of this study is to identify potential antioxidant agents that could be used to treat atrophy. In this study in vitro and in situ screening of different cinnamaldehyde (CNA) derivatives for their antioxidant effects was done along with computational analysis to understand the relationship between their chemical structure and biological activity. Data show that 2-hydroxycinnamaldehyde (2HCNA) worked better than other CNA analogues at physiological pH, while 4-Fluoro-2-methoxycinnamaldehyde (4FoCNA) showed the maximum antioxidant activity under acidic conditions. However, these derivatives (2HCNA and 4FoCNA) were found to be toxic to the cultured myotubes (mature myofiber) under both physiological and pathophysiological conditions. Immunofluorescence, bright-field microscopic and biochemical studies conducted using live C2C12 cells showed that pre-incubation with other CNA analogues i.e. 2-methoxycinnamaldehyde (2MeCNA) and 2-benzyloxycinnamaldehyde (2BzCNA) not only maintained the normal morphology of myotubes but also protected them from H2O2-induced atrophy. These compounds (2MeCNA and 2BzCNA) showed higher stability and antioxidant potential, as indicated by computer simulation data analyzed by Density Functional Theory (DFT) based molecular modeling. Overall, the chemical, biological, and computational studies reveal the therapeutic potential of CNA analogues (BzCNA and MeCNA) against oxidative-stress induced muscle atrophy in C2C12 cells.

A research update: Significance of cytokine storm and diaphragm in COVID-19.

Emerging research on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) shows that it is spreading to multiple organs in addition to the respiratory system. Though the SARS-CoV2 enters the human body by binding to ACE2 receptors on pulmonary alveolar cells, recent studies indicate that it is spreading to the central nervous system, cardiac and skeletal muscles leading to various pathological conditions in these organs. In particular, the effects of SARS-CoV-2 on triggering the cytokine storm and its consequential effects on skeletal muscles has generated a lot of discussion. The effects of this virus on muscular function especially in susceptible elderly populations is still being explored. However, its effects on diaphragm, a respiratory muscle which plays an important role in determining lung capacity are not completely explored. Currently, as new evidence on using lung ultrasounds to confirm COVID-19 diagnosis is gaining traction, it is necessary to explore the role of diaphragm in treating COVID-19 patients. This article will review the effects of cytokine storm triggered by the SARS-CoV-2 and its resultant effects on skeletal muscle with a specific focus on the diaphragm in order to identify knowledge gaps in effectively treating COVID-19 patients, especially those who are on a mechanical ventilator.

Postnatal development alters functional compartmentalization of myosin light chain kinase in ovine carotid arteries.

The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC20) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC20. Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of Vmax and Km for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC20. The in situ rate of MLC20 phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the Vmax values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC20, and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

Efficient and modified 2-NBDG assay to measure glucose uptake in cultured myotubes.

Under type-2 diabetes, insulin resistance develops in skeletal muscles as a key defect and to study the disorder, its manifestation, and possible solution, measurement of glucose uptake is a fundamental necessity. Of various approaches (i.e. scintillation counting, flow cytometry, fluorometry and spectrophotometry) fluorescent labelled glucose analogue, 2-NBDG solution is the most popular one. Although 2-NBDG based assay is the most widely used approach in various cells including skeletal muscle, even then all available protocols possess huge variability which impacts the overall data reproducibility. Moreover, starvation (use of glucose/serum free medium), one of the prerequisite condition for glucose uptake assay, itself induces stress specifically during longer pre-incubation periods and alters muscle cell metabolism and morphology, but the fact has not been duly considered. Therefore in the present article, using specific skeletal muscle cells i.e. C2C12 myotubes, we have re-established the conditions like pre-incubation time period, concentrations of insulin, glucose and serum/BSA while maintaining the cultured myotubes in morphologically healthy state. Our lab standardized protocols were observed to be effective in studying insulin resistance condition induced by diverse stresses (oxidative & inflammation) in myotubes. Comparative study conducted with already established protocols demonstrates that the present method is more efficient, effective and better improvised for studying glucose uptake in C2C12.

Cinnamaldehyde regulates H2 O 2 -induced skeletal muscle atrophy by ameliorating the proteolytic and antioxidant defense systems.

Skeletal muscle atrophy/wasting is associated with impaired protein metabolism in diverse physiological and pathophysiological conditions. Elevated levels of reactive oxygen species (ROS), disturbed redox status, and weakened antioxidant defense system are the major contributing factors toward atrophy. Regulation of protein metabolism by controlling ROS levels and its associated catabolic pathways may help in treating atrophy and related clinical conditions. Although cinnamaldehyde (CNA) enjoys the established status of antioxidant and its role in ROS management is reported, impact of CNA on skeletal muscle atrophy and related pathways is still unexplored. In the current study, the impact of CNA on C2C12 myotubes and the possible protection of cultured cells from H 2 O 2 -induced atrophy is examined. Myotubes were treated with H 2 O 2 in the presence and absence of CNA and the changes in the antioxidative, proteolytic systems, and mitochondrial functions were scored. Morphological analysis showed significant protective effects of CNA on length, diameter, and nuclei fusion index of myotubes. The evaluation of biochemical markers of atrophy; creatine kinase, lactate dehydrogenase, succinate dehydrogenase along with the study of muscle-specific structural protein (i.e., myosin heavy chain-fast [MHCf] type) showed significant protection of proteins by CNA. CNA pretreatment not only checked the activation of proteolytic systems (ubiquitin-proteasome E3-ligases [MuRF1/Atrogin1]), autophagy [Beclin1/LC3B], cathepsin L, calpain, caspase), but also prevented any alteration in the activities of antioxidative defense enzymes (catalase, glutathione- S-transferase, glutathione-peroxidase, superoxide dismutase, glutathione reductase). The results suggest that CNA protects myotubes from H 2 O 2 -induced atrophy by inhibiting/resisting the amendments in proteolytic systems and maintains cellular redox-balance.

Efficacy and risk profile of anti-diabetic therapies: Conventional vs traditional drugs-A mechanistic revisit to understand their mode of action.

An increasing array of anti-diabetic drugs are available today, yet Type-2 diabetes mellitus (T2DM) - remains a life threatening disease, causing high mortality and morbidity in developing and developed countries. As of now, no effective therapy is available for the complete eradication/cure of diabetes and its associated complications. Therefore, it is time to re-think and revisit molecular pathways and targets of each existing drug in order to identify multiple targets from different signaling pathways that may be manipulated simultaneously to treat or manage T2DM effectively. Bearing this goal in mind, the article reviews the mechanisms of action of available anti-diabetic drugs with in-depth mechanistic analysis of each therapy. The conventional and herbal strategies are analysed and compared for their benefits and the associated possible side effects. This critical information is necessary not only for the development of better, novel and potent anti-diabetic therapy in future but also for best possible combinational therapies and strategies with the available drugs.

Development and evaluation of a calcium alginate based oral ceftriaxone sodium formulation.

The purpose of this work was to develop a multiparticulate system exploiting the pH-sensitive property and biodegradability of calcium alginate beads for intestinal delivery of ceftriaxone sodium (CS). CS was entrapped in beads made of sodium alginate and sodium carboxymethylcellulose (CMC), acacia, HPMC K4M and HPMC K15M as drug release modifiers. Beads were prepared using calcium chloride as a cross-linking agent, followed by enteric coating with cellulose acetate phthalate (CAP). The beads were then evaluated for entrapment efficiency using HPLC, in vitro drug release examined in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8), swellability, particle size and surface characterization using optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Thermal gravimetric analysis (TGA) was utilized to check the polymer matrix strength and thermal stability. The drug entrapment efficiency of the optimized formulation was determined to be 75 ± 5 %. Swelling properties of drug-loaded beads were found to be in a range of 0.9-3.4. Alginate beads coated with CAP and containing CMC as a second polymer exhibited sustained release. The drug release followed first-order kinetics via non-Fickian diffusion and erosion mechanism. The particle size of the beads was between 1.04 ± 0.20 and 2.15 ± 0.36 mm. TGA, AFM, and SEM data showed composition and polymer-dependent variations in cross-linking, thermal stability, surface structure, morphology, and roughness. The physico-chemical properties of the developed formulation indicate suitability of the formulation to deliver CS orally.

Skeletal muscle atrophy: Potential therapeutic agents and their mechanisms of action.

Over the last two decades, new insights into the etiology of skeletal muscle wasting/atrophy under diverse clinical settings including denervation, AIDS, cancer, diabetes, and chronic heart failure have been reported in the literature. However, the treatment of skeletal muscle wasting remains an unresolved challenge to this day. About nineteen potential drugs that can regulate loss of muscle mass have been reported in the literature. This paper reviews the mechanisms of action of all these drugs by broadly classifying them into six different categories. Mechanistic data of these drugs illustrate that they regulate skeletal muscle loss either by down-regulating myostatin, cyclooxygenase2, pro-inflammatory cytokines mediated catabolic wasting or by up-regulating cyclic AMP, peroxisome proliferator-activated receptor gamma coactivator-1α, growth hormone/insulin-like growth factor1, phosphatidylinositide 3-kinases/protein kinase B(Akt) mediated anabolic pathways. So far, five major proteolytic systems that regulate loss of muscle mass have been identified, but the majority of these drugs control only two or three proteolytic systems. In addition to their beneficial effect on restoring the muscle loss, many of these drugs show some level of toxicity and unwanted side effects such as dizziness, hypertension, and constipation. Therefore, further research is needed to understand and develop treatment strategies for muscle wasting. For successful management of skeletal muscle wasting either therapeutic agent which regulates all five known proteolytic systems or new molecular targets/proteolytic systems must be identified.

Characterization and evaluation of 5-fluorouracil-loaded solid lipid nanoparticles prepared via a temperature-modulated solidification technique.

The aim of this research was to advance solid lipid nanoparticle (SLN) preparation methodology by preparing glyceryl monostearate (GMS) nanoparticles using a temperature-modulated solidification process. The technique was reproducible and prepared nanoparticles without the need of organic solvents. An anticancer agent, 5-fluorouracil (5-FU), was incorporated in the SLNs. The SLNs were characterized by particle size analysis, zeta potential analysis, differential scanning calorimetry (DSC), infrared spectroscopy, atomic force microscopy (AFM), transmission electron microscopy (TEM), drug encapsulation efficiency, in vitro drug release, and in vitro cell viability studies. Particle size of the SLN dispersion was below 100 nm, and that of redispersed lyophilizates was ~500 nm. DSC and infrared spectroscopy suggested that the degree of crystallinity did not decrease appreciably when compared to GMS. TEM and AFM images showed well-defined spherical to oval particles. The drug encapsulation efficiency was found to be approximately 46%. In vitro drug release studies showed that 80% of the encapsulated drug was released within 1 h. In vitro cell cultures were biocompatible with blank SLNs but demonstrated concentration-dependent changes in cell viability to 5-FU-loaded SLNs. The 5-FU-loaded SLNs can potentially be utilized in an anticancer drug delivery system.

A comparative study of the sulfation of bile acids and a bile alcohol by the Zebra danio (Danio rerio) and human cytosolic sulfotransferases (SULTs).

The current study was designed to examine the sulfation of bile acids and bile alcohols by the Zebra danio (Danio rerio) SULTs in comparison with human SULTs. A systematic analysis using the fifteen Zebra danio SULTs revealed that SULT3 ST2 and SULT3 ST3 were the major bile acid/alcohol-sulfating SULTs. Among the eleven human SULTs, only SULT2A1 was found to be capable of sulfating bile acids and bile alcohols. To further investigate the sulfation of bile acids and bile alcohols by the two Zebra danio SULT3 STs and the human SULT2A1, pH-dependence and kinetics of the sulfation of bile acids/alcohols were analyzed. pH-dependence experiments showed that the mechanisms underlying substrate recognition for the sulfation of lithocholic acid (a bile acid) and 5α-petromyzonol (a bile alcohol) differed between the human SULT2A1 and the Zebra danio SULT3 ST2 and ST3. Kinetic analysis indicated that both the two Zebra danio SULT3 STs preferred petromyzonol as substrate compared to bile acids. In contrast, the human SULT2A1 was more catalytically efficient toward lithocholic acid than petromyzonol. Collectively, the results imply that the Zebra danio and human SULTs have evolved to serve for the sulfation of, respectively, bile alcohols and bile acids, matching the cholanoid profile in these two vertebrate species.

Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries.

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.

Postnatal maturation modulates relationships among cytosolic Ca2+, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries.

The present study tests the hypothesis that age-related changes in patterns of agonist-induced myofilament Ca(2+) sensitization involve corresponding differences in the relative contributions of thick- and thin-filament regulation to overall myofilament Ca(2+) sensitivity. Posterior communicating cerebral arteries from term fetal and nonpregnant adult sheep were used in measurements of cytosolic Ca(2+), myosin light chain (MLC) phosphorylation, and contractile tensions induced by varying concentrations of K(+) or serotonin [5-hydroxytryptamine (5-HT)]. The results were used to assess the relative contributions of the relationships between cytosolic Ca(2+) and MLC phosphorylation (thick-filament reactivity), along with the relationships between MLC phosphorylation and contractile tension (thin-filament reactivity), to overall myofilament Ca(2+) sensitivity. For K(+)-induced contractions, both fetal and adult arteries exhibited similar basal myofilament Ca(2+) sensitivity. Despite this similarity, thick-filament reactivity was greater in fetal arteries, whereas thin-filament reactivity was greater in adult arteries. In contrast, 5-HT-induced contractions exhibited increased myofilament Ca(2+) sensitivity compared with K(+)-induced contractions for both fetal and adult cerebral arteries, and the magnitude of this effect was greater in fetal compared with adult arteries. When interpreted together with our previous studies of 5-HT-induced myofilament Ca(2+) sensitization, we attributed the present effects to agonist enhancement of thick-filament reactivity in fetal arteries mediated by G protein receptor activation of a PKC-independent but RhoA-dependent pathway. In adult arteries, agonist stimulation enhanced thin-filament reactivity was also probably mediated through G protein-coupled activation of RhoA-dependent and PKC-independent mechanisms. Overall, the present data demonstrate that agonist-enhanced myofilament Ca(2+) sensitivity can be partitioned into separate thick- and thin-filament effects, the magnitudes of which are different between fetal and adult cerebral arteries.

Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries.

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K(+) (K(max)) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca(2+) concentration at stretch ratios >2.0 in adult arteries, but increased Ca(2+) concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %K(max) values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 +/- 2.3%). Conversely, adult %K(max) values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 +/- 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.

Chronic hypoxic decreases in soluble guanylate cyclase protein and enzyme activity are age dependent in fetal and adult ovine carotid arteries.

The present study tests the hypothesis that chronic hypoxia enhances reactivity to nitric oxide (NO) through age-dependent increases in soluble guanylate cyclase (sGC) and protein kinase G (PKG) activity. In term fetal and adult ovine carotids, chronic hypoxia had no significant effect on mRNA levels for the beta1-subunit of sGC, but depressed sGC abundance by 16% in fetal and 50% in adult arteries, through possible depression of rates of mRNA translation (15% in fetal and 50% in adult) and/or increased protein turnover. Chronic hypoxia also depressed the catalytic activity of sGC, but only in fetal arteries (63%). Total sGC activity was reduced by chronic hypoxia in both fetal (69%) and adult (37%) carotid homogenates, but this effect was not observed in intact arteries when sGC activity was measured by timed accumulation of cGMP. In intact arteries treated with 300 microM 3-isobutyl-1-methylxanthine (IBMX), chronic hypoxia dramatically enhanced sGC activity in fetal (186%) but not adult (89%) arteries. This latter observation suggests that homogenization either removed an sGC activator, released an sGC inhibitor, or altered the phosphorylation state of the enzyme, resulting in reduced activity. In the absence of IBMX, chronic hypoxia had no significant effect on rates of cGMP accumulation. Chronic hypoxia also depressed the ability of the cGMP analog, 8-(p-chlorophenylthio)-cGMP, to promote vasorelaxation in both fetal (8%) and adult (12%) arteries. Together, these results emphasize the fact that intact and homogenized artery studies of sGC activity do not always yield equivalent results. The results further suggest that enhancement of reactivity to NO by chronic hypoxia must occur upstream of PKG and can only be possible if changes in cGMP occurred in functional compartments that afforded either temporal or chemical protection to the actions of phosphodiesterase. The range and age dependence of hypoxic effects observed also suggest that some responses to hypoxia must be compensatory and homeostatic, with reactivity to NO as the primary regulated variable.