Relaxin-3 Ameliorates Diabetic Cardiomyopathy by Inhibiting Endoplasmic Reticulum Stress.

Background: This study is aimed at investigating whether relaxin-3 exhibits protective effects against cardiomyopathy in diabetic rats by suppressing ERS. Methods: Eighty male SD rats were randomly divided into two groups: controls (n = 20) and diabetes (n = 60). The streptozotocin-treated rats were randomly divided into three groups: diabetic group (DM), low-dose relaxin-3 group (0.2 µg/kg/d), and high-dose relaxin-3 group (2 µg/kg/d). The myocardial tissues and collagen fiber were observed by hematoxylin and eosin (H&E) and Masson staining. Serum brain natriuretic peptide (BNP), troponin (TNI), myoglobin, interleukin (IL-17), interleukin (IL)-1α, and tumor necrosis factor (TNF)-α were determined by ELISA. The protein expression of glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the heart tissue of each group was detected by Western blot analysis. Results: (1) HE and Masson staining indicated that relaxin-3 could attenuate myocardial lesions and myocardial collagen volume fraction. (2) BNP, TnI, and myoglobin in the DM group at four and eight weeks were significantly higher than in the controls (P < 0.01). The relaxin-3-treated groups showed significantly reduced serum BNP, TnI, and myoglobin levels compared with the DM group (P < 0.05). (3) IL-17, IL-1α, and TNF-α levels in the DM rats at 4 weeks were higher than in the controls (P < 0.05). Low or high dose of relaxin-3-treated groups showed reduced serum IL-17 and TNF-α levels compared with the DM group at four and eight weeks (P < 0.05). (4) CHOP and GRP78 protein expression was increased in the DM group at four and eight weeks compared with the controls (P < 0.01), and small and large doses of relaxin-3 significantly reduced GRP78 and CHOP protein expression. Conclusions: Exogenous relaxin-3 ameliorates diabetic cardiomyopathy by inhibiting ERS in diabetic rats.

A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Ulinastatin Alleviates Rhabdomyolysis-Induced Acute Kidney Injury by Suppressing Inflammation and Apoptosis via Inhibiting TLR4/NF-κB Signaling Pathway.

Acute kidney injury (AKI) is an important complication of rhabdomyolysis (RM), but there is lack of effective treatments. Ulinastatin (UTI) is a broad-spectrum serine protease inhibitor isolated and purified from human urine with strong anti-inflammatory and cytoprotective actions. The aim of this research was to investigate the effect and potential mechanism of UTI on RM-induced AKI (RM-AKI). We established RM-induced AKI model and myoglobin (Mb)-stimulated NRK-52E cell model. In vivo, twenty-four rats were randomly divided into three groups (n = 8): control, RM-AKI, and RM-AKI + UTI. In vitro, the NRK-52E cells were divided into six groups according to the different treatment method. Mb-stimulated NRK-52E cells were treated with UTI or si-TLR4 transfection to characterize the mechanisms of UTI in RM-AKI. Indicators of the kidney injury, cell viability, cell cycle, oxidative stress, inflammation, apoptosis, and TLR4/NF-κB signaling pathway were assessed. In vivo and in vitro, UTI significantly decreased the expression of TLR4 and p65. In vivo, UTI significantly improved renal function and reduced inflammatory reaction and kidney injury. In vitro, UTI protected NRK-52E cells from Mb stimulation by suppressing cell cytotoxicity, cell cycle inhibition, overproduction of ROS, inflammation, and apoptosis. Additionally, UTI played a protective role by downregulating the TLR4 expression. The results indicate that UTI alleviates RM-AKI by suppressing the inflammatory response and apoptosis via inhibiting TLR4/NF-κB signaling pathway. Our study provides a new mechanism for the protective effect of UTI on RM-AKI.

Effect of dietary leucine supplementation on skeletal muscle fiber type transformation in weaning piglets.

To investigate the effects of dietary leucine supplementation on muscle fiber type transformation in weaning piglets, 54 21-day-old male DLY (Duroc × Landrace × Yorkshire) weaned piglets were randomly divided into control, 0.25% and 0.5% leucine groups. The experiment lasted for 42 d. The results showed that dietary supplementation of 0.25% leucine significantly increased the protein expressions of slow MyHC, myoglobin and Troponin I-SS and the mRNA expressions of MyHC I, MyHC IIa, Tnni1, Tnnc1, Tnnt1 and myoglobin, while decreased the protein level of fast MyHC and the mRNA level of MyHC IIb in longissimus dorsi (LD) muscle. Furthermore, 0.25% leucine significantly increased succinic dehydrogenase (SDH) activity and decreased lactate dehydrogenase (LDH) activity. In addition, our data found that 0.25% leucine significantly increased serum adiponectin (AdipoQ) concentration, and the protein levels of AdipoQ, adiponectin receptor 1 (AdipoR1), phosphorylated AMP-activated protein kinase (p-AMPK) and PPAR-γ coactivator-1α (PGC-1α) and the mRNA levels of AdipoQ, AdipoR1 and AMPKα2. Together, our findings indicate that leucine promotes porcine skeletal muscle fiber type transformation from fast-twitch to slow-twitch, and the effect may be mediated by AdipoQ-AMPK-PGC-1α signaling pathway.

Selected root plant supplementation reduces indices of exercise-induced muscle damage: A systematic review and meta-analysis.

This systematic review and meta-analysis examined the effects of selected root plants (curcumin, ginseng, ginger and garlic) on markers of muscle damage and muscular performance measures following muscle-damaging protocols. We included 25 studies (parallel and crossover design) with 353 participants and used the PEDro scale to appraise each study. Forest plots were generated to report on standardised mean differences (SMD) and p-values at 24 and 48 hours following the muscle-damaging protocols. The meta-analysis showed that the supplemental (SUPP) condition showed significantly lower levels of indirect muscle damage markers (creatine kinase, lactate dehydrogenase and myoglobin) and muscle soreness at 24 hours and 48 hours (p < 0.01) than the placebo (PLA) condition. The inflammatory markers were significantly lower for the SUPP condition than the PLA condition at 24 hours (p = 0.02), although no differences were identified at 48 hours (p = 0.40). There were no significant differences in muscular performance measures between the SUPP and PLA conditions at 24 hours and 48 hours (p > 0.05) post-exercise. According to our qualitative data, a number of studies reported a reduction in oxidative stress (e.g., malondialdehyde, superoxide dismutase) with a concomitant upregulation of anti-oxidant status, although other studies showed no effects. Accordingly, selected root plants minimised the level of several biomarkers of muscle damage, inflammation and muscle soreness during periods of exercise-induced muscle damage. However, the benefits of these supplements in ameliorating oxidative stress, increasing anti-oxidant status and accelerating recovery of muscular performance appears equivocal, warranting further research in these outcome measures.

Loading Effect of Chitosan Derivative Nanoparticles on Different Antigens and Their Immunomodulatory Activity on Dendritic Cells.

Drug carrier nanoparticles (NPs) were prepared by the polyelectrolyte method, with chitosan sulfate, with different substituents and quaternary ammonium chitosan, including C236-HACC NPs, C36-HACC NPs, and C6-HACC NPs. To evaluate whether the NPs are suitable for loading different antigens, we chose bovine serum albumin (BSA), ovalbumin (OVA), and myoglobin (Mb) as model antigens to investigate the encapsulation effect of the NPs. The characteristics (size, potential, and encapsulation efficiency) of the NPs were measured. Moreover, the NPs with higher encapsulation efficiency were selected for the immunological activity research. The results showed that chitosan derivative NPs with different substitution sites had different loading effects on the three antigens, and the encapsulation rate of BSA and OVA was significantly better than that of Mb. Moreover, the NPs encapsulated with different antigens have different immune stimulating abilities to DCS cells, the immune effect of OVA-coated NPs was significantly better than that of BSA-coated NPs and blank NPs, especially C236-HACC-OVA NPs. Furthermore, we found that C236-HACC-OVA NPs could increase the phosphorylation level of intracellular proteins to activate cell pathways. Therefore, C236-HACC NPs are more suitable for the loading of antigens similar to the OVA structure.

RIG-I, a novel DAMPs sensor for myoglobin activates NF-κB/caspase-3 signaling in CS-AKI model.

BACKGROUND: Acute kidney injury (AKI) is the main life-threatening complication of crush syndrome (CS), and myoglobin is accepted as the main pathogenic factor. The pattern recognition receptor retinoicacid-inducible gene I (RIG-I) has been reported to exert anti-viral effects function in the innate immune response. However, it is not clear whether RIG-I plays a role in CS-AKI. The present research was carried out to explore the role of RIG-I in CS-AKI. METHODS: Sprague-Dawley rats were randomly divided into two groups: the sham and CS groups (n = 12). After administration of anesthesia, the double hind limbs of rats in the CS group were put under a pressure of 3 kg for 16 h to mimic crush conditions. The rats in both groups were denied access to food and water. Rats were sacrificed at 12 h or 36 h after pressure was relieved. The successful establishment of the CS-AKI model was confirmed by serum biochemical analysis and renal histological examination. In addition, RNA sequencing was performed on rat kidney tissue to identify molecular pathways involved in CS-AKI. Furthermore, NRK-52E cells were treated with 200 µmol/L ferrous myoglobin to mimic CS-AKI at the cellular level. The cells and cell supernatant samples were collected at 6 h or 24 h. Small interfering RNAs (siRNA) was used to knock down RIG-I expression. The relative expression levels of molecules involved in the RIG-I pathway in rat kidney or cells samples were measured by quantitative Real-time PCR (qPCR), Western blotting analysis, and immunohistochemistry (IHC) staining. Tumor necrosis factor-α (TNF-α) was detected by ELISA. Co-Immunoprecipitation (Co-IP) assays were used to detect the interaction between RIG-I and myoglobin. RESULTS: RNA sequencing of CS-AKI rat kidney tissue revealed that the different expression of RIG-I signaling pathway. qPCR, Western blotting, and IHC assays showed that RIG-I, nuclear factor kappa-B (NF-κB) P65, p-P65, and the apoptotic marker caspase-3 and cleaved caspase-3 were up-regulated in the CS group (P < 0.05). However, the levels of interferon regulatory factor 3 (IRF3), p-IRF3 and the antiviral factor interferon-beta (IFN-ß) showed no significant changes between the sham and CS groups. Co-IP assays showed the interaction between RIG-I and myoglobin in the kidneys of the CS group. Depletion of RIG-I could alleviate the myoglobin induced expression of apoptosis-associated molecules via the NF-κB/caspase-3 axis. CONCLUSION: RIG-I is a novel damage-associated molecular patterns (DAMPs) sensor for myoglobin and participates in the NF-κB/caspase-3 signaling pathway in CS-AKI. In the development of CS-AKI, specific intervention in the RIG-I pathway might be a potential therapeutic strategy for CS-AKI.

Polymeric Micelles Loading Proteins through Concurrent Ion Complexation and pH-Cleavable Covalent Bonding for In Vivo Delivery.

Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half-life, and adverse immune responses. Thus, protein delivery approaches based on stimuli-responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH-cleavable covalent bonding between proteins and block copolymers directed to pH-triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH-sensitive moiety, since the CDMamide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)-poly(l-lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin-loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half-life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.

Initiating fibro-proliferation through interfacial interactions of myoglobin colloids with collagen in solution.

This work examines fibro-proliferation through interaction of myoglobin (Mb), a globular protein with collagen, an extracellular matrix fibrous protein. Designed colloids of Mb at pH 4.5 and 7.5 have been mixed with collagen solution at pH 7.5 and 4.5 in different concentrations altering their surface charges. For the Mb colloids, 100-200nm sizes have been measured from Transmission electron micrographs and zeta sizer. CD spectra shows a shift to beta sheet like structure for the protein in the colloids. Interaction at Mb/Collagen interface studied using Dilational rheology, Quartz crystal microbalance with dissipation and Differential Scanning calorimetry show that the perturbation is not only by the charge compensation arising from the difference in pH of the colloids and collagen, but also by the organized assembly of collagen at that particular pH. Results demonstrate that positive Mb colloids at pH 4.5, having more% of entrained water stabilize the collagen fibrils (pH 7.5) around them. Ensuing dehydration leads to effective cross-linking and inherently anisotropic growth of fibrils/fibres of collagen. In the case of Mb colloids at pH 7.5, the fibril formation seems to supersede the clustering of Mb suggesting that the fibro-proliferation is both pH and hydrophilic-hydrophobic balance dependent at the interface.

Myoglobin facilitates angiotensin II-induced constriction of renal afferent arterioles.

Vasoconstriction plays an important role in the development of acute kidney injury in rhabdomyolysis. We hypothesized that myoglobin enhances the angiotensin II (ANG II) response in afferent arterioles by increasing superoxide and reducing nitric oxide (NO) bioavailability. Afferent arterioles of C57Bl6 mice were isolated perfused, and vasoreactivity was analyzed using video microscopy. NO bioavailability, superoxide concentration in the vessel wall, and changes in cytosolic calcium were measured using fluorescence techniques. Myoglobin treatment (10-5 M) did not change the basal arteriolar diameter during a 20-min period compared with control conditions. NG-nitro-l-arginine methyl ester (l-NAME, 10-4 M) and l-NAME + myoglobin reduced diameters to 94.7 and 97.9% of the initial diameter, respectively. Myoglobin or l-NAME enhanced the ANG II-induced constriction of arterioles compared with control (36.6 and 34.2%, respectively, vs. 65.9%). Norepinephrine responses were not influenced by myoglobin. Combined application of myoglobin and l-NAME further facilitated the ANG II response (7.0%). Myoglobin or l-NAME decreased the NO-related fluorescence in arterioles similarly. Myoglobin enhanced the superoxide-related fluorescence, and tempol prevented this enhancement. Tempol also partly prevented the myoglobin effect on the ANG II response. Myoglobin increased the fura 2 fluorescence ratio (cytosolic calcium) during ANG II application (10-12 to 10-6 M). The results suggest that the enhanced afferent arteriolar reactivity to ANG II is mainly due to a myoglobin-induced increase in superoxide and associated reduction in the NO bioavailability. Signaling pathways for the augmented ANG II response include enhanced cytosolic calcium transients. In conclusion, myoglobin may contribute to the afferent arteriolar vasoconstriction in this rhabdomyolysis model.

Zinc-Substituted Myoglobin Is a Naturally Occurring Photo-antimicrobial Agent with Potential Applications in Food Decontamination.

Zinc-substituted myoglobin (ZnMb) is a naturally occurring photosensitizer that generates singlet oxygen with a high quantum yield. Using a combination of photophysical and fluorescence imaging techniques, we demonstrate the interaction of ZnMb with Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. An efficient antibacterial action against S. aureus was observed, with a reduction up to 99.9999% in the number of colony-forming units, whereas no sizable effect was detected against E. coli. Because ZnMb is known to form during the maturation of additive-free not-cooked cured ham, the use of this protein as a built-in photodynamic agent may constitute a viable method for the decontamination of these food products from Gram-positive bacteria.

An evaluation of the antioxidant protein α1-microglobulin as a renal tubular cytoprotectant.

α1-Microglobulin (A1M) is a low-molecular-weight heme-binding antioxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought "proof of concept" in this regard. Cultured proximal tubule (HK-2) cells or isolated mouse proximal tubule segments were challenged with a variety of prooxidant insults: 1) hemin, 2) myoglobin; 3) "catalytic" iron, 4) H2O2/Fenton reagents, 5) a Ca(2+) ionophore, 6) antimycin A, or 7) hypoxia (with or without rA1M treatment). HK-2 injury was gauged by the percent lactate dehydrogenase release and 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake. In vivo protection was sought in rA1M-treated mice subjected to 1) graded myohemoglobinura (2, 4, 8, or 9 ml/kg glycerol injection), 2) purified myoglobinemia/uria, or 3) endotoxemia. In vivo injury was assessed by blood urea nitrogen, creatinine, and the expression of redox-sensitive genes (heme oxygenase-1, neutrophil gelatinase-associated lipocalin, and monocyte chemoattractant protein-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder) or 10% serum induced equal protection. rA1M failed to mitigate any nonhemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (by Western blot analysis). Surprisingly, rA1M exerted select injury-promoting effects (increased in vitro catalytic iron/antimycin toxicities and increased in vivo monocyte chemoattractant protein-1/neutrophil gelatinase-associated lipocalin mRNA expression after glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g., albumin) binders.

Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue.

Restricted oxygen diffusion can result in central cell necrosis in engineered tissue, a problem that is exacerbated when engineering large tissue constructs for clinical application. Here we show that pre-treating human mesenchymal stem cells (hMSCs) with synthetic membrane-active myoglobin-polymer-surfactant complexes can provide a reservoir of oxygen capable of alleviating necrosis at the centre of hyaline cartilage. This is achieved through the development of a new cell functionalization methodology based on polymer-surfactant conjugation, which allows the delivery of functional proteins to the hMSC membrane. This new approach circumvents the need for cell surface engineering using protein chimerization or genetic transfection, and we demonstrate that the surface-modified hMSCs retain their ability to proliferate and to undergo multilineage differentiation. The functionalization technology is facile, versatile and non-disruptive, and in addition to tissue oxygenation, it should have far-reaching application in a host of tissue engineering and cell-based therapies.

[Preliminary study on the pathogenic mechanism of myoglobin-induced endoplasmic reticulum stress and apoptosis in crush syndrome].

OBJECTIVE: To explore the pathogenic mechanism of myoglobin-induced endoplasmic reticulum stress (ERS) and apoptosis in tubular epithelial cells in acute kidney injury (AKI) mouse model of crush syndrome. METHODS: Eighteen C56BL/6 mice were randomly divided into control group, modeling 8 h group and modeling 24 h group. The AKI model of crush syndrome was established by intramuscular injection of 50% glycerol saline solution into thigh (8 microL/g), while equivalent volume of physiological saline was injected in control group. AKI was diagnosed when serum creatinine (sCr) level increased to double value of control group. The mice in the experimental groups were sacrificed at the time points of 8 h and 24 h after injection respectively. Serum Cr was detected and renal tissues was observed under electron microscopy. Apoptosis was detected by TUNEL technique. Marker proteins and mRNA of apoptosis and ERS were detected by immunohistochemistry and real-time PCR. Human kidney proximal tubular cell (HK-2) cells cultured in vitro were randomly divided into control, intervention 6 h and intervention 12 h groups. Control group were incubated in standard cell culture (DMEM/F12) and the two intervention groups were incubated in special DMEM/F12 in which ferrohemoglobin was added. After 6 h and 12 h incubation, the cells were collected and apoptosis was detected by flow cytometry. RESULTS: AKI model of crush syndrome was successfully established, which was proved with sCr doubling at the 8 h after the intramuscular injection of glycerol saline. Swelling of endoplasmic reticulum and mitochondria in proximal tubular epithelial cells was more obvious in the two model groups than that in control group. TUNEL staining showed the percentage of positive cells in AKI groups was higher than that in control group (P<0.05). Immunohistochemistry and real-time PCR showed the expressions of caspase3, caspase12 and CHOP in AKI groups were higher than those in control group (P<0.05). Flow cytometry showed cell apoptosis ratio was higher in model groups than control groiap (P<0.05). CONCLUSION: Myoglobin induced ERS and apoptosis may be involved in the pathogenesis of AKI in crush syndrome.

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution.

Glycation accelerates fibrillization of the amyloidogenic W7FW14F apomyoglobin.

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.

Myoglobin-induced apoptosis: two pathways related to endoplasmic reticulum stress.

Myoglobin plays an important role in rhabdomyolysis-induced acute kidney injury (AKI), but the underlying mechanisms are still unclear. The present study investigates myoglobin-induced apoptosis in HK-2 cells (human renal proximal tubule cells) to discover some of the mechanisms involved in rhabdomyolysis related AKI. Metmyoglobin is reduced to ferrous myoglobin by ascorbic acid, and then the HK-2 cells are incubated with ferrous myoglobin. Cell viability is measured by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and cell injury is tested by supernatant lactose dehydrogenase (LDH). Cell apoptosis is evaluated by fluorescent microscopy of Hoechst staining and by flow cytometry of Annexin V/PI double staining. The apoptosis related protein expression is determined by Western blot. HK-2 cells were incubated with 200 µM ferrous myoglobin for 24 h, the cell viability decreased and supernatant LDH release increased. Hoechst staining indicated more apoptosis after incubation. Molecular chaperone glucose-related protein 78 (GRP78), cytochrome C, caspase-9 started to increase within 3 h after incubation while caspase-4, caspase-8 showed no significant change. (iii) When the inositol triphosphate receptor (IP3R) calcium channel was blocked by 2-aminoethoxydiphenyl-borinate (2-APB), caspase-9 was completely inhibited, GRP78 and caspase-4 increased dramatically, and caspase-3 expression was not affected. The apoptosis in HK-2 cells showed no significant change. Apoptosis in HK-2 cells incubated with ferrous myoglobin is an endoplasmic reticulum stress induced, IP3R calcium channel mediated, caspase-9 dependent intrinsic pathway. When the intrinsic pathway was inhibited using an IP3R calcium channel blocker, endoplasmic reticulum stress increased, resulting in the activation of caspase-4 that cleaved caspase-3 and generated a substitutive pathway of apoptosis.

The synthetic polyphenol tert-butyl-bisphenol inhibits myoglobin-induced dysfunction in cultured kidney epithelial cells.

Abstract Rhabdomyolysis caused by severe burn releases extracellular myoglobin (Mb) that accumulates in the kidney and urine (maximum [Mb] approximately 50 microM) (termed myoglobinuria). Extracellular Mb can be a pro-oxidant. This study cultured Madin-Darby-canine-kidney-Type-II (MDCK II) cells in the presence of Mb and tested whether supplementation with a synthetic tert-butyl-polyphenol (tert-butyl-bisphenol; t-BP) protects these renal cells from dysfunction. In the absence of t-BP, cells exposed to 0-100 microM Mb for 24 h showed a dose-dependent decrease in ATP and the total thiol (TSH) redox status without loss of viability. Gene expression of superoxide dismutases-1/2, haemoxygenase-1 and tumour necrosis factor increased and receptor-mediated endocytosis of transferrin and monolayer permeability decreased significantly. Supplementation with t-BP before Mb-insult maintained ATP and the TSH redox status, diminished antioxidant/pro-inflammatory gene responses, enhanced monolayer permissiveness and restored transferrin uptake. Overall, bolstering the total antioxidant capacity of the kidney may protect against oxidative stress induced by experimental myoglobinuria.

Myoglobin induces vascular cell adhesion molecule-1 expression through c-Src kinase-activator protein-1/nuclear factor-kappaB pathways.

BACKGROUND/AIMS: It is not clear whether a sublethal dose of myoglobin induces some pathophysiological changes in tubular cells, potentially affecting tubular injury or tubular regeneration. We investigated the effect of a low dose of myoglobin on vascular cell adhesion molecule-1 (VCAM-1) expression and elucidated the underlying signaling pathways. We further examined the effect of losartan and simvastatin on myoglobin-induced VCAM-1 expression and the signaling pathways. METHODS: Activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 was assessed by electrophoretic mobility shift assay. Phosphorylation of protein kinases was examined by Western blot analysis. VCAM-1 mRNA and protein were measured by Northern blot analysis and cell ELISA. RESULTS: A sublethal dose of myoglobin (100 microg/ml) induced VCAM-1 expression via activation of AP-1 and NF-kappaB, which was mediated through activation of c-Src kinase, followed by mitogen-activated protein kinases (p38, ERK 1/2, JNK-1) and the I kappaB kinase - I kappaB-alpha. Inhibitors of protein kinase C and tyrosine kinase, antioxidants and intracellular calcium chelator suppressed myoglobin-induced activation of c-Src kinase. Losartan and simvastatin suppressed myoglobin-induced VCAM-1 expression via inhibition of c-Src kinase. CONCLUSION: VCAM-1 expression via c-Src kinase-AP-1/NF-kappaB pathways might be one of the possible mechanisms linking myoglobin to tubular injury. Losartan and simvastatin might be beneficial in attenuating myoglobin-induced tubular injury.

Phenylalanine substitution at site B10 (L29F) inhibits metmyoglobin formation and myoglobin-mediated lipid oxidation in washed fish muscle: mechanistic implications.

Wild-type sperm whale myoglobin (WT Mb) and L29F mutant were used to examine the effect of metMb formation rate on Mb-mediated lipid oxidation in washed cod muscle. MetMb formation was 15-fold slower in L29F compared to WT Mb at 2 degrees C (pH 5.7). The electrostatic interaction of bound O(2) and the partial positive edge of the phenyl ring of phenylalanine(29) inhibits deoxyMb-mediated metMb formation and may displace protons that promote oxyMb oxidation. Ferrous L29F was a poor promoter of lipid oxidation in washed cod during extended storage, whereas ferrous WT Mb oxidized the substrate readily. This diminishes a role for free radicals produced by the reaction of oxyMb with peroxides. MetL29F was an effective promoter of lipid oxidation. Mb mutants with low hemin affinity (H97A) were better promoters of microsomal lipid oxidation than mutants with higher hemin affinity (WT Mb and V68T). The much higher heme affinity of oxyMb compared to metMb partly explains the poor ability of ferrous L29F to oxidize lipids in washed cod at post-mortem pH during 2 degrees C storage. The relative roles of cross-linked Mb and hypervalent Mb species to promote lipid oxidation in washed cod at post-mortem pH are discussed.