Myosin light chain (MLC) isoforms play an integral role in modulating maximal shortening velocity and power output in skeletal, cardiac, and smooth muscles. It is thought that the expression of MLCs is regulated, at least in part, by muscle contractile activity. Recent data has suggested that insulin resistance may affect the ability of skeletal muscle to adapt to an overload stimulus. How insulin resistance may affect the regulation of MLC expression following an overload stimulus is not well understood. PURPOSE: The purpose of the study was to investigate the effect of mechanical overload (MOV) on MLC1S (Slow), MLC1F (Fast), and MLC3 expression in the insulin resistant Obese Zucker (OZ) (Leprfa) rat plantaris. METHODS: Young (2 mo, n = 5) male Lean Zucker (LZ) and young (2 mo, n = 5) male OZ rats underwent unilateral surgical ablation of the gastrocnemius muscle to MOV the fast-twitch plantaris muscle for 8 weeks. A 20 mg proximal section from both the OZ and LZ MOV and Contralateral Control Plantaris (CCP) muscle was analyzed for changes in MLC expression using SDS-PAGE. RESULTS: MOV increased MLC1S expression by 50.65% (p = 0.02) in the LZ plantaris compared to the LZ CCP. There were no significant differences in MLC1F and MLC3 between the LZ MOV and LZ CCP. Obese Zucker MOV and OZ CCP muscle MLC1S, MLC1F, and MLC3 did not differ significantly. Furthermore, there were no significant differences observed between the OZ MOV, LZ MOV, OZ CCP, and LZ CCP muscle MLC1S, MLC1F, and MLC3 isoforms, respectively. CONCLUSION: These results suggest that insulin resistance may affect the ability of skeletal muscle to alter MLC isoform expression in response to MOV.
BACKGROUND: Ischemic studies, a known stimulator of intracellular ROS, have suggested that Heat Shock Protein (HSP) 27 may play a role in cardioprotection. We speculate that age-associated increases in ROS may also increase HSPs and that this may be a compensatory mechanism to circumvent the augmented ROS seen in the aging heart. PURPOSE: Here we determine the influence of aging on HSPs in the heart of adult (6-month), aged (30-month) and very aged (36-month) Fischer 344/NNiaHSd X Brown Norway/BiNia (F344/NXBN) rats. METHODS: Immunoblotting and immunohistochemical analyses were performed on hearts samples to examine the expression of HSP and to determine changes in (-O2·) expression and localization. RESULTS: Compared to adults, increases in (-O2·) as determined by oxidation of hydroethidine to ethidium (Et) were increased 168.6 ± 5.2% in 36-month heart and this finding was highly correlated with increases in left ventricular wall thickness (r = 0.669; P < 0.01) and total protein nitration (r = 0.663, P < 0.01). Compared to 6-month animals, cardiac HSP27 content was 76.6 ± 14.0% and 82.5 ± 17.4% higher in the 30- and 36-month hearts, respectively. Similarly, the content of HSP70 in the heart was 47.5 ± 6.1% and 90.2 ± 3.1% higher in the 30- and 36-month hearts. Regression analysis showed that increases in cardiac (-O2·) with aging were significantly correlated with changes in the cardiac HSP content. CONCLUSION: These results suggest that HSP regulation in the aging F344/NXBN heart may be potentiated by increase in ROS. Further, HSPs may be play a pivotal role in unraveling the molecular events that lead to age-associated alterations in cardiac oxidative stress. (Supported by funds from NSF Grant 0314742)
PURPOSE: Bed rest, microgravity, prolonged disuse and hindlimb suspension (HLS) cause debilitating alterations in skeletal muscle function including changes in protein metabolism, fiber type composition and atrophy. Here we examined the efficacy of histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) in preventing muscle atrophy and muscle fiber type composition. METHODS: Male Fischer 344/NNiaHSD X Brown Norway / BiNia (F344XBN) rats were divided into 4 groups: Control, Control+TSA, HLS and HLS+TSA. Rats in Control+TSA and HLS+TSA groups underwent 14-day TSA treatment (500ug/kg/day-s/c). Control and HLS rats did not receive any treatment. Immunoblot analysis was used to examine the alterations in expression of total- and phospho-Nf-κB-p65, phospho-Akt (Ser308 and Ser473). RESULTS: The expression levels of Nf-κB-p65 and phospho-Nf-κB-p65 were 75±4% and 685±47% of Control in 14-day HLS group, respectively, while those in 14-day HLS+TSA group were 101±1 % and 285±8% of Control, respectively. The expression of phospho-Akt(Ser308) and phospho-Akt (Ser473) was 245±6% and 90±17% of Control in 14-day HLS group, respectively, while those in 14-day HLS+TSA group were 307±6% and 136±20% of Control, respectively. CONCLUSIONS: TSA appears to reverse the elevated expression of phospho-Nf-κB-p65, which is a key regulator of muscle atrophy during HLS with no significant effect on phospho-Akt expression. This suggests that TSA may attenuate disuseinduced muscle atrophy via inhibition of pro-atrophy Nf-κB signaling, without significant effect on the pro-growth Akt signaling. Supported by funds from NSF-EPSCoR Grant 0314742 to MU, WV-NASA, and NIH Grant 027103-1 to EB
Background: Age associated cardiovascular disease is thought to be caused in part by the gradual oxidative damage to biomolecules. We have previously reported that aging in the Fisher344 X Brown Norway (FBN) rat aorta is characterized by increased levels of ROS and alterations in oxidative stress induced cell signaling. Acetaminophen was found to scavenge free radicals in recent ischemia-reperfusion studies. It remains unknown if chronic acetaminophen administration influences ROS signaling in the aging inferior vena cava (IVC). PURPOSE: Here we examined if chronic treatment with a therapeutic dose of acetaminophen attenuates age-associated alterations in ROS-related signaling in the IVC. METHODS: FBN rats (27 month; n=8) were subjected to 6 months of treatment with a therapeutic dose of acetaminophen (30mg/kg/day), with age-matched untreated FBN rats as controls. Immunoblotting analysis was used to examine the expression and activation of signaling molecules, which were previously shown to be affected by oxidative stress in the IVC. RESULTS: Immunoblotting analysis revealed that when compared to IVC from 6 month control animals, the total levels of NFκB p65 in 33 month-old control and treated IVC were 50.43±11.41% and 26.52±7.94% lower, respectively. Corresponding phospho-Erk1/2 levels were 21.51±7.66%, 94.97±17.16% higher and phospho-AMPK levels were 148.94±17.72% and 88.53±27.40% higher, respectively. CONCLUSION: Chronic acetaminophen treatment altered the age associated expression of ROS related signaling molecules in FBN rat IVC, suggesting that acetaminophen may have a protective effect against oxidative stress. (Supported by NIH Grant 027103-1 to EB, NSF-EPSCoR Grant 0314742 to MU, McNeil Pharmaceuticals Grant 991-546-518 to EB and EW)
BACKGROUND: Recent efforts have suggested that actomyosin may be useful for the transport molecular cargo across an inorganic surface. The transportation between any two points is dependent upon three separate, but linked processes: cargo loading, cargo transport and cargo unloading. PURPOSE: Assembly and disassembly of the transport mechanism may be useful for loading and unloading of molecular cargo. Here we investigate the effect of the heavy meromyosin (HMM) concentration upon the size of actin filaments bundled by the actin binding protein fascin. METHODS: Motility assay was performed using HMM bound to nitrocellulosecoated coverslip and actin bundles labeled with rhodamine-phalloidin. Movement of bundles was observed under a fluorescence microscope. Bundle size was analyzed by measuring the fluorescence intensity of rhodamine-labeled bundles. RESULTS: Increasing HMM concentration decreased the fluorescence intensity of bundles: At an actin to fascin ratio of 1.5 to 1, the intensity of moving bundles was reduced by 10, 8 and 28%, respectively, in 120, 150 and 180 ìg/ml HMM, as compared with that at 90 ìg/ml HMM. CONCLUSION: These data suggest that manipulation of HMM concentration can be used to alter bundle size and disassembly. These findings may be useful in designing strategies to control cargo unloading. Supported by NSF-EPSCoR Grant # 0314742 to MU and NIH Grant # 027103-1 to EB
Iron-induced cardiovascular disease is the leading cause of death in iron-overloaded patients. Deferasirox is a novel, once daily oral iron chelator that was recently approved for the treatment of transfusional iron overload. Here, we investigate whether deferasirox is capable of removing cardiac iron and improving iron-induced pathogenesis of the heart using the iron overload gerbil model. Animals were randomly divided into three groups: control, iron overload, and iron overload + deferasirox treatment. Iron-dextran was given 100 mg/kg per 5 days i.p for 10 weeks. Deferasirox treatment was taken post iron loading and was given at 100 mg/kg/day p.o for 1 or 3 months. Cardiac iron concentration was determined by inductively coupled plasma atomic emission spectroscopy. Compared with the untreated group, deferasirox treatment for 1 and 3 months decreased cardiac iron concentration 17.1% (P = 0.159) and 23.5% (P < 0.05), respectively. These treatment-associated reductions in cardiac iron were paralleled by decreases in tissue ferritin expression of 20% and 38% at 1 and 3 months, respectively (P < 0.05). Using oxyblot analysis and hydroethidine fluorescence, we showed that deferasirox significantly reduces cardiac protein oxidation and superoxide abundance by 36 and 47.1%, respectively (P < 0.05). Iron-induced increase in oxidative stress was also associated with increased phosphorylation of ERK-, p38-, and JNK-mitogen-activated protein kinase (MAPK). Interestingly, deferasirox treatment significantly diminished the phosphorylation of all three MAPK subfamilies. These results suggest that deferasirox may confer a cardioprotective effect against iron induced injury.
The factors that regulate vascular mechanotransduction and how this process may be altered with aging are poorly understood and have not been widely studied. Recent data suggest that increased tissue loading can result in the release of prostaglandin F2 alpha (PGF2alpha) and other reports indicate that aging diminishes the ability of the aged aorta to activate mitogen activated protein kinase (MAPK) signaling in response to increased loading. Using ex vivo incubations, here we investigate whether aging affects the ability of the aorta to induce phosphorylation of extracellular signal-regulated kinase 1/2 (ERK(1/2)-MAPK), p38-MAPK, and Jun N-terminal kinase (JNK-MAPK) activation following stimulation with a PGF2alpha analog, fluprostenol. Compared to aortas from 6-mo animals, the amounts of ERK(1/2)- and p38-MAPK remained unchanged with aging, while the level of JNK-MAPK protein increased by 135% and 100% at 30- and 36-mo, respectively. Aging increased the basal phosphorylation of ERK(1/2) (115% and 47%) and JNK (29% and 69%) (p <0.05) in 30- and 36-mo aortas, while p38 phosphorylation levels remained unaltered. Compared to age-matched controls, fluprostenol induced phosphorylation of ERK(1/2) (310%, 286%, and 554%), p38-MAPK (unchanged, 48%, and 148%), and JNK (78%, 88%, and 95%) in 6-, 30- and 36-mo aortas, respectively. These findings suggest that aging does not affect the ability of the rat aorta to activate ERK(1/2)-, p38-MAPK, and JNK-MAPK phosphorylation in response to PGF2alpha stimulation.
Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s) underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB) is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention.Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-beta), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6-months.These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.
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