Effect of 40 Hz Magnetic Field Application in Posttraumatic Muscular Atrophy Development on Muscle Mass and Contractions in Rats.

Muscle atrophy refers to the deterioration of muscle tissue due to a long-term decrease in muscle function. In the present study, we simulated rectus femoris muscle atrophy experimentally and investigated the effect of pulsed electromagnetic field (PEMF) application on the atrophy development through muscle mass, maximal contraction force, and contraction-relaxation time. A quadriceps tendon rupture with a total tenotomy was created on the rats' hind limbs, inhibiting knee extension for 6 weeks, and this restriction of the movement led to the development of disuse atrophy, while the control group underwent no surgery. The operated and control groups were divided into subgroups according to PEMF application (1.5 mT for 45 days) or no PEMF. All groups were sacrificed after 6 weeks and had their entire rectus femoris removed. To measure the contraction force, the muscles were placed in an organ bath connected to a transducer. As a result of the atrophy, muscle mass and strength were reduced in the operated group, while no muscle mass loss was observed in the operated PEMF group. Furthermore, measurements of single, incomplete and full tetanic contraction force and contraction time (CT) did not change significantly in the operated group that received the PEMF application. The PEMF application prevented atrophy resulting from 6 weeks of immobility, according to the contraction parameters. The effects of PEMF on contraction force and CT provide a basis for further studies in which PEMF is investigated as a noninvasive therapy for disuse atrophy development. © 2022 Bioelectromagnetics Society.

Pulsed magnetic field maintains vascular homeostasis against H2O2-induced oxidative stress.

Pulsed magnetic fields (PMFs) have significant therapeutic effects on many disorders. However, the effects of PMF on vascular homeostasis remain unclear. Therefore, in the present study, we investigated the role of in vivo PMF in maintaining vascular homeostasis during H2O2-induced oxidative stress. For this purpose, rats were exposed to PMF (40 Hz, 1.5 mT) for 1 h for a period of 30 days, following which their thoracic aortas were excised. H2O2 was exogenously applied to the aortic rings. Constrictions were measured in a tissue bath using an electrophysiological technique. Bcl-2 and endothelial nitric oxide synthase (eNOS) protein levels were determined by Western blotting. We found lesser H2O2-induced vasoconstriction in the PMF group than in the control group in endothelium-intact (E+) rings. As H2O2 also induces apoptosis, after incubation with H2O2 (40 min) to induce early apoptosis, we added KCl and measured KCl-induced contractions. All the groups, endothelium intact or denuded (E-) showed decreased responses; however, we still observed the effect of PMF in the E+ group due to increased endothelial activity. In addition, PMF increased the expression of the eNOS protein, which might be a key target of PMF. Our results suggest that in vivo application of PMF protects vascular responses through endothelium-mediated mechanisms during oxidative stress. Therefore, PMF might play a protective role against vascular diseases.

BK channel openers NS1619 and NS11021 reverse hydrogen peroxide-induced membrane potential changes in skeletal muscle.

Large conductance calcium-activated potassium (BK) channels play a crucial role in the repolarization and after-hyperpolarization phases of the cell membrane. The channel openers are also used in treatment of some diseases, including hypo/hyperkalemic periodic paralysis. However, little is known about the effects of BK channels and the channel activators on membrane potentials in skeletal muscle. In addition, the effects of reactive oxygen species (ROS) on BK channels in skeletal muscle are also unknown. Therefore, the aim of this study was to determine the effects of BK channel openers and ROS on membrane potentials in skeletal muscle fibers. For this purpose, resting membrane potentials and action potentials (AP) of frog gastrocnemius muscles were recorded in the presence of commonly used BK channel openers NS1619 and NS11021, H2O2 (a type of ROS), and both using intracellular microelectrode technique. The channel activators significantly and dose-dependently decreased amplitude and increased rise time of AP but did not impact repolarization. The presence of H2O2 plus NS1619 or NS11021 resulted in significant change because the channel openers completely reversed the deleterious effects of hydrogen peroxide on the repolarization phase of AP in skeletal muscle fibers. In the present study, the contributions of BK channel activation and the modulatory role of H2O2 on membrane potentials was demonstrated in skeletal muscle fibers, for the first time. Moreover, it should be noted that BK channel openers should be used in the treatment of reactive oxygen species-induced skeletal muscle diseases.

The effect of very low dose pulsed magnetic waves on cochlea / Efeito de ondas magnéticas pulsadas de dosagem extremamente baixa na cóclea

Abstract Introduction: In daily life biological systems are usually exposed to magnetic field forces at different intensities and frequencies, either directly or indirectly. Despite negative results, the therapeutic use of the low dose magnetic field has been found in recent studies. The effect of magnetic field forces on cochlear cells is not clear in the literature. Objective: In our study, we first applied in vivo pulsed magnetic fields to laboratory rats to investigate the effects on cochlea with distortion product otoacoustic emission test followed by histopathological examinations. Methods: Twelve rats were included in this study, separated into two groups as study group and control group. The rats in the study group were exposed to 40 Hz pulsed magnetic field for 1 h/day for 30 days; the hearing of the rats was controlled by otoacoustic emission test. Also, their cochleas were removed and histochemical examination was performed by Caspase-3, Caspase-9, and TUNEL methods. Results: A statistically significant difference was determined (p < 0.05) when the hearing thresholds of the groups obtained by using 5714 Hz and 8000 Hz stimuli were compared by Kruskal-Wallis test. A significant reaction was observed in the study group, especially in the outer ciliated cells during immunohistochemical examinations by using Caspase-3 and Caspase-9 methods. A significantly positive difference was determined in the study group, especially at the outer ciliated cells and the support cells of the corti organ, when compared to the control group (p < 0.05) by the TUNEL method. Conclusion: According to the results of our study, the very low dose magnetic field, which is considered to be used for therapeutic purposes recently, can cause both auditory function defects and histopathologic damage in cochlear cells.

Resumo Introdução: Os sistemas biológicos são geralmente expostos a forças de campo magnético em diferentes intensidades e frequências, direta ou indiretamente, na vida diária. Apesar dos resultados negativos, o uso terapêutico do campo magnético de baixa dose tem sido encontrado em estudos recentes. O efeito das forças do campo magnético sobre as células cocleares não está claro na literatura. Objetivo: Em nosso estudo, aplicamos pela primeira vez campos magnéticos pulsados in vivo em ratos de laboratório para investigar os efeitos na cóclea através do teste de emissão otoacústica por produto de distorção e análises histopatológicas. Método: Doze ratos foram incluídos neste estudo, os quais foram separados em dois grupos, grupo de estudo e grupo controle. Os ratos do grupo de estudo foram expostos a campo magnético pulsado de 40 Hz por 1 hora/dia por 30 dias, e a audição dos ratos foi controlada por testes de emissão otoacústica. Além disso, suas cócleas foram colhidas e o exame histoquímico foi feito pelos métodos caspase-3, caspase-9 e TUNEL. Resultados: Foi determinada uma diferença estatisticamente significante (p < 0,05) quando os limiares auditivos dos grupos obtidos por meio dos estímulos de 5714 Hz e 8000 Hz foram comparados pelo teste de Kruskal-Wallis. Uma reação significante foi observada no grupo de estudo, especialmente nas células ciliadas externas nas análises imuno-histoquímicas, com os métodos caspase-3 e caspase-9. Uma diferença significantemente positiva foi determinada no grupo de estudo, especialmente nas células ciliadas externas e nas células de suporte do órgão de Corti, quando comparadas com o grupo controle (p < 0,05) pelo método TUNEL. Conclusão: De acordo com os resultados do nosso estudo, o campo magnético de dose baixa, que tem sido considerado para uso terapêutico recentemente, pode causar defeitos na função auditiva e danos histopatológicos nas células cocleares.

The effect of very low dose pulsed magnetic waves on cochlea.

INTRODUCTION: In daily life biological systems are usually exposed to magnetic field forces at different intensities and frequencies, either directly or indirectly. Despite negative results, the therapeutic use of the low dose magnetic field has been found in recent studies. The effect of magnetic field forces on cochlear cells is not clear in the literature. OBJECTIVE: In our study, we first applied in vivo pulsed magnetic fields to laboratory rats to investigate the effects on cochlea with distortion product otoacoustic emission test followed by histopathological examinations. METHODS: Twelve rats were included in this study, separated into two groups as study group and control group. The rats in the study group were exposed to 40Hz pulsed magnetic field for 1h/day for 30 days; the hearing of the rats was controlled by otoacoustic emission test. Also, their cochleas were removed and histochemical examination was performed by Caspase-3, Caspase-9, and TUNEL methods. RESULTS: A statistically significant difference was determined (p<0.05) when the hearing thresholds of the groups obtained by using 5714Hz and 8000Hz stimuli were compared by Kruskal-Wallis test. A significant reaction was observed in the study group, especially in the outer ciliated cells during immunohistochemical examinations by using Caspase-3 and Caspase-9 methods. A significantly positive difference was determined in the study group, especially at the outer ciliated cells and the support cells of the corti organ, when compared to the control group (p<0.05) by the TUNEL method. CONCLUSION: According to the results of our study, the very low dose magnetic field, which is considered to be used for therapeutic purposes recently, can cause both auditory function defects and histopathologic damage in cochlear cells.

The interplay between plasma membrane and endoplasmic reticulum Ca(2+)ATPases in agonist-induced temporal Ca(2+) dynamics.

A change in the intracellular free Ca(2+) concentration ([Ca(2+)]i) functions as a transmitter for signal transduction and shows a broad temporal pattern. Even genetically homogeneous cell types show different Ca(2+) response patterns under permanent agonist stimulation. In Ca(2+) signaling, the dynamics of the Ca(2+) release from the Ca(2+) channels during continuous agonist stimulation and the simultaneous effect of the pumps are unclear. In this study, the dynamic interaction of the Ca(2+) ATPases in the plasma membrane (PMCA) and the endoplasmic reticulum membrane (SERCA) during continuous ACh stimulation is monitored using Fluo-3 and Fura-2 loaded HEK 293 cells. We characterize Ca(2+) release patterns at the sub-maximal and maximal stimulation doses in the absence of extracellular Ca(2+). We analyze the responses regarding their types, oscillation frequency and response times. La(3+) (PMCA blocker) do not change the frequency and time courses in sub-maximal ACh treatment, while with the maximal stimulation oscillation frequency increase as oscillations superimpose on robust release, and response time of [Ca(2+)]i is elongated. A similar effect of La(3+) is observed in quantal Ca(2+) release phenomenon. In the presence of CPA, a SERCA blocker, oscillations are completely abolished, but response time does not change. We also observe that during continuous receptor stimulation, Ca(2+) release do not cease. These data may suggest that Ca(2+) release continues during agonist stimulation, but SERCA and PMCA form a new steady state and return [Ca(2+)]i to its physiological concentration.

Beta-blocker timolol alleviates hyperglycemia-induced cardiac damage via inhibition of endoplasmic reticulum stress.

Current data support that pharmacological modulators of endoplasmic reticulum stress (ERS) have therapeutic potential for diabetic individuals. Therefore, we aimed to examine whether timolol, having free radical-scavenger action, besides being a ß-blocker, exerts a cardioprotective effect via inhibition of ERS response in diabetic rats in a comparison with an antioxidant N-acetylcysteine (NAC). Histopathological data showed that either timolol- or NAC-treatment of diabetic rats prevented the changes in mitochondria and nucleus of the cardiac tissue while they enhanced the cellular redox-state in heart as well. The levels of ER-targeted cytoprotective chaperones GRP78 and calnexin, unfolded protein response signaling protein CHO/Gadd153 besides the levels of calpain, BCL-2, phospho-Akt, PUMA, and PML in the hearts from diabetic rats, treated with either timolol or NAC, are found to be similar among these groups, although all these parameters were markedly preserved in the untreated diabetics compared to those of the controls. Taken into consideration how important a balanced-ratio between anti-apoptotic and pro-apoptotic proteins for the maintenance mitochondria/ER function, our results suggest that ERS in diabetic rat heart is mediated by increased oxidative damage, which in turn triggers cardiac dysfunction. Moreover, we also demonstrated that timolol treatment of diabetic rats, similar to NAC treatment, induced a well-controlled redox-state and apoptosis in cardiac myocardium. We, thus for the first time, report that cardioprotective effect of timolol seems to be associated with normalization of ER function due to its antioxidant action in cardiomyocytes even under hyperglycemia.

Di-peptidyl peptidase-4 inhibitor sitagliptin protects vascular function in metabolic syndrome: possible role of epigenetic regulation.

Metabolic syndrome (MetS) is a complex medical disorder characterized by insulin resistance, hypertension, and high risk of coronary disease and stroke. Microvascular rarefaction and endothelial dysfunction have also been linked with MetS, and recent evidence from clinical studies supports the efficacy of incretin-based antidiabetic therapies for vascular protection in diabetes. Previous studies pointed out the importance of dipeptidyl peptidase-4 (DPP-4) inhibition in endothelial cells due to getting protection against metabolic pathologies. We therefore aimed to investigate the acute effects of a DPP-4 inhibitor, sitagliptin, on vascular function in rats with high-sucrose diet-induced MetS. In order to elucidate the mechanisms implicated in the effects of DPP-4 inhibition, we tested the involvement of NO pathway and epigenetic regulation in the MetS. Acute use of sitagliptin protects the vascular function in the rats with MetS in part due to NO pathway via restoring the depressed aortic relaxation responses mediated by receptors. Application of sitagliptin enhanced the depressed phosphorylation levels of both the endothelial NO synthase and the apoptotic status of protein kinase B, known as Akt, in endothelium-intact thoracic aorta from rats with MetS. One-hour application of sitagliptin on aortic rings from rats with MetS also induced remarkable histon posttranslational modifications such as increased expression of H3K27Me3, but not of H3K27Me2, resulting in an accumulation of the H3K27Me3. Our findings suggest that, in addition to its well-known hypoglycemic action, sitagliptin may also have beneficial effects on hyperglycemia-induced vascular changes in an endotheium-dependent manner. These present results with sitagliptin aside from the glycaemic control, may demonstrate its important role in the treatment of patients with MetS.

Role of ROCK upregulation in endothelial and smooth muscle vascular functions in diabetic rat aorta.

BACKGROUND: The RhoA/ROCK signaling pathway mediates vascular smooth muscle contraction while endogenous NO induces vasodilation through its inhibition. Since myosin light chain phosphatase (MLCP) and eNOS are targeted by RhoA/ROCK upregulation then turn to lead abnormalities in vasculature, we aimed to examine whether less endothelial NO-production and inhibited eNOS together with an upregulation of RhoA/ROCK signaling pathway in thoracic aorta can play an important role in vascular dysfunction under hyperglycemia. METHODS: We used streptozotocin-injected rats, as a model of type 1 diabetes, and their lean controls to investigate the role of ROCK upregulation in the function of toracic aorta by using electrophysiological and biochemical techniques. RESULTS: The protein level of ROCK isoform ROCK2 was found to be 2.5-fold higher in endothelium-intact aortic rings of the diabetic rats compared to those of the controls while its level in endothelium-denuded rings was similar among these two groups. Phosphorylation level of eNOS in endothelium-intact rings from the diabetics was 50% less compared to that of the control. ROCK inhibitors, either Y27632 or HA1077, induced concentration-dependent relaxation with a marked left-shift in phenylephrine pre-contracted endothelium-intact rings from either diabetics or high glucose incubated controls while pretreatment of these rings with L-NAME abolished this shift, fully. Moreover, phosphorylation levels of both MLCP and MLC in endothelium-denuded rings were markedly higher in the diabetics than the controls. CONCLUSION: We demonstrated that diabetes-induced vascular dysfunction can arise due to either inbition of eNOS, thereby less endothelial NO-production, either directly or indirectly, in part, due to an upregulation of ROCK2 by hyperglycemia. Additionally, our data demonstrate that high phosphorylation levels of both MLC and MLCP in endothelium-denuded rings can be due to a less endothelial NO-production dependent ROCK upregulation in the smooth muscle cells under hyperglycemia, as well.

Cell adhesion modulates 5-HT(1D) and P2Y receptor signal trafficking differentially in LTK-8 cells.

In this study, we investigated adhesion-induced changes in cellular responses to serotonin 5-HT(1D) and purinergic P2Y receptor stimulation. We demonstrated that detachment of LTK-8 cells increased 5-HT(1D) receptor-mediated intracellular Ca(2+) and extracellular signal regulated kinase (ERK) phosphorylation responses without affecting the adenylate cyclase response. Additionally, detachment enabled 5-HT(1D) receptor stimulation to inhibit P2Y receptor-induced [Ca(2+)](i) mobilization. Such a cross talk between the two receptor systems was not observed in attached cells. P2Y receptor-induced Ca(2+) response was insensitive to adhesion state of the cells, while ERK phosphorylation response was enhanced upon detachment. Integrity of the actin cytoskeleton did not appear to play a role in adhesion sensitivity of 5-HT(1D)-mediated responses, as treatment of attached cells with cytochalasin D did not mimic detachment-induced effects. Effects of detachment were reversed immediately after re-attachment of the suspended cells on poly-l-lysine coated cover slips, suggesting that the involvement of integrins or focal adhesion complexes is unlikely. Taken collectively, our results demonstrate that not only cellular responses induced by different G protein-coupled receptors, but also different responses induced by a particular G protein-coupled receptor, can be affected differentially by the adhesion status of cells. This suggests an important role for cell adhesion in controlling the coupling of a single G protein-coupled receptor to different intracellular responses.

The role of gender differences in beta-adrenergic receptor responsiveness of diabetic rat heart.

Since the mechanisms responsible for gender differences in cardiac contractile function have not been fully elucidated, we focused to determine the effect of gender difference on beta-adrenergic receptors (beta-ARs) signal transduction in ventricular cardiomyocytes from insulin-dependent diabetic (streptozotocin-induced) rats. Dose-response curves of left ventricular developed pressure (LVDP) to isoproterenol (ISO) in females showed that there was only a approximately 30% decrease in the maximum response without a significant shift in EC50 in diabetic females. On the other hand, diabetes induced a clear rightward shift in the potency (5-10 folds) without a significant change in the maximum response in the males. In order to further determine of the underlying mechanism for this difference, we measured cAMP production and obtained dose-response curves with ISO stimulation in isolated cardiomyocytes. In diabetic females, there was no obvious change in the cAMP dose-response curve. On the other hand, there was a significant decrease in the maximum response without any apparent change in the potency of diabetic males. Our findings indicate that male and female rats are affected differently by diabetes in terms of LVDP responses to beta-ARs stimulation. Also, the difference between their beta-ARs induced cAMP responses may underlie this disparity.