Mechanosensitive channels in the mechanical component of the exercise pressor reflex.

The cardiovascular response is appropriately regulated during exercise to meet the metabolic demands of the active muscles. The exercise pressor reflex is a neural feedback mechanism through thin-fiber muscle afferents activated by mechanical and metabolic stimuli in the active skeletal muscles. The mechanical component of this reflex is referred to as skeletal muscle mechanoreflex. Its initial step requires mechanotransduction mediated by mechanosensors, which convert mechanical stimuli into biological signals. Recently, various mechanosensors have been identified, and their contributions to muscle mechanoreflex have been actively investigated. Nevertheless, the mechanosensitive channels responsible for this muscular reflex remain largely unknown. This review discusses progress in our understanding of muscle mechanoreflex under healthy conditions, focusing on mechanosensitive channels.

Effects of Dietary Intake of Medium-chain Triacylglycerols on Energy Restriction-induced Weight Control and Loss of Skeletal Muscle in Rats.

Dietary intake of medium-chain triacylglycerols (MCTs) is known to alleviate obesity. MCTs have also been suggested to beneficially influence protein metabolism. This study evaluated the effects of dietary intake of MCTs on energy restriction-induced weight control and loss of skeletal muscle. Rats were divided into the following groups: 1) AL-LCT group that received the AIN-93G-based control diet containing long-chain triacylglycerols (LCTs) ad libitum, 2) ER-LCT group fed the control diet with 30% energy restriction, and 3) ER-MCT group fed a diet containing MCTs with 30% energy restriction. After the 4-wk dietary treatment, both energy-restricted groups had significantly lower body weight than the AL-LCT group and rats in the ER-MCT group were significantly lighter than those in the ER-LCT group. In contrast, the extent of energy restriction-induced loss of skeletal muscle was not significantly different between the two energy-restricted groups, resulting in an increase in muscle mass relative to body weight in the ER-MCT group. Despite maintaining the lower body weight, dietary intake of MCTs did not further influence signaling pathways involved in protein synthesis or breakdown. These results suggest that intake of MCTs could be a valuable dietary intervention to maintain a lower body weight and increase relative muscle mass without negative effects on skeletal muscle protein metabolism.

Blockade of endogenous insulin receptor signaling in the nucleus tractus solitarius potentiates exercise pressor reflex function in healthy male rats.

Insulin not only regulates glucose and/or lipid metabolism but also modulates brain neural activity. The nucleus tractus solitarius (NTS) is a key central integration site for sensory input from working skeletal muscle and arterial baroreceptors during exercise. Stimulation of the skeletal muscle exercise pressor reflex (EPR), the responses of which are buffered by the arterial baroreflex, leads to compensatory increases in arterial pressure to supply blood to working muscle. Evidence suggests that insulin signaling decreases neuronal excitability in the brain, thus antagonizing insulin receptors (IRs) may increase neuronal excitability. However, the impact of brain insulin signaling on the EPR remains fully undetermined. We hypothesized that antagonism of NTS IRs increases EPR function in normal healthy rodents. In decerebrate rats, stimulation of the EPR via electrically induced muscle contractions increased peak mean arterial pressure (MAP) responses 30 min following NTS microinjections of an IR antagonist (GSK1838705, 100 µM; Pre: Δ16 ± 10 mmHg vs. 30 min: Δ23 ± 13 mmHg, n = 11, p = .004), a finding absent in sino-aortic baroreceptor denervated rats. Intrathecal injections of GSK1838705 did not influence peak MAP responses to mechano- or chemoreflex stimulation of the hindlimb muscle. Immunofluorescence triple overlap analysis following repetitive EPR stimulation increased c-Fos overlap with EPR-sensitive nuclei and IR-positive cells relative to sham operation (p < .001). The results suggest that IR blockade in the NTS potentiates the MAP response to EPR stimulation. In addition, insulin signaling in the NTS may buffer EPR stimulated increases in blood pressure via baroreflex-mediated mechanisms during exercise.

Neural discharge of muscle afferents and pressor response to mechanical stimulation are associated with muscle deformation velocity in rats.

Skeletal muscle reflexes play a crucial role in determining the magnitude of the cardiovascular response to exercise. However, evidence supporting an association between the magnitude of the pressor response and the velocity of muscle deformation has remained to be elucidated. Thus, we investigated the impact of different muscle deformation rates on the neural discharge of muscle afferents and pressor and sympathetic responses in Sprague-Dawley rats. In an ex vivo muscle-nerve preparation, action potentials elicited by sinusoidal mechanical stimuli (137 mN) at different frequencies (0.01, 0.05, 0.1, 0.2, and 0.25 Hz) were recorded in mechanosensitive group III and IV fibers. The afferent response magnitude to sine-wave stimulation significantly varied at different frequencies (ANOVA, P = 0.01). Specifically, as compared with 0.01 Hz (0.83 ± 0.96 spikes/s), the response magnitudes were significantly greater at 0.20 Hz (4.07 ± 5.04 spikes/s, P = 0.031) and 0.25 Hz (4.91 ± 5.30 spikes/s, P = 0.014). In an in vivo decerebrated rat preparation, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch (1 kg) of hindlimb skeletal muscle at different velocities of loading (slow, medium, and fast) were measured. Pressor responses to passive stretch were significantly associated with the velocity of muscle deformation (ANOVA, P < 0.001). The MAP response to fast stretch (Δ 56 ± 12 mmHg) was greater than slow (Δ 33 ± 11 mmHg, P = 0.006) or medium (Δ 30 ± 11 mmHg, P < 0.001) stretch. Likewise, the RSNA response was related to deformation velocity (ANOVA, P = 0.024). These findings suggest that the muscle neural afferent discharge and the cardiovascular response to mechanical stimulation are associated with muscle deformation velocity.

Antagonism of TRPV4 channels partially reduces mechanotransduction in rat skeletal muscle afferents.

Mechanical distortion of working skeletal muscle induces sympathoexcitation via thin fibre afferents, a reflex response known as the skeletal muscle mechanoreflex. However, to date, the receptor ion channels responsible for mechanotransduction in skeletal muscle remain largely undetermined. Transient receptor potential vanilloid 4 (TRPV4) is known to sense mechanical stimuli such as shear stress or osmotic pressure in various organs. It is hypothesized that TRPV4 in thin-fibre primary afferents innervating skeletal muscle is involved in mechanotransduction. Fluorescence immunostaining revealed that 20.1 ± 10.1% of TRPV4 positive neurons were small dorsal root ganglion (DRG) neurons that were DiI-labelled, and among them 9.5 ± 6.1% of TRPV4 co-localized with the C-fibre marker peripherin. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons demonstrated that mechanically activated current amplitude was significantly attenuated after the application of the TRPV4 antagonist HC067047 compared to control (P = 0.004). Such reductions were also observed in single-fibre recordings from a muscle-nerve ex vivo preparation where HC067047 significantly decreased afferent discharge to mechanical stimulation (P = 0.007). Likewise, in an in vivo decerebrate rat preparation, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch of hindlimb muscle were significantly reduced by intra-arterial injection of HC067047 (ΔRSNA: P = 0.019, ΔMAP: P = 0.002). The findings suggest that TRPV4 plays an important role in mechanotransduction contributing to the cardiovascular responses evoked by the skeletal muscle mechanoreflex during exercise. KEY POINTS: Although a mechanical stimulus to skeletal muscle reflexively activates the sympathetic nervous system, the receptors responsible for mechanotransduction in skeletal muscle thin fibre afferents have not been fully identified. Evidence suggests that TRPV4 is a mechanosensitive channel that plays an important role in mechanotransduction within various organs. Immunocytochemical staining demonstrates that TRPV4 is expressed in group IV skeletal muscle afferents. In addition, we show that the TRPV4 antagonist HC067047 decreases the responsiveness of thin fibre afferents to mechanical stimulation at the muscle tissue level as well as at the level of dorsal root ganglion neurons. Moreover, we demonstrate that intra-arterial HC067047 injection attenuates the sympathetic and pressor responses to passive muscle stretch in decerebrate rats. These data suggest that antagonism of TRPV4 attenuates mechanotransduction in skeletal muscle afferents. The present study demonstrates a probable physiological role for TRPV4 in the regulation of mechanical sensation in somatosensory thin fibre muscle afferents.

Dapagliflozin Attenuates Sympathetic and Pressor Responses to Stress in Young Prehypertensive Spontaneously Hypertensive Rats.

BACKGROUND: SGLT2i (sodium-glucose cotransporter 2 inhibitor), a class of anti-diabetic medications, is shown to reduce blood pressure (BP) in hypertensive patients with type 2 diabetes. Mechanisms underlying this action are unknown but SGLT2i-induced sympathoinhibition is thought to play a role. Whether SGLT2i reduces BP and sympathetic nerve activity (SNA) in a nondiabetic prehypertension model is unknown. METHODS: Accordingly, we assessed changes in conscious BP using radiotelemetry and alterations in mean arterial pressure and renal SNA during simulated exercise in nondiabetic spontaneously hypertensive rats during chronic administration of a diet containing dapagliflozin (0.5 mg/kg per day) versus a control diet. RESULTS: We found that dapagliflozin had no effect on fasting blood glucose, insulin, or hemoglobin A1C levels. However, dapagliflozin reduced BP in young (8-week old) spontaneously hypertensive rats as well as attenuated the age-related rise in BP in adult spontaneously hypertensive rat up to 17-weeks of age. The rises in mean arterial pressure and renal SNA during simulated exercise (exercise pressor reflex activation by hindlimb muscle contraction) were significantly reduced after 4 weeks of dapagliflozin (Δmean arterial pressure: 10±7 versus 25±14 mm Hg, Δrenal SNA: 31±17% versus 68±39%, P<0.05). Similarly, rises in mean arterial pressure and renal SNA during mechanoreflex stimulation by passive hindlimb stretching were also attenuated by dapagliflozin. Heart weight was significantly decreased in dapagliflozin compared with the control group. CONCLUSIONS: These data demonstrate a novel role for SGLT2i in reducing resting BP as well as the activity of skeletal muscle reflexes, independent of glycemic control. Our study may have important clinical implications for preventing hypertension and hypertensive heart disease in young prehypertensive individuals.

Effects of a Very High-Carbohydrate Diet and Endurance Exercise Training on Pancreatic Amylase Activity and Intestinal Glucose Transporter Content in Rats.

We previously reported that the combination of a very high-carbohydrate diet and endurance training increased glucose transporter 4 and glycogen concentration in skeletal muscle. However, it remains unclear whether they also affect the digestive and absorptive capacity in the pancreas and small intestine, which are suggested to be rate-limiting steps in the delivery of exogenous carbohydrates to skeletal muscle and muscle glycogen synthesis. Thus, we aimed to evaluate the effects of a very high-carbohydrate diet and endurance training on pancreatic amylase activity and intestinal glucose transporters in rats and to examine the relationship between these adaptations and their influence on muscle glycogen concentration. Male Sprague-Dawley rats (n=29) were fed a high-carbohydrate diet (59% carbohydrate) or a very high-carbohydrate diet (76% carbohydrate) for 4 wk. Half of the rats in each dietary group were subjected to 6-h swimming exercise training (two 3-h sessions separated by 45 min of rest) for 4 wk. Although there was no significant effect of diet or endurance training on sodium-dependent glucose transporter 1 and glucose transporter 2 contents in the intestine, the rats fed a very high-carbohydrate diet in combination with endurance training had substantially higher pancreatic amylase activity and muscle glycogen concentration. Furthermore, there was a positive correlation between pancreatic amylase activity and muscle glycogen concentration (r=0.599, p=0.001). In conclusion, intake of a very high-carbohydrate diet and endurance training synergistically elevated carbohydrate digestive capacity, which partially accounted for the higher muscle glycogen accumulation.

Insulin potentiates the response to capsaicin in dorsal root ganglion neurons in vitro and muscle afferents ex vivo in normal healthy rodents.

Systemic insulin administration evokes sympathoexcitatory actions, but the mechanisms underlying these observations are unknown. We reported that insulin sensitizes the response of thin-fibre primary afferents, as well as the dorsal root ganglion (DRG) that subserves them, to mechanical stimuli. However, little is known about the effects of insulin on primary neuronal responses to chemical stimuli. TRPV1, whose agonist is capsaicin (CAP), is widely expressed on chemically sensitive metaboreceptors and/or nociceptors. The aim of this investigation was to determine the effects of insulin on CAP-activated currents in small DRG neurons and CAP-induced action potentials in thin-fibre muscle afferents of normal healthy rodents. Additionally, we investigated whether insulin potentiates sympathetic nerve activity (SNA) responses to CAP. In whole-cell patch-clamp recordings from cultured mice DRG neurons in vitro, the fold change in CAP-activated current from pre- to post-application of insulin (n = 13) was significantly (P < 0.05) higher than with a vehicle control (n = 14). Similar results were observed in single-fibre recording experiments ex vivo as insulin potentiated CAP-induced action potentials compared to vehicle controls (n = 9 per group, P < 0.05). Furthermore, insulin receptor blockade with GSK1838705 significantly suppressed the insulin-induced augmentation in CAP-activated currents (n = 13) as well as the response magnitude of CAP-induced action potentials (n = 9). Likewise, the renal SNA response to CAP after intramuscular injection of insulin (n = 8) was significantly (P < 0.05) greater compared to vehicle (n = 9). The findings suggest that insulin potentiates TRPV1 responsiveness to CAP at the DRG and muscle tissue levels, possibly contributing to the augmentation in sympathoexcitation during activities such as physical exercise. KEY POINTS: Evidence suggests insulin centrally activates the sympathetic nervous system, and a chemical stimulus to tissues activates the sympathetic nervous system via thin fibre muscle afferents. Insulin is reported to modulate putative chemical-sensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, it is demonstrated that insulin potentiates the responsiveness of thin fibre afferents to capsaicin at muscle tissue levels as well as at the level of dorsal root ganglion neurons. In addition, it is demonstrated that insulin augments the sympathetic nerve activity response to capsaicin in vivo. These data suggest that sympathoexcitation is peripherally mediated via insulin-induced chemical sensitization. The present study proposes a possible physiological role of insulin in the regulation of chemical sensitivity in somatosensory thin fibre muscle afferents.

The Safety of Very-long-term Intake of a Ketogenic Diet Containing Medium-chain Triacylglycerols.

We previously reported that consuming a ketogenic diet containing medium-chain triacylglycerols (MCTs) might be a valuable dietary strategy for endurance athletes. However, the long-term safety of the diet has not been established, and there is a concern that a higher intake of MCTs increases the liver triacylglycerol content. In this study, we found that consuming an MCT-containing ketogenic diet for 24 weeks decreased, rather than increased, the liver triacylglycerol concentration and did not aggravate safety-related blood biomarkers in male Wistar rats. Our results may therefore suggest that the long-term intake of a ketogenic diet containing MCTs may have no deleterious effects on physiological functions.

Effects of Dietary Fat Restriction on Endurance Training-induced Metabolic Adaptations in Rat Skeletal Muscle.

Endurance exercise training enhances muscle fat oxidation while concomitantly reducing carbohydrate (glycogen) utilization during exercise, thereby delaying the onset of fatigue. This study examined the effects of dietary fat restriction on endurance training-induced metabolic adaptations in rat skeletal muscle. Male Sprague-Dawley rats were placed on either a control diet (CON: 19.2% protein, 21.6% fat, and 59.2% carbohydrate as a percentage of total energy) or a fat-restricted diet (FR: 21.5% protein, 2.4% fat, and 76.1% carbohydrate as a percentage of total energy) for 4 wks. Half the rats in each dietary group performed daily 6-h swimming exercise (two 3-h sessions separated by 45 min of rest) on 5 days each wk. Endurance training significantly increased the expression of ß-hydroxyacyl CoA dehydrogenase (ßHAD), a key enzyme of fat oxidation, and pyruvate dehydrogenase kinase 4 (PDK4), an inhibitory regulator of glycolytic flux, in the skeletal muscle of rats fed the CON diet. However, such endurance training-induced increases in muscle ßHAD and PDK4 were partially suppressed by the FR diet, suggesting that a FR diet may diminish the endurance training-induced enhancement of fat oxidation and reduction in glycogen utilization during exercise. We then assessed the muscle glycogen utilization rate during an acute bout of swimming exercise in the trained rats fed either the CON or the FR diet and consequently found that rats fed the FR diet had a significantly higher muscle glycogen utilization rate during exercise compared with rats fed the CON diet. In conclusion, dietary fat restriction may attenuate the endurance training-induced metabolic adaptations in skeletal muscle.

Effects of a Ketogenic Diet Containing Medium-Chain Triglycerides and Endurance Training on Metabolic Enzyme Adaptations in Rat Skeletal Muscle.

Long-term intake of a ketogenic diet enhances utilization of ketone bodies, a particularly energy-efficient substrate, during exercise. However, physiological adaptation to an extremely low-carbohydrate diet has been shown to upregulate pyruvate dehydrogenase kinase 4 (PDK4, a negative regulator of glycolytic flux) content in skeletal muscle, resulting in impaired high-intensity exercise capacity. This study aimed to examine the effects of a long-term ketogenic diet containing medium-chain triglycerides (MCTs) on endurance training-induced adaptations in ketolytic and glycolytic enzymes of rat skeletal muscle. Male Sprague-Dawley rats were placed on either a standard diet (CON), a long-chain triglyceride-containing ketogenic diet (LKD), or an MCT-containing ketogenic diet (MKD). Half the rats in each group performed a 2-h swimming exercise, 5 days a week, for 8 weeks. Endurance training significantly increased 3-oxoacid CoA transferase (OXCT, a ketolytic enzyme) protein content in epitrochlearis muscle tissue, and MKD intake additively enhanced endurance training-induced increases in OXCT protein content. LKD consumption substantially increased muscle PDK4 protein level. However, such PDK4 increases were not observed in the MKD-fed rats. In conclusion, long-term intake of ketogenic diets containing MCTs may additively enhance endurance training-induced increases in ketolytic capacity in skeletal muscle without exerting inhibitory effects on carbohydrate metabolism.

Effects of long-term exercise training for different durations on pancreatic amylase activity and intestinal glucose transporter content in rats.

Long-term endurance training for a relatively short duration (~1 h) is reported to increase pancreatic amylase activity in rats, suggesting that chronic exercise training enhances carbohydrate digestive capacity. However, it remains unknown whether longer exercise training duration results in greater adaptation in the pancreas and small intestine. Thus, this study aimed to examine the effects of long-term endurance training for a longer duration on pancreatic amylase activity and intestinal glucose transporter content in rats. Male Sprague-Dawley rats were subjected to swimming exercise training for 1 h (Ex-1h group) or 6 h (Ex-6h group, two 3-h sessions separated by 1 h of rest) each day, 5 days a week, for 6 weeks. Sedentary rats were used as a control (Con group). Total pancreatic amylase activity in the Ex-6h group was significantly lower than that in the Con and Ex-1h groups immediately after the last training session. After 24 h of recovery, total pancreatic amylase activity was significantly higher in the Ex-1h group (~46%) than in the Con group, and a further increase was observed in the Ex-6h group (~98%). In addition, the Ex-6h group, but not the Ex-1h group, showed significantly greater intestinal sodium-dependent glucose transporter 1 (SGLT1) content compared with the Con group after 24 h of recovery. However, no significant difference was observed in glucose transporter 2 (GLUT2) content among the three groups. In conclusion, chronic endurance exercise training for a longer duration results in larger increases in pancreatic amylase activity and intestinal SGLT1 content in rats.

Effects of topical application of ibudilast for seasonal allergic conjunctivitis in patients wearing soft contact lenses.

PURPOSE: To assess the influence of topical application of ibudilast for seasonal allergic conjunctivitis in patients wearing soft contact lenses (SCLs). MATERIALS AND METHODS: There were 16 SCL wearers (32 eyes) with allergic conjunctivitis due to cedar pollen, who were studied from February to April 2007. Before enrollment, informed consent to participation in this study was obtained from all subjects. A frequent replacement SCL (2 Week Pure) was worn for 2 weeks, and ibudilast was applied topically four times daily during this period. The severity of allergic symptoms and the severity of SCL-related symptoms were assessed by scoring using two questionnaires, and before and after topical application of ibudilast results were compared. The severity of objective ocular findings was also scored and compared in the same way. After the final examination, the SCLs were collected and immersed in physiologic saline. Then morphologic changes and drug adsorption were investigated. RESULTS: Among the allergic symptoms, itching and a dry sensation improved after topical application of ibudilast ophthalmic solution (both P<0.05). Phlyctenular conjunctivitis was noted in one eye after topical application, but there were no significant differences of SCL-related symptoms and objective ocular findings between before and after application. There were also no morphologic changes of the contact lenses, and the ibudilast concentration in the lenses was below the detection limit. CONCLUSIONS: These results suggest that topical application of ibudilast while using 2 Week Pure lenses can improve subjective symptoms without influencing drug adsorption or lens morphology.