Thin Muscle Afferents Research Articles

Role of Piezo channels in group IV muscle afferent fiber mechanosensation in rats

During exercise, skeletal muscle afferentnerves are activated by both mechanical and chemical stimuli within contracting muscle, subsequently contributing to an increase in blood pressure (termed the exercise pressor reflex; EPR). Although metabolically-sensitive receptors mediating the EPR have been described, the mechano-gated channels responsible for mechanosensation in thin muscle afferents (i.e. group III and IV fibers) remain to be elucidated. Piezo channels are known to be mechanically-activated ion channels that play crucial roles in several mechanotransduction processes in various organs. Moreover, Piezo channels are expressed in sensory tissues including dorsal root ganglion associated with skeletal muscle group III and IV afferents. Evidence suggests that antagonism of Piezo channels reduces the pressor response to activation of the EPR in rats. However, whether antagonism of Piezo channels affects muscle afferent discharge during mechanical stimulation remains unknown. PURPOSE The purpose of this study was to determine the impact of GsMTx4, a selective Piezo channel inhibitor, on neuronal responses to mechanical stimulation in group IV muscle afferents. It was hypothesized that GsMTx4 reduces the neural response to mechanical stimulation in sensory afferent fibers innervating skeletal muscle. METHODS Sprague-Dawley rats were used in this experiment (8-12 wks old). Using the ex vivo extensor digitorum longus muscle-nerve preparation, the responsiveness of mechanically activated group IV fibers was assessed by obtaining single-fiber recordings. Action potentials elicited by a mechanical stimulus (0-196 mN ramped stimulus) were recorded before and 20-min after injection of either Krebs-buffer solution (control), gadolinium (a non-specific mechano-gated channel antagonist, 10 mM) or GsMTx4 (250 μM) near the receptive field. RESULTS Conduction velocities of the group IV fibers assessed ranged between 0.32 and 0.77 m/s (average 0.56±0.10 m/s, n=22). The percentage of group IV fibers unable to generate an action potential during mechanical stimulation after injection of Krebs-buffer, Gadolinium and GsMTx4 were 0% (0 out of 6 fibers), 29% (2 out of 7 fibers) and 22% (2 out of 9 fibers), respectively. Fibers whose action potentials were partially attenuated by Krebs-buffer, Gadolinium and GsMTx4 were 50% (3 out of 6 fibers), 29% (2 out of 7 fibers) and 56% (5 out of 9 fibers), respectively. Compared to control, the reduction in neural responsiveness to mechanical stimulation tended to be greater in GsMTx4 (-1±49 vs. -48±38 %, P=0.08) albeit similar to Gadolinium (-71±33%, P=0.32). CONCLUSIONS The data demonstrate, for the first time, that GsMTx4 reduces the neuronal response to mechanical stimulation in group IV muscle afferent fibers of rats. These findings suggest that Piezo channels may significantly contribute to mechanosensation in sensory afferent fibers innervating skeletal muscle.

Alteration of Metabosensitive Afferent Response With Aging: Exercised versus Non-exercised Rats

This study was designed to evaluate the effect of aging on the activity of metabosensitive afferent fibers (thin muscle afferents from group III and IV) and to determine if physical activity performed at old age may influence the afferent discharge. Afferents from tibialis anterior and soleus muscles were recorded on non-exercised rats aged of 3, 6, 12, and 20 months and on animals aged of 12 and 20 months performing a daily incremental treadmill exercise protocol during the last 8 weeks preceding the recordings. Metabosensitive afferent fibers were activated with potassium chloride (KCl) and lactic acid (LA) injections into the blood stream or by muscle electrically-induced fatigue (EIF). Results indicated that aging is associated to a decrease in the magnitude of the responses to chemical injections and EIF. Unfortunately, physical activity did not allow restoring the metabosensitive afferents responses. These results indicate an alteration of the thin afferent fibers with aging and should be taken into account regarding the management of muscle fatigue and potential alterations of exercise pressor reflex (EPR) occurring with aging.

Insulin Potentiates Neuronal Responses to Chemical Stimulation in Thin Muscle Afferents and Dorsal Root Ganglia

The cardiovascular response to physical activity is abnormally exaggerated in patients with type 2 diabetes mellitus (T2D). Recent studies in patients with T2D have suggested that this exaggerated responsiveness is mediated, in part, by the skeletal muscle metaboreflex. However, the mechanisms causing augmentations in muscle metaboreflex function in this disease remain to be elucidated. Chronic hyperinsulinemia associated with peripheral insulin resistance is one of the pathophysiological characteristics of T2D. Evidence suggests that transient receptor potential vanilloid receptors, which contribute to metaboreflex activation, are more responsive to stimuli in the presence of insulin. Given that metaboreflex afferent fibers reside in skeletal muscle, it is suggested that hyperinsulinemia may underlie the skeletal muscle metaboreflex overactivity manifest in T2D. PURPOSE: To examine the impact of insulin on neuronal responses to chemical stimulation in thin muscle afferents and dorsal root ganglia (DRG) of normal healthy rodents. It was hypothesized that insulin potentiates the activity of metabolically sensitive afferent neurons. METHODS: Chemically activated neurons were assessed by single-fiber recordings from rat muscle-nerve preparations in vitro and by whole cell patch-clamp recordings from cultured mice DRG neurons. The magnitude of responses to capsaicin stimulation and the capsaicin activated current were recorded, respectively. RESULTS: Compared to control conditions, thin muscle afferent response magnitude was significantly increased by insulin exposure (0.03±0.03 vs. 0.25±0.02 imp, n=3, P<0.05). In DRG cell culture, total charge transfer by capsaicin activated current was largely augmented by insulin administration (403±159 % changes from control conditions, n=3, P=0.11). CONCLUSIONS: These data demonstrate that thin muscle afferent as well as DRG neuronal responses to capsaicin are augmented by insulin exposure in normal healthy animals. The data support the concept that chronic hyperinsulinemia may potentiate skeletal muscle metaboreflex function in T2D contributing to the abnormal cardiovascular response to exercise characteristic of this disease. Supported by the Lawson & Rogers Lacy Research Fund in Cardiovascular Disease and JSPS KAKENHI JP17K01769

Neural Responses to Mechanical Stimulation in Thin Muscle Afferents and Dorsal Root Ganglia are Sensitized by Insulin

In patients with type 2 diabetes mellitus (T2D), the circulatory responses to physical activity are abnormally exaggerated. Recent studies in patients with T2D have suggested that this exaggerated cardiovascular response is mediated, in part, by the exercise pressor reflex (EPR) which is a primary contributor to autonomic cardiovascular control during exercise. However, the mechanisms underlying this abnormal EPR function in T2D remain unknown. The pathophysiology of T2D is characterized by insulin resistance induced hyperinsulinemia. Further, evidence suggests that insulin increases the sensitivity of transient receptor potential vanilloid receptors localized to muscle afferent neurons that contribute to EPR activation. Importantly, sensitization of muscle receptors associated with the mechanically sensitive component of the EPR (i.e. the muscle mechanoreflex) has been shown to mediate reflex overactivity in other cardiovascular disease states such as heart failure and hypertension. Given this information, it is logical to suggest that excess levels of insulin may potentiate mechanoreflex function in T2D.PURPOSETo examine the effect of insulin on neuronal responses to mechanical stimulation in thin muscle afferents and dorsal root ganglia (DRG) of normal healthy rodents. We hypothesized that insulin sensitizes the activity of mechanically sensitive afferent neurons.METHODSMechanically activated neurons were assessed before (control) and after exposure to insulin 1) by single‐fiber recordings from rat muscle‐nerve preparations in vitro and 2) by whole cell patch‐clamp recordings from cultured mice DRG neurons. The magnitude of the response to mechanical stimulation as well as the mechanical threshold were assessed.RESULTSCompared to control, the mechanical threshold of thin muscle afferents was significantly decreased after insulin administration (58±16 vs. 15±17 mN, n=8, P&lt;0.05) while the response magnitude was not changed (66±22 vs. 68±16 spikes, n=8). In DRG cell culture, mechanically activated current was significantly augmented by insulin (−93 ±12 vs. −190±43 pA, n=16, P&lt;0.05) whereas the mechanical threshold was significantly decreased (4.6±0.4 vs. 2.6±0.3 steps, n=17, P&lt;0.05). Further, after pretreatment of DRG insulin receptors with the inhibitor GSK1838705, insulin failed to augment the mechanically activated current (−91±19 vs. −92±19 pA, n=14) or to reduce the mechanical threshold (3.5 ± 0.4 vs. 3.4 ± 0.5 steps, n=11).CONCLUSIONSThese data demonstrate that thin muscle afferent and DRG neuronal responses to mechanical stimulation are sensitized by the presence of insulin in normal healthy animals. Importantly, the findings provide support for the concept that chronic hyperinsulinemia enhances skeletal muscle mechanoreflex function in T2D contributing to the abnormal cardiovascular response during exercise in this disease.Support or Funding InformationSupported by the Lawson &amp; Rogers Lacy Research Fund in Cardiovascular Disease and JSPS KAKENHI JP17K01769This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endomorphins potentiate acid-sensing ion channel currents and enhance the lactic acid-mediated increase in arterial blood pressure: effects amplified in hindlimb ischaemia.

Chronic limb ischaemia, characterized by inflammatory mediator release and a low extracellular pH, leads to acid-sensing ion channel (ASIC) activation and reflexively increases mean arterial pressure; endomorphin release is also increased under inflammatory conditions. We examined the modulation of ASIC currents by endomorphins in sensory neurons from rats with freely perfused and ligated femoral arteries: peripheral artery disease (PAD) model. Endomorphins potentiated sustained ASIC currents in both groups of dorsal root ganglion neurons, independent of mu opioid receptor stimulation or G protein activation. Intra-arterial administration of lactic acid (to simulate exercising muscle and evoke a pressor reflex), endomorphin-2 and naloxone resulted in a significantly greater pressor response than lactic acid alone, while administration of APETx2 inhibited endomorphin's enhancing effect in both groups. These results suggest a novel role for endomorphins in modulating ASIC function to effect lactic acid-mediated reflex increase in arterial pressure in patients with PAD. Chronic muscle ischaemia leads to accumulation of lactic acid and other inflammatory mediators with a subsequent drop in interstitial pH. Acid-sensing ion channels (ASICs), expressed in thin muscle afferents, sense the decrease in pH and evoke a pressor reflex known to increase mean arterial pressure. The naturally occurring endomorphins are also released by primary afferents under ischaemic conditions. We examined whether high affinity mu opioid receptor (MOR) agonists, endomorphin-1 (E-1) and -2 (E-2), modulate ASIC currents and the lactic acid-mediated pressor reflex. In rat dorsal root ganglion (DRG) neurons, exposure to E-2 in acidic solutions significantly potentiated ASIC currents when compared to acidic solutions alone. The potentiation was significantly greater in DRG neurons isolated from rats whose femoral arteries were ligated for 72h. Sustained ASIC current potentiation was also observed in neurons pretreated with pertussis toxin, an uncoupler of G proteins and MOR. The endomorphin-mediated potentiation was a result of a leftward shift of the activation curve to higher pH values and a slight shift of the inactivation curve to lower pH values. Intra-arterial co-administration of lactic acid and E-2 led to a significantly greater pressor reflex than lactic acid alone in the presence of naloxone. Finally, E-2 effects were inhibited by pretreatment with the ASIC3 blocker APETx2 and enhanced by pretreatment with the ASIC1a blocker psalmotoxin-1. These findings have uncovered a novel role of endomorphins by which the opioids can enhance the lactic acid-mediated reflex increase in arterial pressure that is MOR stimulation-independent and APETx2-sensitive.

Interaction between cutaneous and muscle afferent activity in polysynaptic reflex pathways: a human experimental study

Interactions between the input from cutaneous and nociceptive muscle afferents in polysynaptic reflex pathways were investigated in man. Interaction was tested by evoking reflexes before, during, and after a period of muscle pain induced by intramuscular injection of hypertonic saline. Muscle pain was induced either in the ankle flexor (tibialis anterior, TA) or in the extensor (soleus, SOL) muscles by injection of 1 ml hypertonic saline. Electrical skin stimulation (1.1×initial reflex threshold) at the dorsum of the foot over the tarsal joint was used to elicit cutaneo-muscular polysynaptic reflexes in the knee flexor semitendinosus (ST). The injected hypertonic saline evoked a robust muscle pain (the subjects made a continuous score of the muscle pain on a 0–10 cm VAS scale, and the mean VAS area was 1229±251 cm×s and lasting 390±30 s). In five of 12 subjects, the infusion of hypertonic saline into TA evoked referred pain to the dorsal aspect of the ankle. A significant inhibition (17±8.2%, P<0.05) of the ST-reflex by pain in SOL was observed. Pain in TA facilitated (92±36%, P<0.05) the short-latency part (50–70 ms post stimulation) of the reflex. The muscle pain did not modulate the perceived sensory intensity of the electrical stimuli. The findings indicate an interaction of input from thin muscle afferents and cutaneous group A-fibre afferents in polysynaptic segmental reflex pathways, which seems to depend on the location of the muscle pain.

Powerful respiratory stimulation by thin muscle afferents

The ventilatory responses to electrical stimulation of phrenic afferents were examined in spontaneously breathing dogs at different levels of sodium pentobarbital anesthesia. High intensity stimulation (activation of all the afferents, including thin fibers) increased ventilation ( V ̇ e). The increase in V ̇ e was comparable to that of breathing 10% CO 2 and was inversely related to anesthesia level. Under light anesthesia, V ̇ e increased to 282±36% of the control value when the phrenic nerve was stimulated at 130 times the twitch threshold ( n=15; P<0.01). The increase in V ̇ e was due to increases in breathing rate (193±19%) and tidal volume (V t) (143±8%). On the other hand, inspiratory time (T i) decreased. Thus, average airflow rate (V t/T i) increased to 204±23%. After administration of 20 and 40% of the initial dose of pentobarbital, V ̇ e response was attenuated to 157±21 and 121±4%, respectively. We conclude that thin muscle afferents are capable of eliciting pronounced ventilatory stimulation. The small responses observed earlier were likely due to depth of anesthesia.

Responses in muscle afferent fibres of slow conduction velocity to contractions and ischaemia in the cat.

The aim of the study was to find out to what extent muscle receptors with slowly conducting afferent fibres (group III and IV) are activated by muscular contractions of moderate force, and what kind of muscle afferents could mediate the pain of ischaemic exercise. In chloralose-anaesthetized cats, the impulse activity of single afferent units from the triceps surae muscle was recorded from dorsal root filaments during muscular contractions with intact blood supply and after occlusion of the muscle artery. Two types of responses were observed to contractions without muscular ischaemia. One was characterized by sudden onset and a graded response amplitude to contractions of increasing force. In most cases stretching the muscle was also an effective stimulus. Units showing this response behaviour were labelled c.s.m (contraction-sensitive with mechanical mechanism of activation). The other response type had a more delayed onset and often outlasted the exercise period; because of the unknown mechanism of activation, units of this kind were labelled c.s.x. The proportion of c.s.m receptors was significantly higher amongst group III than amongst group IV units. During ischaemic contractions of comparable force the c.s.m and c.s.x receptors exhibited an unchanged or a decreased response amplitude. Under these conditions another receptor type (N, for nociceptive) was activated which did not respond to contractions with intact blood supply. Vigorous activations during ischaemic work were only observed in group IV receptors. The majority of the 131 group III and IV units tested did not respond to contractions at all. These contraction-insensitive (c.i.) endings probably comprised different receptor populations (nociceptors, thermoreceptors, low-threshold mechanoreceptors). It is concluded that the various central nervous effects of muscular exercise without ischaemia which are known to be due to raised activity in thin muscle afferents (e.g. cardiopulmonary adjustments, spinal locomotor reflexes) are probably produced by the c.s.m and c.s.x types. The pain of ischaemic contractions is most likely mediated by the N receptors most of which possess non-myelinated afferent fibres.