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The Journal of Physiology logoLink to The Journal of Physiology
. 2016 Jan 5;594(3):641–655. doi: 10.1113/JP271714

The mechano‐gated channel inhibitor GsMTx4 reduces the exercise pressor reflex in decerebrate rats

Steven W Copp 1,✉, Joyce S Kim 1, Victor Ruiz‐Velasco 2, Marc P Kaufman 1
PMCID: PMC4930067  PMID: 26608396

Abstract

Key points

  • Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano‐gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex.

  • We determined the effect of GsMTx4, an inhibitor of mechano‐gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats.

  • GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically‐sensitive thin fibre muscle afferents.

  • Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia.

  • Our findings suggest that mechanically‐sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats.

Abstract

Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano‐gated channels and exerts multiple off‐target effects. We tested the hypothesis that GsMTX4, a more selective mechano‐gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano‐gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24  ±  5, GsMTx4: 12 ± 5 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to tendon stretch, a purely mechanical stimulus, but had no effect on the pressor responses to intra‐arterial injection of α,β‐methylene ATP or lactic acid. Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 2, GsMTx4: 14 ± 3 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to electrically‐induced intermittent hindlimb muscle contractions. By contrast, injection of 10 μg of GsMTx4 into the jugular vein had no effect on the pressor, cardioaccelerator, or renal sympathetic nerve responses to contraction. Quantitative RT‐PCR and western blot analyses indicated that both Piezo1 and Piezo2 channel isoforms were natively expressed in rat dorsal root ganglia tissue. We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano‐gated Piezo channels.

Key points

  • Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano‐gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex.

  • We determined the effect of GsMTx4, an inhibitor of mechano‐gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats.

  • GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically‐sensitive thin fibre muscle afferents.

  • Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia.

  • Our findings suggest that mechanically‐sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats.

Abbreviations

DRG

dorsal root ganglia

HR

heart rate

MAP

mean arterial pressure

NaV channel

voltage‐gated sodium channel

qRT‐PCR

quantitative RT‐PCR

RSNA

renal sympathetic nerve activity

TTI

tension‐time index

Introduction

Exercise elicits multiple cardiovascular and autonomic adjustments that include increases in blood pressure, heart rate and sympathetic nervous system activity (Rowell, 1993). The exercise pressor reflex (Mitchell et al. 1983) is a feedback mechanism that contributes importantly to the cardiovascular and autonomic adjustments to exercise in both humans (Strange et al. 1993; Amann et al. 2010) and animals (Coote et al. 1971; McCloskey & Mitchell, 1972; Mitchell et al. 1977; Stebbins et al. 1988; O'Leary & Sheriff, 1995). The afferent arm of the reflex is comprised of thinly myelinated group III and unmyelinated group IV afferents, whose sensory endings are located within the interstitium of the contracting muscles and are stimulated primarily by mechanical and metabolic stimuli, respectively (McCloskey & Mitchell, 1972; Kaufman et al. 1983; Kaufman & Rybicki, 1987; Adreani et al. 1997). Activation of the exercise pressor reflex contributes to the increases in contracting skeletal muscle perfusion during exercise (O'Leary & Sheriff, 1995; O'Leary et al. 1999; Amann et al. 2011).

The exercise pressor reflex is evoked by both metabolic (Alam & Smirk, 1937; McCloskey & Mitchell, 1972; O'Leary et al. 1999; Augustyniak et al. 2001) and mechanical stimuli (Hollander & Bouman, 1975; Stebbins et al. 1988; Victor et al. 1989; Hayes & Kaufman, 2001; Middlekauff & Chiu, 2004). The proteins comprising the metabolically‐sensitive receptors that play a role in evoking the exercise pressor reflex include acid sensing ion channel 3 (ASIC3) (Hayes et al. 2008), purinergic 2X (P2X) (Kindig et al. 2007; Greaney et al. 2014; Stone et al. 2014), bradykinin B2 (Pan et al. 1993; Leal et al. 2013) and EP4 (Yamauchi et al. 2013) receptors. By contrast to the proteins comprising the metabolically‐sensitive receptors, the proteins comprising the mechanically‐sensitive receptors that play a role in evoking the exercise pressor reflex are unknown.

In cats and rats, the role played by the mechanoreflex has been investigated primarily by use of the mechano‐gated channel inhibitor gadolinium (Hayes & Kaufman, 2001; Smith et al. 2005; Hayes et al. 2009; Mizuno et al. 2011). Gadolinium is a trivalent lanthanide that inhibits multiple classes of mechano‐gated channels, including TREK‐1, TRAAK (Maingret et al. 2000) and Piezo (Coste et al. 2010). Gadolinium has also been shown to block voltage‐gated L‐ T‐ and N‐type Ca2+ channels (Biagi & Enyeart, 1990; Boland et al. 1991; Lacampagne et al. 1994), as well as voltage‐gated sodium channels (NaV) and voltage‐independent leak channels (Elinder & Arhem, 1994; Hamill & McBride, 1996). Therefore, studies using gadolinium to investigate the mechanically‐sensitive component of the exercise pressor reflex may be confounded by the multiple non‐selective targets of gadolinium (Hamill & McBride, 1996). In addition, the mechanism of action of gadolinium is not clear and may depend on its concentration (Hamill & McBride, 1996). Consequently, the use of gadolinium provides no information about the identity of the protein comprising the channel(s) responsible for evoking the mechanical component of the exercise pressor reflex.

The toxin of the tarantula spider Grammostola spatulata, GsMTx4, has also been shown to inhibit mechano‐gated cation channels (Suchyna et al. 2000; Bae et al. 2011; Gottlieb & Sachs, 2012; Lee et al. 2014; Cahalan et al. 2015; Jin et al. 2015). Park et al. (2008) found, for example, that i.p. injection of recombinant GsMTx4 in rats significantly increased the paw withdrawal threshold to mechanical pressure but had no effect on motor function or the behavioural responses of the rats to thermal stimuli. More recently, GsMTx4 was shown to be a particularly potent inhibitor of mechano‐gated Piezo channels. Specifically, GsMTx4 was shown to inhibit both Piezo1 (Bae et al. 2011) and Piezo2 (Sachs, 2014) isoforms, whereas GsMTx4 did not inhibit TREK‐1 (Bae et al. 2011). Thus, GsMTx4 appears to inhibit specific classes of mechano‐gated channels, especially Piezo channels, with greater selectivity than that of gadolinium. Those findings prompted us to determine the effects of GsMTx4 on the exercise pressor reflex. Specifically, we tested the hypothesis that the injection of GsMTx4 into the arterial supply of the hindlimb of decerebrate rats reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to both tendon stretch and intermittent contractions of the hindlimb muscles.

Methods

Ethical approval

All procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Penn State College of Medicine and were conducted in accordance with the ethical standards mandated by the Journal of Physiology (Drummond et al. 2010; Grundy, 2015). Experiments were performed on adult (∼12–15 weeks old) male Sprague–Dawley rats (n = 33, body weight range 330–502 g; Charles River Laboratories, Malvern, PA, USA). Rats were housed in accredited facilities with food and water provided ad libitum. At the end of each experiment, the decerebrated rats (see below) were killed with an i.v. injection of saturated potassium chloride (>3 ml kg−1) and the chest was opened bilaterally.

Surgical procedures

In vivo experiments were performed in 30 rats. On the day of the experiment, rats were anaesthetized with 4% isoflurane (balance O2). Adequate depth of anaesthesia was confirmed by the absence of toe‐pinch and blink reflexes. The trachea was cannulated and the lungs were mechanically ventilated (Harvard Apparatus, Holliston, MA, USA) with the gaseous anaesthetic until the decerebration was completed (see below). The right jugular vein and both carotid arteries were cannulated with PE‐50 catheters for the injection of fluids and measurement of arterial blood pressure (P23 XL; Statham, Oxnard, CA, USA), respectively. Heart rate (HR) was calculated beat to beat from the arterial pressure pulse with a Gould (Valley View, OH, USA) Biotach. In 19 rats, the left superficial epigastric artery was cannulated with a PE‐8 catheter whose tip was placed near its junction with the femoral artery. In those 19 rats, a reversible snare (2‐0 silk suture) was placed around the left iliac artery and vein (i.e. proximal to the location of the catheter placed in the superficial epigastric artery). For rats in which the effects of GsMTx4 on the responses to hindlimb muscle contraction (n = 19; see below) were tested, the left sciatic nerve was exposed.

In all rats, the left calcaneal bone was severed and the triceps surae (gastrocnemius, soleus and plantaris) muscles were exposed. The severed end of the calcaneal tendon was linked by string to a force transducer (model FT10; Grass Technologies, Warwick, RI, USA), which, in turn, was attached to a rack‐and‐pinion. In all rats, bundles from the left renal sympathetic nerve were exposed using a retroperitoneal approach. The bundles were then glued (Kwik‐Sil; World Precision Instruments, Sarasota, FL, USA) onto a pair of thin stainless steel recording electrodes, which, in turn, were connected to a high impedance probe (Grass model HZP) and amplifier (Grass P511). Multi‐unit signals from the renal sympathetic nerve fibres were filtered at 100 Hz (low frequency) and 1 kHz (high frequency). At the end of each experiment, hexamethonium (20 mg kg−1) was injected into the jugular vein to abolish renal sympathetic nerve activity (RSNA), thereby demonstrating that the activity was postganglionic.

Rats were then placed in a Kopf stereotaxic frame and spinal unit with clamps placed around the pelvis and rostral lumbar vertebrae. Dexamethasone (0.2 mg i.v.) was injected to minimize brainstem oedema. A precollicular decerebration was performed and all neural tissue rostral to the section was aspirated. After the decerebration was completed, anaesthesia was terminated and the lungs were mechanically ventilated with room air. The rats were given at least 60 min to recover and stabilize prior to the initiation of any experimental protocol. Experiments were performed in decerebrate, unanaesthetized rats because anaesthesia has been shown to depress the exercise pressor reflex in this species (Smith et al. 2001). Arterial blood gases and pH were measured periodically throughout the experiment with a blood gas analyser (ABL 80 FLEX; Radiometer, Copenhagen, Denmark) and maintained within normal limits (P aC O2: 35–45 mmHg, PaO2: ∼100 mmHg, pH 7.35–7.45) by adjusting ventilation and/or administering i.v. sodium bicarbonate (8.5%). Core temperature was measured by a rectal probe and maintained at ∼37–38ºC by a heating lamp.

Experimental protocols

In six rats, we compared the pressor, cardioaccelerator and renal sympathetic nerve responses to calcaneal (achilles) tendon stretch, as well as to both α,β‐methylene ATP and lactic acid injection, before and after the injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb. Prior to initiating the protocol, we paralysed the decerebrate rats with pancuronium bromide (1 mg kg−1 i.v.). Triceps surae muscle tension was set at ∼80–100 g and baseline data were collected for 30 s. We then stretched the calcaneal tendon for 30 s by rapidly turning the rack and pinion. After completing the stretch manoeuvre, we waited for arterial pressure, heart rate and RSNA to return to their baseline values. We then tightened the snare placed around the left iliac artery and vein. Next, we injected α,β‐methylene ATP (20 μg kg−1 in 0.2 ml of saline) and lactic acid (0.2 ml of a 24 mm concentration in saline), separately and in random order, into the arterial supply of the left hindlimb via the left superficial epigastric artery catheter. The drug injections were performed as a bolus over ∼2 s and were separated by at least 5 min. Following the initial stretch and drug injections, the snare on the left iliac artery and vein was tightened and 10 μg of GsMTx4 (dissolved in 0.1 ml of saline) was then injected into the arterial supply of the hindlimb via the left superficial epigastric artery catheter. Ten minutes after injection, the snare was released. Tightening the snare in this manner partially trapped GsMTx4 within the arterial supply of the hindlimb. Twenty minutes after releasing the snare (i.e. 30 min after the injection of GsMTx4), the stretch manoeuvre was repeated, followed by the injections of α,β‐methylene ATP and lactic acid.

In an additional group of eight rats, we compared the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent hindlimb muscle contractions before and after the injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb. Baseline triceps surae muscle tension was set as described above and the sciatic nerve was stimulated (40 Hz, 0.01 ms pulse duration, 500 ms train duration, ≤2 × motor threshold) with shielded stimulating electrodes for 30 s. Following recovery from the control contraction (∼10 min), the snare was tightened and 10 μg of GsMTx4 was injected into left superficial epigastric artery catheter. Twenty minutes after releasing the snare (i.e. 30 min after the injection of GsMTx4), the contractions were repeated.

The 10 μg dose of GsMTx4 and timing of subsequent stretch and contraction maneouvres (30 min after GsMTx4 injection) that were used in the present investigation were based on the study of Park et al. (2008) in which rat paw withdrawal thresholds were significantly increased in response to a mechanical stimulus 30 min after the i.p. injection of ∼40 μg of GsMTx4. We used 10 μg of GsMTx4 in the present study because GsMTx4 was injected directly into the arterial supply of the hindlimb.

Control procedures and experiments

In additional groups of rats, we performed the same stretch (n = 5) or contraction (n = 6) protocols as described above, except that 10 ug of GsMTx4 was injected into the jugular vein. We injected the toxin i.v. to determine whether circulation of GsMTx4 to central nervous system sites could explain the reduced pressor, cardioaccelerator and RSNA responses to stretch and intermittent contractions that we observed after injection of GsMTx4 into the arterial supply of the hindlimb (see Results).

In another group of rats (n = 5), we compared the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent hindlimb muscle contractions before and after the injection of 1 μg of GsMTx4 into the arterial supply of the hindlimb. In these experiments, intermittent contractions and the method and timing of GsMTx4 injection were performed as described above.

In all rats in which we tested the effects of injection of GsMTx4 into the arterial supply of the hindlimb (n = 19 total), we injected blue dye at the end of the experiment in the same manner as GsMTx4 was injected, aiming to determine that the toxin had access to the triceps surae muscles.

Subsequent to all of the experiments in which the sciatic nerve was stimulated to induce muscle contractions (n = 19), rats were paralysed with pancuronium bromide (1 mg kg−1 i.v.) and the sciatic nerve was stimulated with the same parameters as those used to induce muscle contraction. This was carried out to ensure that the pressor response to contractions was not the result of electrical activation of the axons of thin fibre afferents in the sciatic nerve. Stimulation of the sciatic nerve after pancuronium bromide injection did not increase blood pressure, HR or RSNA in any of the experiments in the present study.

Quantitative RT‐PCR (qRT‐PCR) and western blotting assays

In this set of experiments, DRG (L4 and L5) and lung samples from three rats were obtained to determine the expression and mRNA levels of Piezo1 and Piezo2. Total RNA and protein were isolated using the NucleoSpin RNA/Protein Kit (Macherey‐Nagel, Bethlehem, PA, USA) in accordance with the manufacturer's instructions and as described previously (Hassan et al. 2014). The synthesis of cDNA was performed with the High Capacity cDNA RT Kit (Thermo Fisher Scientific, Waltham, MA, USA) and qRT‐PCR experiments were carried out with TaqMan Gene Expression assays (Thermo Fisher Scientific) specific for rat Piezo1, Piezo2 and GAPDH (reference gene) on a 7900HT PCR system (Thermo Fisher Scientific). The results were analysed with the comparative threshold method. The mean C t values from triplicate reactions were normalized to the mean C t values obtained for GAPDH. Gene expression differences were determined by calculating the ratio of Piezo1/GAPDH or Piezo2/GAPDH and using lung (Coste et al. 2010) as the tissue calibrator.

Western blot experiments were performed with the Wes System (Protein Simple, San Jose, CA, USA) and tissue samples and reagents were prepared in accordance with the manufacturer's instructions. The protein samples (0.2 μg μl−1), primary and secondary antibodies, blocking reagent, wash buffer and chemiluminescent substrate were loaded on to the provided microplate. The anti‐Piezo1 rabbit antibody (Alomone Labs, Jerusalem, Israel) was used at a 1:50 dilution and the anti‐Piezo2 rabbit antibody (a kind gift from Dr Ardem Patapoutian, The Scripps Research Institute, La Jolla, CA, USA) (Woo et al. 2014) was diluted at 1:400. Data analysis was performed with Compass software (ProteinSimple, San Jose, CA, USA) and the images were exported to iDraw software (Indeeo Inc., Palo Alto, CA, USA).

Statistical analysis

All variables were displayed continuously in real‐time with a Spike2 data acquisition system (Cambridge Electronic Design, Cambridge, UK) and stored on a computer hard drive for future off‐line analysis. The original RSNA recordings were integrated and corrected for background noise recorded following hexamethonium administration. Baseline mean arterial pressure (MAP), HR and RSNA values were determined from the 30 s baseline period that preceded either stretch or contraction. The peak pressor and cardioaccelerator responses to stretch and intermittent contraction were calculated as the difference between the peak MAP and HR value, wherever they occurred during the stretch or contractions, and the corresponding baseline MAP and HR. The onset latency of the increase in RSNA in response to contraction in the control condition was taken as the time from the initial rise in the tension trace to the first RSNA burst that was obviously elevated above baseline RSNA. Second‐by‐second time courses of the pressor and RSNA responses were plotted as their change (absolute increase for MAP and relative increase for RSNA) from baseline during the 30 s stretch or contraction periods. For RSNA, second‐by‐second integrated RSNA values were analysed and expressed as the percentage change from baseline. The tension‐time indexes (TTI, in kg·s) for stretch and intermittent contraction were calculated by subtracting the area under the tension signal trace for the 30 s baseline period from the area under the tension signal trace for the 30 s stretch or contractions periods.

All data are expressed as the mean ± SEM. For peak and TTI data, comparisons were performed with paired Student's t tests. For time course data, comparisons were performed with two‐way (condition and time) repeated measures ANOVAs with Holm–Sidak post hoc tests used as indicated from the ANOVA result. P < 0.05 was considered statistically significant.

Results

Effects of GsMTx4 on responses to tendon stretch and drug injections

Stretching the achilles tendon for 30 s markedly increased blood pressure and RSNA and modestly increased HR (Fig. 1). The peak pressor response to stretch occurred within the first 5 s of its onset and MAP remained elevated above baseline levels throughout the duration of the stretch. The stretch‐induced increase in RSNA occurred within the first 2 s of its onset and was followed by a sharp reduction in RSNA back to, and most often transiently below, baseline levels (see RSNA tracing in Fig. 1). The transient reduction of RSNA below baseline levels most probably reflects strong arterial baroreflex‐mediated buffering of RSNA during tendon stretch. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb (n = 6) had no effect on baseline MAP or HR. The injection of GsMTx4 into the arterial supply of the hindlimb, however, significantly reduced the peak pressor and cardioaccelerator responses to tendon stretch (Fig. 1; see also Fig. 2, left column). Second‐by‐second analysis revealed that GsMTx4 significantly reduced the pressor response during the third to fifth seconds and significantly reduced RSNA during the second second of tendon stretch (Fig. 1; see also Fig. 3, left column). There were no differences in the TTI (Fig. 2) or the tension time course (Fig. 3) during tendon stretch between the control and GsMTx4 conditions.

Figure 1.

Figure 1

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Original data from one rat showing a dramatic effect of injecting 10 μg of GsMTx4 into the arterial supply of the hindlimb (i.a.) on arterial blood pressure (ABP) and HR responses and RSNA during the first ～10 s of tendon stretch

The sharp reduction in RSNA following the initial increase in RSNA during tendon stretch probably reflects arterial baroreflex‐mediated buffering of RSNA.

Figure 2.

Figure 2

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Effects of GsMTx4 on peak responses to tendon stretch

Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb (i.a., n = 6) (left) significantly reduced the peak pressor (Δ MAP) and cardioaccelerator (Δ HR) responses to tendon stretch. Injection of GsMTx4 into the jugular vein (i.v., n = 5) (right) had no effect on the peak pressor or cardioaccelerator responses to tendon stretch. The TTIs were not different between conditions for either group of rats. Numbers within mean bars are the corresponding baseline values. Asterisks indicate a statistically significant difference from control.

Figure 3.

Figure 3

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Effects of GsMTx4 on the time course of the responses to tendon stretch

Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb (i.a., n = 6) (left) significantly reduced the pressor response and RSNA at specific time points during tendon stretch. The horizontal black line and/or asterisks indicate a statistically significant difference from control. Injection of GsMTx4 into the jugular vein (i.v., n = 5) had no effect on the pressor response and RSNA during tendon stretch. The tension development was not different between control and GsMTx4 conditions at any time point in either group of rats.

The injection of GsMTx4 into the arterial supply of the hindlimb (n = 6) had no effect on the pressor response to injection of α,β‐methylene ATP or lactic acid (Fig. 4) and these findings are consistent with GsMTx4 not blocking metabolically‐sensitive channels.

Figure 4.

Figure 4

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Effects of GsMTx4 on the pressor responses to drug injections

Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb (i.a., n = 6) had no effect on the peak pressor responses that resulted from the injection of α,β‐methelyne ATP or lactic acid into the arterial supply of the hindlimb.

Effects of GsMTx4 on responses to intermittent contractions

Intermittent muscle contractions markedly increased MAP, HR and RSNA. The increases in MAP and HR persisted throughout the 30 s contraction period. RSNA increased with an onset latency of 170 ± 26 ms and was elevated above baseline for the first few seconds of contraction but then returned to baseline levels for the duration of the contraction period. We also often found that RSNA bursts were synchronized to the rising phase of the tension trace during contractions (Fig. 5). The injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb had no effect on baseline blood pressure, whereas the baseline HR increased slightly following GsMTx4 injection (Fig. 6, left). The injection of GsMTx4 into the arterial supply of the hindlimb significantly reduced the peak pressor and cardioaccelerator responses to intermittent hindlimb muscle contractions. Second‐by‐second analysis revealed that GsMTx4 significantly reduced the pressor response during the fifth to thirtieth seconds and significantly reduced the RSNA response during the first to third seconds of intermittent contractions (Fig. 7, left). The injection of GsMTx4 into the arterial supply of the hindlimb also appeared to reduce the synchronization of RSNA bursts to tension development during muscle contractions (Fig. 5). There were no differences in the TTI (Fig. 6, bottom) or the tension time courses (Fig. 7, bottom) between the control and GsMTx4 conditions.

Figure 5. An example of original data from one rat showing the effects of 10 μg of GsMTx4 on the RSNA, blood pressure (BP) and HR .

Figure 5

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A, baseline immediately before contraction in the control condition. B, during intermittent contraction of the triceps surae muscles in the control condition. C, baseline immediately before contraction post GsMTx4. D, during intermittent contraction of the triceps sure muscles post GsMTx4. Data are from the same time points before and during contraction in the control and GsMTx4 conditions. The synchronization between RSNA and the rising phase of the tension (T) trace in the control condition comprises evidence for intermittent contractions providing a primarily mechanical stimulus, which was effectively abolished following GsMTx4. GsMTx4 reduced the peak HR response to contraction from 32 to 26 beats min–1 in this rat.

Figure 6.

Figure 6

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Effects of GsMTx4 on peak responses to intermittent contraction

Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb (i.a., n = 8) (left) significantly reduced the peak pressor and cardioaccelerator responses to intermittent contractions. Injection of 10 μg of GsMTx4 into the jugular vein (i.v., n = 6) (right) had no effect on the peak pressor or cardioaccelerator responses to intermittent contractions. The TTIs were not different between control and GsMTx4 conditions for either group of rats. Numbers within mean bars are the corresponding baseline values. Asterisks indicate a statistically significant difference from control.

Figure 7.

Figure 7

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Effects of GsMTx4 on the time course of the responses to intermittent contraction

Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb (i.a., n = 8) (left) significantly reduced the pressor and RSNA responses at specific time points during intermittent contractions. The injection of GsMTx4 into the jugular vein (i.v., n = 6) (right) had no effect on the pressor and RSNA responses to intermittent contractions. The tension values in both groups represent the average tension over each 1 s period, which is why the plots do not appear to represent intermittent contractions. Inset: RSNA responses for all eight rats in which GsMTx4 was injected i.a., whereas the larger panel shows the responses with one rat removed from the analysis. The data are depicted both ways to demonstrate that the differences between the control and GsMTx4 conditions are not solely a result of one rat that had very large (i.e. > 800% increase above baseline) control RSNA responses. The horizontal black lines and/or asterisks indicate statistically significant differences from the control.

Control experiments

By contrast to intra‐arterial injection, i.v. injection of 10 μg of GsMTx4 had no effect on the peak pressor or cardioaccelerator responses or the time courses of the pressor response and RSNA to either tendon stretch (n = 5) (Figs 2 and 3, right) or intermittent contractions (n = 6) (Figs 6 and 7, right). There were no differences in the TTIs or the tension time courses between the control and GsMTx4 conditions for either group of rats in which GsMTx4 was injected i.v. The results of these i.v. injection experiments indicate that, when GsMTx4 was injected into the arterial supply of the hindlimb, it did not circulate to the brainstem or spinal cord to exert its effect.

The injection of 1 μg of GsMTx4 (n = 5) into the arterial supply of the hindlimb had no effect on the peak pressor (control: 20 ± 1, GsMTx4: 18 ± 5 mmHg, P = 0.54) or cardioaccelerator (control: 18 ± 3, GsMTx4: 13 ± 2 beats min–1, P = 0.07) responses to intermittent contractions. Similarly, injection of 1 μg of GsMTx4 had no effect on the time courses of the pressor and renal sympathetic nerve responses to intermittent contractions (data not shown). There was no difference in the TTI between the control (12 ± 2 kg s) and GsMTx4 (11 ± 1 kg s, P = 0.77 vs. control) conditions.

Piezo1 and Piezo2 mRNA and protein expression

We next measured the expression levels of both Piezo1 and Piezo2 channels in DRG and lung tissue in three rats. Both isoforms were natively expressed in DRG and lung tissue. When compared with lung tissue, however, Piezo1 mRNA was ∼73% lower, whereas Piezo2 mRNA was 36% higher. Western blotting analysis further indicated that Piezo1 protein levels were lower than Piezo2 protein levels (Fig. 8).

Figure 8. Detection of Piezo1 and Piezo2 mRNA and protein expression in rat DRG by qRT‐PCR and western blotting assays, respectively .

Figure 8

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Top: Piezo1 and Piezo2 gene expression quantification in tissue samples from three rats. The difference in gene expression was determined by calculating the ratio of Piezo1/GAPDH or Piezo2/GAPDH in DRG samples relative to that in lung tissue (the calibrator gene). Bottom: western blot for Piezo1 and Piezo2 channels in DRG tissue from the same rats in which mRNA levels were measured. Each lane was loaded with ∼0.2 μg μl−1 protein per lane. Arrows indicate the approximate molecular masses (kDa) of Piezo1 and Piezo2. All images shown were captured at the same exposure.

Discussion

We determined the effects of GsMTx4, a relatively selective mechano‐gated channel inhibitor and particularly potent inhibitor of Piezo channels, on the exercise pressor reflex. Consistent with our hypothesis, we found that injection of GsMTx4 into the arterial supply of the hindlimb reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to both tendon stretch and intermittent contractions of the triceps surae muscles of decerebrate, unanaesthetized rats. By contrast, injection of GsMTx4 into the arterial supply of the hindlimb had no effect on the pressor responses to α,β‐methylene ATP or lactic acid injection, and these findings indicate that GsMTx4 did not affect metabosensitive channels. qRT‐PCR and western blot analyses confirmed the presence of both known Piezo isoforms, namely Piezo1 and Piezo2, in rat DRG tissue. Collectively, our findings indicate that GsMTx4 reduced the exercise pressor reflex and that the reduction was probably attributable, at least in part, to the effects of GsMTx4 on mechanically‐sensitive Piezo channels.

GsMTx4 inhibits mechano‐gated channels by inserting into the lipid bilayer immediately adjacent to channel proteins and forcing them to a closed state (Suchyna et al. 2004). GsMTx4 was found to inhibit mechano‐gated Piezo1 (Bae et al. 2011; Gottlieb & Sachs, 2012; Bae et al. 2013; Miyamoto et al. 2014) and Piezo2 (Sachs, 2014) channels with at least some degree of selectivity because it did not inhibit mechanically‐sensitive TREK‐1 channels (Bae et al. 2011). Both Piezo1 and Piezo2 isoforms (previously known as FAM38a and FAM38b, respectively) are well‐conserved across species (including rodents and humans) and are expressed in a variety of tissues, including DRG tissue (Coste et al. 2010; Delmas et al. 2011; Bagriantsev et al. 2014). The robust mechanically‐sensitive nature of Piezo channels is highlighted by the findings that in vitro Piezo channels opened in response to a light mechanical stimulus, and overexpressing Piezo1 and Piezo2 in multiple cell types increased mechanically‐activated currents by 17‐ to 300‐fold (Coste et al. 2010).

Our finding that both Piezo1 and Piezo2 are natively expressed in rat DRG tissue raises the possibility that both play a role in evoking the mechanically‐sensitive component of the exercise pressor reflex. Nevertheless, because Piezo2 mRNA levels were markedly greater than Piezo1 mRNA levels in rat (present investigation) and mouse (Coste et al. 2010) DRG tissue, the former may play a greater role than the latter in evoking the reflex. In addition, Piezo1 and Piezo2 may have synergistic roles in evoking the exercise pressor reflex. For example, synergistic effects between Piezo1 and Piezo2 were found in articular chondrocytes (Lee et al. 2014) but not in Merkel cells (Ranade et al. 2014). The potential relative and/or synergistic contributions played by Piezo1 and Piezo2, as well as the contributions of other classes of mechano‐gated channels, in evoking the exercise pressor reflex need to be investigated further.

One important consideration of our findings is that GSMTx4 has been found to inhibit several NaV channels. Specifically, Redaelli et al. (2010) reported IC50 values of GsMTx4 for NaV channels, which ranged from 2.6 μm (for NaV 1.7) to 10 μm (for NaV 1.3), raising the possibility that GsMTx4 may have reduced the exercise pressor reflex in the present study by blocking NaV channels. We injected GsMTx4 into the arterial supply of the lower rat hindlimb near the junction of the femoral and popliteal artery and, consequently, knowledge of the precise concentration of GsMTx4 near the sensory endings of the thin fibre afferents innervating the triceps surae muscles is not possible. Nevertheless, assuming that the volume of the rat lower hindlimb is ∼10 ml, the concentration of GsMTx4 in our experiments may have been as low as ∼250 nm, which is ∼10 times less than the IC50 for the NaV 1.7. Importantly, NaV 1.7 channels have been shown to play a role in the axonal transmission of the thin fibre afferents evoking the exercise pressor reflex (Stone et al. 2015 a). We also found that the injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the pressor, cardioaccelerator and sympathetic nerve responses to tendon stretch and muscle contractions but had no effect on the pressor responses to α,β‐methylene ATP or lactic acid injection. If GsMTx4 had blocked NaV channels in the present study, the pressor responses to α,β‐methylene ATP and lactic acid injection would have also been reduced.

We found that the injection of GsMTx4 into the arterial supply of the hindlimb reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to 30 s of sustained tendon stretch, which is a purely mechanical stimulus (Stebbins et al. 1988) that includes a rapid (i.e. ∼2 s) phase of dynamic stretch followed by a sustained phase of static stretch. Specifically, GsMTx4 reduced the pressor response to the dynamic phase of stretch but had no effect on the pressor response to the static phase of stretch. By contrast, Hayes & Kaufman (2001) found, in cats, that gadolinium reduced the pressor response to sustained tendon stretch throughout the duration of the stretch. Our data may therefore indicate that Piezo channels evoke the pressor response to the dynamic phase of tendon stretch, whereas other mechano‐gated channels evoke the pressor response to the static phase of tendon stretch. That conclusion is consistent with the rapid adaptation kinetics of Piezo channels reported by Coste et al. (2010).

We also found that the injection of GsMTx4 into the arterial supply of the hindlimb reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to 30 s of intermittent muscle contractions, which is a mixed mechanical and metabolic stimulus. The synchronization between RSNA and the rising phase of the tension trace during intermittent contractions was first reported in cats (Victor et al. 1989) and indicates that intermittent contractions produced a robust mechanical stimulus with each muscle contraction. Despite the synchronization between RSNA and tension development, average second‐by‐second RSNA returned to baseline levels during the latter part of the contraction period, a finding that has also been reported for the static contraction of rat hindlimb muscles (Koba et al. 2011) and probably reflects arterial baroreflex‐mediated control of RSNA. Nevertheless, GsMTx4 reduced the initial large RSNA bursts at the onset of the contraction period and reduced the synchronization of the RSNA bursts to muscle tension development. Moreover, GsMTx4 reduced the pressor response throughout the contraction period, which is probably attributable to the consistent dynamic mechanical stimuli evoked by intermittent contractions.

The present study has two limitations. First, we did not specifically identify the presence of Piezo1 and Piezo2 channels on group III and/or IV muscle afferents. In mouse DRG tissue, Piezo2 mRNA was expressed in 60% of the neurons that expressed peripherin, which identifies group IV fibres, and 28% of the neurons that expressed neurofilament 200, which identifies group III fibres (Coste et al. 2010). Our recent electrophysiological finding in rats showing that group IV afferents displayed considerable mechanosensitivity (Stone et al. 2015 b) appears to be consistent with the possibility that many of these unmyelinated muscle afferents have Piezo2 channels. Second, we do not know whether Piezo1 and Piezo2 channels are co‐localized with ASIC3 and/or P2X channels on group III and IV afferents.

In conclusion, we have found that injection of GsMTx4 into the arterial supply of the hindlimb reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to stretch and intermittent contractions of the triceps surae muscles of decerebrate rats. The effects of GsMTx4 were probably partly a result of the inhibition of mechano‐gated Piezo1 and Piezo2 channels, although the important contributions of other classes of mechano‐gated channels cannot be discounted. Our findings are the first to shed light on the identity of the proteins comprising the mechano‐gated channels that evoke the exercise pressor reflex. Moreover, our findings may have important implications in conditions such as hypertension (Leal et al. 2008), heart failure (Middlekauff et al. 2001; Middlekauff et al. 2004; Smith et al. 2005) and peripheral arterial disease (Muller et al. 2012; Lu et al. 2013) in which the mechanically‐sensitive component of the exercise pressor reflex has been found to contribute to the exaggerated pressor responses to exercise observed in those pathophysiological states.

Additional information

Competing interests

The authors declare that they have no competing interests.

Author contributions

SWC, VRV and MPK conceived and designed the study. SWC, JSK, VR and MPK acquired, analysed or interpreted the data. SWC, JSK, VR and MPK drafted the work or revised it critically for intellectual content. The experiments were performed in the laboratories of Marc Kaufman and Victor Ruiz‐Velasco. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was supported by National Institutes of Health Grants HL‐096570 and AR‐059397.

Acknowledgements

We are grateful to Dr Ardem Patapoutian of The Scripps Research Institute (La Jolla, CA, USA) for the generous gift of the Piezo2 antibody.

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