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We investigated the role played by bradykinin 2 (B2) receptors in the exaggerated exercise pressor reflex in rats with a femoral artery ligated for 72 h to induce simulated peripheral artery disease (PAD). We hypothesized that in decerebrate, unanesthetized rats with a ligated femoral artery, hindlimb arterial injection of HOE-140 (100 ng, B2 receptor antagonist) would reduce the pressor response to 30 s of electrically induced 1 Hz hindlimb skeletal muscle contraction, and 30 s of 1 Hz hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). We hypothesized no effect of HOE-140 in sham-operated “freely perfused” rats. In both freely perfused ( n = 4) and “ligated” ( n = 4) rats, we first confirmed efficacious B2 receptor blockade by demonstrating that HOE-140 injection significantly reduced ( P < 0.05) the peak increase in mean arterial pressure (peak ΔMAP) in response to hindlimb arterial injection of bradykinin. In subsequent experiments, we found that HOE-140 reduced the peak ΔMAP response to muscle contraction in ligated ( n = 14; control: 23 ± 2; HOE-140: 17 ± 2 mmHg; P = 0.03) but not freely perfused rats ( n = 7; control: 17 ± 3; HOE-140: 18 ± 4 mmHg; P = 0.65). Furthermore, HOE-140 had no effect on the peak ΔMAP response to stretch in ligated rats ( n = 14; control: 37 ± 4; HOE-140: 32 ± 5 mmHg; P = 0.13) but reduced the integrated area under the blood pressure signal over the final ∼20 s of the maneuver. The data suggest that B2 receptors contribute to the exaggerated exercise pressor reflex in rats with simulated PAD, and that contribution includes a modest role in the chronic sensitization of the mechanically activated channels/afferents that underlie mechanoreflex activation.

Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fibre muscle afferents, which, in turn, generates reflex increases in sympathetic nerve activity (SNA) and blood pressure (the exercise pressor reflex; EPR). EPR activation in patients and animals with heart failure with reduced ejection fraction (HF-rEF) results in exaggerated increases in SNA and promotes exercise intolerance. In the healthy decerebrate rat, a subtype of acid sensing ion channel (ASIC) on the sensory endings of thin fibre muscle afferents, namely ASIC1a, has been shown to contribute to the metabolically sensitive portion of the EPR (i.e. metaboreflex), but not the mechanically sensitive portion of the EPR (i.e. the mechanoreflex). However, the role played by ASIC1a in evoking the EPR in HF-rEF is unknown. We hypothesized that, in decerebrate, unanaesthetized HF-rEF rats, injection of the ASIC1a antagonist psalmotoxin-1 (PcTx-1; 100 ng) into the hindlimb arterial supply would reduce the reflex increase in renal SNA (RSNA) evoked via 30 s of electrically induced static hindlimb muscle contraction, but not static hindlimb muscle stretch (model of mechanoreflex activation isolated from contraction-induced metabolite-production). We found that PcTx-1 reduced the reflex increase in RSNA evoked in response to muscle contraction (n = 8; mean (SD) ∫ΔRSNA pre: 1343 (588) a.u.; post: 816 (573) a.u.; P = 0.026) and muscle stretch (n = 6; ∫ΔRSNA pre: 688 (583) a.u.; post: 304 (370) a.u.; P = 0.025). Our data suggest that, in HF-rEF rats, ASIC1a contributes to activation of the exercise pressor reflex and that contribution includes a novel role for ASIC1a in mechanosensation that is not present in healthy rats. KEY POINTS: Skeletal muscle contraction results in exaggerated reflex increases in sympathetic nerve activity in heart failure patients compared to healthy counterparts, which likely contributes to increased cardiovascular risk and impaired tolerance for even mild exercise (i.e. activities of daily living) for patients suffering with this condition. Activation of acid sensing ion channel subtype 1a (ASIC1a) on the sensory endings of thin fibre muscle afferents during skeletal muscle contraction contributes to reflex increases in sympathetic nerve activity and blood pressure, at least in healthy subjects. In this study, we demonstrate that ASIC1a on the sensory endings of thin fibre muscle afferents plays a role in both the mechanical and metabolic components of the exercise pressor reflex in male rats with heart failure. The present data identify a novel role for ASIC1a in evoking the exercise pressor reflex in heart failure and may have important clinical implications for heart failure patients.

NEW FINDINGS What is the central question of this study? Do endoperoxide 4 and thromboxane A2 receptors, which are receptors for cyclooxygenase products of arachidonic metabolism, on thin fibre muscle afferents play a role in the chronic mechanoreflex sensitization present in rats with heart failure with reduced ejection fraction (HF-rEF)? What is the main finding and its importance? The data do not support a role for endoperoxide 4 receptors or thromboxane A2 receptors in the chronic mechanoreflex sensitization in HF-rEF rats. ABSTRACT We investigated the role of cyclooxygenase metabolite-associated endoperoxide 4 receptors (EP4-R) and thromboxane A2 receptors (TxA2 -R) on thin fibre muscle afferents in the chronic mechanoreflex sensitization in rats with myocardial infarction-induced heart failure with reduced ejection fraction (HF-rEF). We hypothesized that injection of either the EP4-R antagonist L-161,982 (1 µg) or the TxA2 -R antagonist daltroban (80 µg) into the arterial supply of the hindlimb would reduce the increase in blood pressure and renal sympathetic nerve activity (RSNA) evoked in response to 30 s of static hindlimb skeletal muscle stretch (a model of isolated mechanoreflex activation) in decerebrate, unanaesthetized HF-rEF rats but not sham-operated control rats (SHAM). Ejection fraction was significantly reduced in HF-rEF (45 ± 11%) compared to SHAM (83 ± 6%; P

The mechanoreflex is exaggerated in patients with peripheral artery disease (PAD) and in a rat model of simulated PAD in which a femoral artery is chronically (∼72 h) ligated. We found recently that, in rats with a ligated femoral artery, blockade of thromboxane A2 (TxA2) receptors on the sensory endings of thin fiber muscle afferents reduced the pressor response to 1 Hz repetitive/dynamic hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). Conversely, we found no effect of TxA2 receptor blockade in rats with freely perfused femoral arteries. Here, we extended the isolated mechanoreflex findings in “ligated” rats to experiments evoking dynamic hindlimb skeletal muscle contractions. We also investigated the role played by inositol 1,4,5-trisphosphate (IP3) receptors, receptors associated with intracellular signaling linked to TxA2 receptors, in the exaggerated response to dynamic mechanoreflex and exercise pressor reflex activation in ligated rats. Injection of the TxA2 receptor antagonist daltroban into the arterial supply of the hindlimb reduced the pressor response to 1 Hz dynamic contraction in ligated but not “freely perfused” rats. Moreover, injection of the IP3 receptor antagonist xestospongin C into the arterial supply of the hindlimb reduced the pressor response to 1 Hz dynamic stretch and contraction in ligated but not freely perfused rats. These findings demonstrate that, in rats with a ligated femoral artery, sensory neuron TxA2 receptor and IP3 receptor-mediated signaling contributes to a chronic sensitization of the mechanically activated channels associated with the mechanoreflex and the exercise pressor reflex.

Passive limb movement and limb muscle stretch in humans and animals are common experimental strategies used to investigate activation of the muscle mechanoreflex independent of contraction-induced metabolite production. Cyclooxygenase (COX) metabolites, however, are produced by skeletal muscle stretch in vitro and have been found to impact various models of mechanoreflex activation. Whether COX metabolites influence the decerebrate rat triceps surae muscle stretch mechanoreflex model remains unknown. We examined the effect of rat triceps surae muscle stretch on the interstitial concentration of the COX metabolite prostaglandin E2 (PGE2). Interstitial PGE2 concentration was increased above baseline values by 4 min of both static (38% increase, P = 0.01) and dynamic (56% increase, P < 0.01) triceps surae muscle stretch ( n = 10). The 4-min protocol was required to collect enough microdialysis fluid for PGE2 detection. The finding that skeletal muscle stretch in vivo was capable of producing COX metabolites prompted the hypothesis that intra-arterial administration of the COX inhibitor indomethacin (1 mg/kg) would reduce the pressor and cardioaccelerator responses evoked during 30 s (the duration most commonly used in the rat mechanoreflex model) of static and dynamic rat triceps surae muscle stretch. We found that indomethacin had no effect ( P > 0.05, n = 9) on the pressor or cardioaccelerator response during 30 s of either static or dynamic stretch. We conclude that, despite the possibility of increased COX metabolite concentration, COX metabolites do not activate or sensitize thin-fiber muscle afferents stimulated during 30 s of static or dynamic hindlimb skeletal muscle stretch in healthy rats.

The primary purpose of this investigation was to determine the role played by endoperoxide 4 receptors (EP4-R) and thromboxane A(2) receptors (TxA(2)-R) during isolated dynamic muscle mechanoreflex activation in rats with heart failure with reduced ejection fraction (HF-rEF) and sham-operated healthy controls. We found that injection of the EP4-R antagonist L-161,982 (1μg) into the arterial supply of the hindlimb had no effect on the peak pressor response to dynamic hindlimb muscle stretch in HF-rEF (n=6, peak ΔMAP pre: 27±7; post: 27±4 mmHg; P=0.99) or sham (n=6, peak ΔMAP pre: 15±3; post: 13±3 mmHg; P=0.67) rats. In contrast, injection of the TxA(2)-R antagonist daltroban (80μg) into the arterial supply of the hindlimb reduced the pressor response to dynamic hindlimb muscle stretch in HF-rEF (n=11, peak ΔMAP pre: 28±4; post: 16±2 mmHg; P=0.02) but not sham (n=8, peak ΔMAP pre: 17±3; post: 16±3; P=0.84) rats. Our data suggest that TxA(2)-Rs on thin fibre muscle afferents contribute to the exaggerated mechanoreflex in HF-rEF.

We investigated the role of cyclooxygenase metabolite-associated endoperoxide 4 receptors (EP4-R) and thromboxane A(2) receptors (TxA(2)-R) on thin fiber muscle afferents in the chronic mechanoreflex sensitization in rats with myocardial infarction-induced heart failure with reduced ejection fraction (HF-rEF). We hypothesized that injection of either the EP4-R antagonist L-161,982 (1 μg) or the TxA(2)-R antagonist daltroban (80 μg) into the arterial supply of the hindlimb would reduce the increase in blood pressure and renal sympathetic nerve activity (RSNA) evoked in response to 30 s of static hindlimb skeletal muscle stretch (a model of isolated mechanoreflex activation) in decerebrate, unanesthetized HF-rEF rats but not sham operated control rats (SHAM). Ejection fraction was significantly reduced in HF-rEF (45±11%) compared to SHAM (83±6%; P

The exercise pressor reflex is a feedback autonomic and cardiovascular control mechanism evoked by mechanical and metabolic signals within contracting skeletal muscles. The mechanically sensitive component of the reflex (the mechanoreflex) is exaggerated in patients with peripheral artery disease (PAD) and in a rat model of simulated PAD in which a femoral artery is chronically ligated. Products of cyclooxygenase enzyme activity have been shown to chronically sensitize the mechanoreflex in PAD, but the identity of the muscle afferent receptors that mediate the sensitization is unclear. We hypothesized that injection of the endoperoxide 4 receptor (EP4-R) antagonist L161982 or the thromboxane A(2) receptor (TxA(2)-R) antagonist daltroban into the arterial supply of the hindlimb would reduce the pressor response to repetitive, dynamic hindlimb skeletal muscle stretch (a model of isolated mechanoreflex activation) in rats with a femoral artery that was ligated ~72 h before the experiment but not in rats with freely perfused femoral arteries. We found that EP4-R blockade had no effect on the pressor response (peak Δmean arterial pressure) to stretch in freely perfused (n = 6, pre: 14 ± 2, post: 15 ± 2 mmHg, P = 0.97) or ligated (n = 8, pre: 29 ± 4, post: 29 ± 6 mmHg, P = 0.98) rats. In contrast, TxA(2)-R blockade had no effect on the pressor response to stretch in freely perfused rats (n = 6, pre: 16 ± 3, post: 17 ± 4 mmHg, P = 0.99) but significantly reduced the response in ligated rats (n = 11, pre: 29 ± 4, post: 17 ± 5 mmHg, P < 0.01). We conclude that TxA(2)-Rs contribute to chronic mechanoreflex sensitization in the chronic femoral artery-ligated rat model of simulated PAD. NEW & NOTEWORTHY We demonstrate that thromboxane A(2) receptors, but not endoperoxide 4 receptors, on the sensory endings of thin fiber muscle afferents contribute to the chronic sensitization of the muscle mechanoreflex in rats with a ligated femoral artery (a model of simulated peripheral artery disease). The data may have important implications for our understanding of blood pressure control during exercise in patients with peripheral artery disease.

Background: Recent evidence suggests prostate cancer independent of treatment has atrophic effects on whole heart and left ventricular (LV) masses, associated with reduced endurance exercise capacity. In a pre-clinical model, we tested the hypothesis that high-intensity training could prevent cardiac atrophy with prostate cancer and alter cardiac protein degradation mechanisms. Methods: Dunning R-3327 AT-1 prostate cancer cells (1×10(5)) were injected into the ventral prostate lobe of 5-6 mo immunocompetent Copenhagen rats (n=24). These animals were randomized into two groups, tumor-bearing exercise (TBEX, n=15) or tumor bearing sedentary (TBS, n=9). Five days after surgery, TBEX animals began exercise on a treadmill (25 m/min, 15° incline) for 45-60 min/day for 18±2 days. Pre-surgery (Pre), and post-exercise training (Post) echocardiographic evaluation (Vivid S6, GE Health Care), using the parasternal short axis view, was used to examine ventricle dimensions. Markers of protein degradation (muscle atrophy F-box, Cathepsin B, Cathepsin L) in the left ventricle were semi-quantified via Western Blot. Results: There were no significant differences in tumor mass between groups (TBEX 3.4±0.7, TBS 2.8±0.6 g, P=0.3), or body mass (TBEX 317±5, TBS 333±7 g, P=0.2). Heart-to-body mass ratio was lower in TBS group compared to TBEX (2.3±0.1 vs. 2.5±0.1 mg/g, P

Mechanical and metabolic signals arising during skeletal muscle contraction reflexly increase sympathetic nerve activity and blood pressure (i.e., the exercise pressor reflex). In a rat model of simulated peripheral artery disease in which a femoral artery is chronically (~72 h) ligated, the mechanically sensitive component of the exercise pressor reflex during 1-Hz dynamic contraction is exaggerated compared with that found in normal rats. Whether this is due to an enhanced acute sensitization of mechanoreceptors by metabolites produced during contraction or involves a chronic sensitization of mechanoreceptors is unknown. To investigate this issue, in decerebrate, unanesthetized rats, we tested the hypothesis that the increases in mean arterial blood pressure and renal sympathetic nerve activity during 1-Hz dynamic stretch are larger when evoked from a previously “ligated” hindlimb compared with those evoked from the contralateral “freely perfused” hindlimb. Dynamic stretch provided a mechanical stimulus in the absence of contraction-induced metabolite production that closely replicated the pattern of the mechanical stimulus present during dynamic contraction. We found that the increases in mean arterial blood pressure (freely perfused: 14 ± 1 and ligated: 23 ± 3 mmHg, P = 0.02) and renal sympathetic nerve activity were significantly greater during dynamic stretch of the ligated hindlimb compared with the increases during dynamic stretch of the freely perfused hindlimb. These findings suggest that the exaggerated mechanically sensitive component of the exercise pressor reflex found during dynamic muscle contraction in this rat model of simulated peripheral artery disease involves a chronic sensitizing effect of ligation on muscle mechanoreceptors and cannot be attributed solely to acute contraction-induced metabolite sensitization. NEW & NOTEWORTHY We found that the pressor and sympathetic nerve responses during dynamic stretch were exaggerated in rats with a ligated femoral artery (a model of peripheral artery disease). Our findings provide mechanistic insights into the exaggerated exercise pressor reflex in this model and may have important implications for peripheral artery disease patients.

Mechanical and metabolic stimuli within contracting skeletal muscles reflexly increase sympathetic nervous system activity and blood pressure. That reflex, termed the exercise pressor reflex, is exaggerated in patients with peripheral artery disease (PAD) and in a rat PAD model with a chronically ligated femoral artery. The cyclooxygenase (COX) pathway contributes to the exaggerated pressor response during rhythmic skeletal muscle contractions in patients with PAD, but the specific mechanism(s) of the COX-mediated exaggeration are not known. In decerebrate, unanesthetized rats with a chronically ligated femoral artery (“ligated” rats), we hypothesized that hindlimb arterial injection of the COX inhibitor indomethacin would reduce the pressor response during 1-Hz dynamic hindlimb skeletal muscle stretch; a model of the activation of the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). In ligated rats ( n = 7), indomethacin reduced the pressor response during stretch (control: 30 ± 4; indomethacin: 12 ± 3 mmHg; P < 0.01), whereas there was no effect in rats with “freely perfused” femoral arteries ( n = 6, control: 18 ± 5; indomethacin: 17 ± 5 mmHg; P = 0.87). In ligated rats ( n = 4), systemic indomethacin injection had no effect on the pressor response during stretch. Femoral artery ligation had no effect on skeletal muscle COX protein expression or activity or concentration of the COX metabolite prostaglandin E2. Conversely, femoral artery ligation increased expression of the COX metabolite receptors endoperoxide 4 and thromboxane A2-R in dorsal root ganglia tissue. We conclude that, in ligated rats, the COX pathway sensitizes the peripheral endings of mechanoreflex afferents, which occurs principally as a result of increased expression of COX metabolite receptors. NEW & NOTEWORTHY We demonstrate that the mechanoreflex is sensitized by the cyclooxygenase (COX) pathway within hindlimb skeletal muscles in the rat chronic femoral artery ligation model of simulated peripheral artery disease (PAD). The mechanism of sensitization appears attributable to increased receptors for COX metabolites on sensory neurons and not increased concentration of COX metabolites. Our data may carry important clinical implications for patients with PAD who demonstrate exaggerated increases in blood pressure during exercise compared with healthy counterparts.

Mechanical signals within contracting skeletal muscles contribute to the generation of the exercise pressor reflex; an important autonomic and cardiovascular control mechanism. In decerebrate rats, the mechanically activated channel inhibitor GsMTx4 was found to reduce the pressor response during static hindlimb muscle stretch; a maneuver used to investigate specifically the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). However, the effect was found only during the initial phase of the stretch when muscle length was changing and not during the later phase of stretch when muscle length was relatively constant. We tested the hypothesis that in decerebrate, unanesthetized rats, GsMTx4 would reduce the pressor response throughout the duration of a 30 sec, 1 Hz dynamic hindlimb muscle stretch protocol that produced repetitive changes in muscle length. We found that the injection of 10 μg of GsMTx4 into the arterial supply of a hindlimb reduced the peak pressor response (control: 15 ± 4, GsMTx4: 5 ± 2 mmHg, P

The primary purpose of this investigation was to determine the role played by endoperoxide 4 receptors (EP4-R) and thromboxane A2 receptors (TxA2-R) during isolated dynamic muscle mechanoreflex activation in rats with heart failure with reduced ejection fraction (HF-rEF) and sham-operated healthy controls. We found that injection of the EP4-R antagonist L-161,982 (1 μg) into the arterial supply of the hindlimb had no effect on the peak pressor response to dynamic hindlimb muscle stretch in HF-rEF (n = 6, peak ∆MAP pre: 27 ± 7; post: 27 ± 4 mm Hg; P = 0.99) or sham (n = 6, peak ∆MAP pre: 15 ± 3; post: 13 ± 3 mm Hg; P = 0.67) rats. In contrast, injection of the TxA2-R antagonist daltroban (80 μg) into the arterial supply of the hindlimb reduced the pressor response to dynamic hindlimb muscle stretch in HF-rEF (n = 11, peak ∆MAP pre: 28 ± 4; post: 16 ± 2 mm Hg; P = 0.02) but not sham (n = 8, peak ∆MAP pre: 17 ± 3; post: 16 ± 3; P = 0.84) rats. Our data suggest that TxA2-Rs on thin fibre muscle afferents contribute to the exaggerated mechanoreflex in HF-rEF.

We investigated the effects of chronic (∼7 weeks) treatment with the angiotensin converting enzyme (ACE) inhibitor Captopril in rats with heart failure with reduced ejection fraction (HF-rEF) on brain blood flow (BF; radiolabeled microspheres) at rest and during submaximal exercise. We hypothesized that middle cerebral, posterior cerebral, and cerebellar BF during submaximal exercise (20 m/min, 5% incline) would be reduced in rats with HF-rEF (n = 10) compared to healthy (SHAM, n = 10) controls and HF-rEF rats chronically treated with Captopril (HF-rEF + Cap., n = 20). During submaximal exercise middle cerebral (HF-rEF + Cap.: 274 ± 12; HF-rEF: 234 ± 23; SHAM: 248 ± 24 ml/min/100 g) and cerebellar (HF-rEF + Cap.: 222 ± 14; HF-rEF: 243 ± 22; SHAM: 214 ± 23 ml/min/100 g) BF increased from rest in all groups with no difference among groups (P > 0.24). Posterior cerebral BF increased from rest in all groups but was lower than SHAM (394 ± 46 ml/min/100 g; P = 0.03) in HF-rEF (298 ± 19 ml/min/100 g) but not HF-rEF + Cap. (356 ± 18 ml/min/100 g; P = 0.14), supporting the concept that ACE inhibition in HF-rEF elevates brain BF increases, at least to the posterior cerebral region, during moderate intensity exercise/physical activity.

We investigated the effect of type 2 diabetes mellitus (T2DM) on respiratory muscle blood flow (BF) during exercise. Using the Goto-Kakizaki (GK) rat model of T2DM, we hypothesized that diaphragm, intercostal and transverse abdominis BFs (radiolabeled microspheres) would be higher in male GK rats (n = 10) compared to healthy male Wistar controls (CON; n = 8) during submaximal exercise (20 m/min, 10 % grade). Blood glucose was significantly higher in GK (246 ± 29 mg/dL) compared to CON (103 ± 4 mg/dL; P0.01). Respiratory muscle BFs were not different at rest (P0.50). From rest to submaximal exercise, respiratory muscle BFs increased in both groups to all muscles (P0.01). During submaximal exercise GK rats had higher diaphragm BFs (GK: 189 ± 13; CON: 138 ± 14 mL/min/100 g, P0.01), and vascular conductance (GK: 1.4 ± 0.1; CON: 1.0 ± 0.1 mL/min/mmHg/100 g; P0.01) compared to CON. There were no differences in intercostal or transverse abdominis BF or VC during exercise (P0.15). These findings suggest that submaximal exercise requires a higher diaphragm BF and VC in T2DM compared to healthy counterparts.