Exaggerated Exercise Pressor Reflex Research Articles

Role of ASIC3 in Evoking the Exercise Pressor Reflex in Type 1 Diabetes Mellitus Rats

Previous studies have suggested that the exercise pressor reflex is exaggerated in individuals with type 1 diabetes mellitus (T1DM); however, the mechanisms responsible for this altered reflex are poorly understood. Acid-sensing ion channel 3 (ASIC3) likely contributes to this exaggeration as previous studies found that peripheral blockade, as well as functional knockout (KO) of the ASIC3 gene, prevented the exaggeration of exercise pressor reflex in rats with cardiovascular-related diseases. However, whether ASIC3 plays a similar role in the exaggerated exercise pressor reflex in T1DM is currently unknown. Therefore, the purpose of this study was to determine whether ASIC3 contributes to the exaggerated exercise pressor reflex in T1DM rats. We hypothesized that the exaggerated exercise pressor reflex would be attenuated in ASIC3 KO T1DM rats compared to their wild-type (WT) counterparts. To test this, we used adult, male and female Wistar-Kyoto ASIC3 KO (n=3; body weight: 348 ± 84 g) and WT counterparts (n=5; body weight: 318 ± 112 g) rats. Streptozotocin (50 mg/kg) was injected intraperitoneally in fasted anesthetized rats to induce T1DM, and a random blood glucose over 300 mg/dl was used to diagnose diabetes. One week later, the exercise pressor reflex was evoked in unanesthetized, decerebrated rats by statically contracting the hindlimb muscles for 30 s. Peak mean arterial pressure (MAP) and heart rate (HR) were calculated as the highest change (Δ) during 30 s contraction baseline. We found that the peak pressor and cardioaccelerator responses to static contraction, although is not statistically significant, appear to be lower in ASIC3 KO (ΔMAP: 17 ± 14 mmHg; ΔHR: 13 ± 12 bpm), compared to WT rats (ΔMAP: 23 ± 11 mmHg; P=0.26 ΔHR:23 ± 9 bpm; P=0.11). Furthermore, we found that within female rats, the peak pressor and heart rate responses were lower in ASIC3 KO (n=1) rat (ΔMAP: 7 mmHg; ΔHR: 15 bpm) compared to WT rats (n=3) (ΔMAP: 27 ± 12 mmHg; ΔHR: 27 ± 11 bpm). Our preliminary findings suggest that ASIC3 may play a role in the exaggerated exercise pressor reflex in T1DM rats and that sex differences may also be present. Further studies involving larger sample sizes will reveal if trends from the current results become significant. This project was supported by NIH R01HL 166323-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

What Is the Role of the Carotid Baroreflex in Exaggerating the Exercise Pressor Reflex in Male UCD-Type 2 Diabetes Mellitus Rats?

Previous studies have shown that exercise evokes an excessive increase in blood pressure in individuals with type 2 diabetes mellitus (T2DM), which is mediated, in part, by an altered exercise pressor reflex. Importantly, in healthy individuals the arterial baroreflex modulates the pressor response to exercise; however, the effects of T2DM on this modulatory effect of the baroreflex remain unclear. Therefore, the purpose of this study was to characterize the arterial baroreflex function in T2DM rats and to determine whether the carotid baroreflex plays a role in exaggerating the exercise pressor reflex. We hypothesized that T2DM rats would present a baroreflex dysfunction that likely contributes to the exaggerated pressor response during muscle contraction. Experiments were performed on adult (8 ± 2 months old), male University of California Davis (UCD)-T2DM ( n=7; body weight: 640 ± 43 g) and healthy Sprague-Dawley rats ( n=3; body weight: 485 ± 29 g). Female rats were not included in this study because they become diabetic when they are much older (>10 months old). Cardiac baroreflex function was characterized in anesthetized T2DM and healthy rats using the modified Oxford technique, which involved sequential bolus infusions of sodium nitroprusside (10 μg/kg) and phenylephrine (10 μg/kg) to induce blood pressure decreases and increases, respectively. Overall cardiac baroreflex gain (i.e., slope of the relationship between systolic blood pressure and R-R interval) was calculated using logistic regression, while gains to blood pressure falling and rising were calculated separately using piecewise and linear regression. The role of the carotid baroreflex in exaggerating the exercise pressor reflex was tested by statically contracting the hindlimb muscles for 30 s after surgical sinoaortic denervation (SAD) in decerebrated T2DM ( n=1; body weight: 555 g) and healthy ( n=1; body weight: 519 g) rats. Carotid barointact T2DM ( n=8; body weight: 521 ± 38 g) and healthy rats ( n=8; body weight: 482 ± 56 g) were included as controls. The pressor responses were calculated as a change (Δ) in peak mean arterial pressure during 30 s contraction relative to baseline. Data are presented as mean ± SD. T2DM rats presented with lower overall baroreflex sensitivity (T2DM: 0.07 ± 0.02 ms/mmHg, healthy: 0.61 ± 0.81 ms/mmHg, p=0.044), as well as sensitivity to increases in blood pressure (T2DM: 0.14 ± 0.10 ms/mmHg , healthy: 0.28 ± 0.15 ms/mmHg, p=0.049) compared to healthy controls. In contrast, cardiac baroreflex gain during decreases in blood pressure were not different between groups (T2DM: 0.16 ± 0.07 ms/mmHg, healthy: 0.10 ± 0.06 ms/mmHg, p=0.109). Notably, SAD did not alter blood pressure response to muscle contraction in the T2DM rat (SAD: Δ32 mmHg vs barointact: Δ32 ± 12 mmHg), but evoked an exaggerated blood pressure response in healthy rat (SAD: Δ34 mmHg vs barointact: Δ12 ± 3 mmHg). Our preliminary findings suggest that despite an attenuated cardiac baroreflex function in T2DM rats, specifically during acute increases in blood pressure, carotid baroreflex dysfunction may not contribute significantly to the exaggerated exercise pressor reflex in diabetes. This project was supported by NIH R01 HL144723. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuro-inflammatory interactions as a mediator of the Exaggerated Exercise Pressor Reflex (EPR) in a mouse model of Peripheral Artery Disease

Introduction and rationale: The Exercise pressor reflex (EPR) is a physiological mechanism in which the contraction of skeletal muscle results in a rise in heart rate (HR) and mean arterial pressure (MAP). In patients with Peripheral Artery Disease (PAD), this reflex is exaggerated, leading to abnormally high elevations in blood pressure during exercise. These cardiovascular responses in patients with PAD have been linked to increased morbidity and mortality, thus limiting the prescription of exercise for these patients. Using a novel murine model of the EPR, our laboratory has demonstrated that mice with femoral artery ligation (a well-established model of PAD), exhibit augmented increases in MAP which is mediated by PIEZO2 positive, mechanosensitive afferent neurons. We have demonstrated that inhibition of PIEZO2 expressing channels normalizes the MAP response to exercise. Patients with PAD also display increased macrophage infiltration in their ischemia-injured limbs, indicating a pro-inflammatory environment. Sensory neurons have been reported to detect various signals produced by immune cells including IL-1β and TNFα. The immune-derived signals can sensitize the peripheral terminals of the sensory neurons, thereby reducing their firing threshold and increasing their responsiveness. Previous studies have also reported the enhancement of Piezo2-mediated mechanosensitive currents in the presence of inflammatory signals. Hypothesis: We, therefore, hypothesize that in mice with femoral artery ligation, muscle inflammation increases the expression and activation of resident ion channels (e.g., Piezo2) in mechanosensitive afferent neurons to mediate the exaggerated EPR in PAD.Study objective: To mechanistically investigate the interaction between muscle inflammation and Piezo2 in mechanosensitive afferent neurons.Methods: We performed sham surgery or ligation of the femoral artery ipsilaterally in C57BL/6 mice. After 72 hours, we harvested the gastrocnemius muscle for histological and molecular analyses.Data and summary and results: Our preliminary findings reveal that there is significantly increased expression of pro-inflammatory genes and infiltrating immune cells in the ischemic muscle when compared to non-ischemic muscle.Specifically, H&E staining revealed the presence and accumulation of inflammatory cells, including macrophages, in the ischemic muscle compared to control, along with observed necrosis. Transcript analysis of the ischemic gastrocnemius muscle showed significantly increased IL-1β and TNFα, two pan-proinflammatory genes that encode cytokines released by macrophages. TNFα is known to induce the synthesis of adhesion molecules (E-selectin and ICAM-1) on endothelial cells, allowing leukocytes to adhere and cross the endothelial wall into the injured tissue. Indeed, we observed significantly increased ICAM-1 and E-selectin transcripts in the ischemic muscle compared to controls. MCP1, a well-established chemokine, was also significantly increased, further emphasizing the recruitment of leukocytes to the ischemic muscle. In addition, inducible NOS ( iNOS), which is known to be expressed in IL-1β- and TNFα-rich proinflammatory environment was also significantly increased in our FAL model compared to Sham. These data establish that the ischemic muscle of a femoral-artery ligated mouse has a high proinflammatory phenotype. We are currently exploring the impact of proinflammatory mediators on Piezo2 channel expression and activity, as well as the effect of macrophage depletion on Piezo2 in afferent neurons. The data from these studies will provide novel insights regarding the influence of neuroimmune interactions on cardiovascular responses to exercise in PAD. These data hold the potential to reveal innovative therapies enabling safe prescription of exercise, thus alleviating the burden of PAD, and improving the quality of life for those affected by this condition. Internal sources. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cyclooxygenase products contribute to the exaggerated exercise pressor reflex evoked by static muscle contraction in male UCD-type 2 diabetes mellitus rats.

Cyclooxygenase (COX) products of arachidonic acid metabolism, specifically prostaglandins, play a role in evoking and transmitting the exercise pressor reflex in health and disease. Individuals with type 2 diabetes mellitus (T2DM) have an exaggerated exercise pressor reflex; however, the mechanisms for this exaggerated reflex are not fully understood. We aimed to determine the role played by COX products in the exaggerated exercise pressor reflex in T2DM rats. The exercise pressor reflex was evoked by static muscle contraction in unanesthetized, decerebrate, male, adult University of California Davis (UCD)-T2DM (n = 8) and healthy Sprague-Dawley (n = 8) rats. Changes (Δ) in peak mean arterial pressure (MAP) and heart rate (HR) during muscle contraction were compared before and after intra-arterial injection of indomethacin (1 mg/kg) into the contracting hindlimb. Data are presented as means ± SD. Inhibition of COX activity attenuated the exaggerated peak MAP (Before: Δ32 ± 13 mmHg and After: Δ18 ± 8 mmHg; P = 0.004) and blood pressor index (BPi) (Before: Δ683 ± 324 mmHg·s and After: Δ361 ± 222 mmHg·s; P = 0.006), but not HR (Before: Δ23 ± 8 beats/min and After Δ19 ± 10 beats/min; P = 0.452) responses to muscle contraction in T2DM rats. In healthy rats, COX activity inhibition did not affect MAP, HR, or BPi responses to muscle contraction. Inhibition of COX activity significantly reduced local production of prostaglandin E2 in T2DM and healthy rats. We conclude that peripheral inhibition of COX activity attenuates the pressor response to muscle contraction in T2DM rats, suggesting that COX products partially contribute to the exaggerated exercise pressor reflex in those with T2DM.NEW & NOTEWORTHY We compared the pressor and cardioaccelerator responses to static muscle contraction before and after inhibition of cyclooxygenase (COX) activity within the contracting hindlimb in decerebrate, unanesthetized type 2 diabetic mellitus (T2DM) and healthy rats. The pressor responses to muscle contraction were attenuated after peripheral inhibition of COX activity in T2DM but not in healthy rats. We concluded that COX products partially contribute to the exaggerated pressor reflex in those with T2DM.

Abstract 12634: Amplified P2x 3 Pathway Activity in IB4 Positive Muscle DRG Neurons and Exercise Pressor Reflex Regulation in Hindlimb Ischemia-Reperfusion

Background: Hindlimb ischemia-reperfusion (IR) represents one of the most prominent pathological conditions observed in peripheral artery disease (PAD). Arterial blood pressure response (BP) during exercise, partly driven by the exaggerated exercise pressor reflex (EPR), is associated with an elevated risk of cardiovascular events in PAD patients. However, the precise underlying mechanisms leading to this exaggerated BP response are poorly elucidated. The purinergic P2X 3 signaling pathway, which plays a key role in modifying the EPR, was the focus of the present study. Objectives: To examine the regulatory role of P2X 3 on the EPR in a rat model of hindlimb IR, which was induced by 6 hours’ ischemia followed by 18 hours’ reperfusion (referred to as IR rats). Results: We found that, in IR rats, there were: 1) upregulation of P2X 3 protein expression in the L4-6 dorsal root ganglion (DRG) (1.48±0.16 in IR rats/n=8 vs. 1.00±0.41 in sham rats/n=9; P<0.05), which was localized in IB4 positive DRG neurons by the method of immunohistochemistry, 2) amplified P2X currents in isolated muscle DRG neurons (P2X transient currents: 1281.64±776.74 pA in IR rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, P<0.05; and sustain currents: 466.94±168.68 pA in IR rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8, P<0.05); 3) amplification of P2X-mediated BP response (29±8 mmHg in IR rats/n=12 vs. 21±4 mmHg in sham rats/n=7; P<0.05); and 4) alleviation of the exaggerated BP response induced by static muscle contraction (18±8 mmHg in IR +A317491/n=8, vs. 29±11 mmHg in IR/n=7, P<0.05) and P2X-mediated BP response by α, β-me-ATP injection (15±4 mmHg in IR+A317491 /n=6 vs. 23±7 mmHg in IR/n=4, P<0.05) following the inhibition of muscle afferents’ P2X 3 receptor by A-317491. We further verified that both A-317491 and siRNA knockdown of P2X 3 significantly decreased the activity of P2X currents in the isolated muscle DRG neurons (all P<0.05 vs. their respective controls). Conclusion: With the above evidence from multiple approaches, we concluded that the P2X 3 signaling pathway activity is amplified following hindlimb IR via the enhancing expression and activity of P2X 3 receptors in IB4 positive muscle DRG afferents. This contributes to the exacerbated EPR responses in PAD.

Abstract P3015: Mechanosensitive, Piezo2 Channels Mediate The Exaggerated Exercise Pressor Reflex In A Mouse Model Of Hind Limb Ischemia.

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to afferent neuron activation by skeletal muscle contraction. Patients with peripheral artery disease (PAD) have an exaggerated EPR, contributing to exercise intolerance, morbidity and mortality. Recently, we developed a mouse model which we used to determine the effect of femoral artery ligation (FAL) on the EPR. We observed that the EPR is exaggerated 72 hrs following FAL in mice. Stimulation of metabolically active afferent neurons was not different between FAL and control. In contrast, the responses induced by static contraction (stimulation of both mechanically and metabolically sensitive afferent fibers) and passive stretch (preferential stimulation of mechanically sensitive afferent neurons) were exaggerated in FAL mice when compared to control. These exaggerated responses were not affected by SB-705498 (a TRPv1 receptor inhibitor) but were nearly abolished by Gadolinium (a mechanical sensitive group III afferent neuron inhibitor) and significantly reduced by GsMTx-4 (a selective inhibitor of the PIEZO2 receptor).. Furthermore, we observed that PIEZO2 expression was upregulated in the dorsal root ganglia (DRG) from FAL mice compared to controls. Importantly, FAL mice displayed a decreased exercise capacity when compared to control mice, verifying that this mouse model mimics human PAD and supporting the notion that an exaggerated EPR contributes to exercise intolerance. These results further indicate that the exaggerated EPR in FAL is mediated, in part, by the PIEZO2 receptor. To explore this, we generated mice with a conditional, DRG targeted, deletion of Piezo2 . In these mice, we observed that the responses to static contraction and passive stretch were significantly reduced compared to control. Moreover, we performed whole-cell voltage-clamp on dissociated DRG observing increased negative pressure induced inward currents in the setting of FAL while these currents were decreased in the Piezo2 -/- DRG. These data indicate that PIEZO2 expression may serve as a therapeutic target in the treatment of PAD.

Exaggerated exercise pressor reflex in male UC Davis type 2 diabetic rats is due to the pathophysiology of the disease and not aging.

Introduction: Studies in humans and animals have found that type 2 diabetes mellitus (T2DM) exaggerates the blood pressure (BP) response to exercise, which increases the risk of adverse cardiovascular events such as heart attack and stroke. T2DM is a chronic disease that, without appropriate management, progresses in severity as individuals grow older. Thus, it is possible that aging may also exaggerate the BP response to exercise. Therefore, the purpose of the current study was to determine the effect of the pathophysiology of T2DM on the exercise pressor reflex independent of aging. Methods: We compared changes in peak pressor (mean arterial pressure; ΔMAP), BP index (ΔBPi), heart rate (ΔHR), and HR index (ΔHRi) responses to static contraction, intermittent contraction, and tendon stretch in UCD-T2DM rats to those of healthy, age-matched Sprague Dawley rats at three different stages of the disease. Results: We found that the ΔMAP, ΔBPi, ΔHR, and ΔHRi responses to static contraction were significantly higher in T2DM rats (ΔMAP: 29 ± 4mmHg; ΔBPi: 588 ± 51mmHg•s; ΔHR: 22 ± 5bpm; ΔHRi: 478 ± 45bpm•s) compared to controls (ΔMAP: 10 ± 1mmHg, p < 0.0001; ΔBPi: 121 ± 19mmHg•s, p < 0.0001; ΔHR: 5 ± 2bpm, p = 0.01; ΔHRi: 92 ± 19bpm•s, p < 0.0001) shortly after diabetes onset. Likewise, the ΔMAP, ΔBPi, and ΔHRi to tendon stretch were significantly higher in T2DM rats (ΔMAP: 33 ± 7mmHg; ΔBPi: 697 ± 70mmHg•s; ΔHRi: 496 ± 51bpm•s) compared to controls (ΔMAP: 12 ± 5mmHg, p = 0.002; ΔBPi: 186 ± 30mmHg•s, p < 0.0001; ΔHRi: 144 ± 33bpm•s, p < 0.0001) shortly after diabetes onset. The ΔBPi and ΔHRi, but not ΔMAP, to intermittent contraction was significantly higher in T2DM rats (ΔBPi: 543 ± 42mmHg•s; ΔHRi: 453 ± 53bpm•s) compared to controls (ΔBPi: 140 ± 16mmHg•s, p < 0.0001; ΔHRi: 108 ± 22bpm•s, p = 0.0002) shortly after diabetes onset. Discussion: Our findings suggest that the exaggerated exercise pressor reflex and mechanoreflex seen in T2DM are due to the pathophysiology of the disease and not aging.

Bradykinin 2 receptors contribute to the exaggerated exercise pressor reflex in a rat model of simulated peripheral artery disease.

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.

Abstract P3134: Mechanosensitive Piezo2 Channel Mediates The Exaggerated Exercise Pressor Reflex In A Mouse Model Of Hind Limb Ischemia.

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to skeletal muscle contraction. These reflexive increases are caused by both mechanical and metabolic stimulation of group III and IV afferent neurons innervating in contracting skeletal muscle. Patients with peripheral artery disease (PAD) have an exaggerated EPR which contributes to exercise intolerance and morbidity and mortality. Recently, we developed a mouse model of EPR, and have validated that the mouse model is similar to our rat model. With this mouse model of EPR, we studied the EPR following femoral artery ligation (FAL), briefly the right hind limb common femoral artery was ligated and transected between the proximal end near the groin and the distal end near the bifurcation of the popliteal artery and the saphenous artery. The same procedure was done without femoral artery ligation and transection for sham control. We observed that the mean arterial pressure (MAP) responses to ventral root stimulation (which excite metabo- and mechano-sensitive afferent neurons) and passive stretch (mechanosensitive stimulation only) were exaggerated 72 hours following FA ligation when compared to sham treated controls. The exaggerated EPR was significantly reduced by intra-arterial administration of Gadolinium (a mechanical sensitive group III afferent neurons inhibitor), GsMTx-4 (a selective Piezo2 inhibitor), but not by SB-705498 (a TRPv1 receptor inhibitor). Additionally, selective stimulation of metabolically sensitive afferent neurons by intra-arterial capsaicin injection (an activator of metabolically sensitive afferent neurons) induced equal, dose-related increases in MAP in both FA ligation and control mice. Finally, immunofluorescence staining visualized that the mechano-sensitive channel Piezo2 is localized in most large diameter neuron cells in mouse lumbar DRG. First, these results indicate that metabolically sensitive afferent neurons are not affected by FAL. Second, we observed that the exaggerated EPR in a murine model of PAD is mediated by mechanically sensitive group III neurons, which is due, in part, to Piezo2 receptor activation. Future studies will be aimed at interrogation of the mechanosensitive mechanisms that mediate this abnormal response to exercise in a model of PAD. The goal of these studies will be to determine molecular targets through which the exaggerated EPR can be normalized to allow for the prescription of exercise in PAD patients.

Exaggerated Activities of P2X3 and ASIC3 Signaling Pathway in Muscle Afferent Following Himblimb Muscle Ischemia‐Reperfusion

BackgroundPeripheral artery disease (PAD) is one of the major cardiovascular concerns and dramatically reduces the blood flow supply in the affected limbs. A combination of revascularization surgery in the femoral artery and supervised exercise intervention is among the most general approaches for the treatment of PAD. The approach of revascularization inevitably induces ischemia‐reperfusion (IR) injury in the tissues with the reperfusion of blood flow. Notably, following muscle IR in rats, we have observed an exaggerated exercise pressor reflex (EPR) indicating a risk of cardiovascular diseases, as the increased blood pressure (BP) response during exercise. In specific, a time‐course study (18, 66 and 114 hours post‐IR) showed that an increment in BP response was most profound in 18 hours post‐IR (IR 18h) among all the time points. However the underlying molecular mechanism leading to the IR‐induced exaggerated EPR has yet poorly understood.AimsWe targeted if the hindlimb muscle IR alters the activities of two key signaling pathway in muscle afferent nerves in regulating the EPR, namely P2X3 and ASIC3.MethodsWestern blot technique was used to determine the protein expression of P2X3 and ASIC3 in L4‐6 dorsal root ganglion (DRG) of sham rats and IR 18h rats. Meanwhile, the mean arterial pressure (MAP) responses to α‐β‐me‐ATP and lactic acid intra‐arterially injected into the hindlimb were examined to assess the P2X3 and ASIC3‐mediated EPR, respectively. Whole‐cell patch clamp was applied to evaluate the P2X and ASIC3 currents in the isolated muscle DRG neurons in both sham and IR 18h rats. All data were presented as mean ± standard deviation.ResultsCompared with sham rats, there were significant increases in both P2X3 and ASIC3 protein expression in L4‐6 DRGs in IR 18h rats (P2X3: 1.56±0.10 in IR 18h rats/n=4 vs. 1.00±0.10 in sham rats/n=6; P&lt;0.05; and ASIC3: 1.42±0.47 in IR 18h rats/n=8 vs. 1.00±0.07 in sham rats/n=10; P&lt;0.05). In addition, compared with sham rats, the MAP responses following the injection of α‐β‐me‐ATP (0.125 mM) and lactic acid (2 and 4 µmol/kg) were significantly amplified in IR 18h rats (α‐β‐me‐ATP: 29±8 mmHg in IR 18h rats/n=5 vs. 20±5 mmHg in sham rats/n=2; P&lt;0.05; Lactic acid: 2 µmol/kg, 28±8 mmHg in IR 18h rats/n=15 vs. 21±7 mmH in sham rats/n=8, P&lt;0.05; 4 µmol/kg, 37±10 mmHg in IR 18h rats/n=15 vs. 25±6 mmH in sham rats/n=9, P&lt;0.01). In the isolated DRG neurons, amplitude of both P2X and ASIC3 currents was significantly higher in IR 18h rats than that in sham rats. i.e., P2X transient currents: 1281.64±776.74 pA in IR 18h rats/n=25, vs. 717.91±556.2 pA in sham rats/n=9, (P&lt;0.05); and sustain currents: 466.94±168.68 pA in IR 18h rats/n=11, vs. 271.49±200.90 pA in sham rats/n=8 (P&lt;0.05).ConclusionThe activities of the P2X3 and ASIC3 signaling pathways in the muscle afferents are exaggerated following muscle IR. Results of this study shed light upon the future potential intervention strategy to minimize the IR‐induced exaggerated EPR response in the post‐revascularization PAD patients.

Thromboxane A2 receptors contribute to the exaggerated exercise pressor reflex in male rats with heart failure.

Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fiber muscle afferents and produce reflex increases in sympathetic nerve activity and blood pressure during exercise (i.e., the exercise pressor reflex; EPR). The EPR is exaggerated in patients and animals with heart failure with reduced ejection fraction (HF‐rEF) and its activation contributes to reduced exercise capacity within this patient population. Accumulating evidence suggests that the exaggerated EPR in HF‐rEF is partially attributable to a sensitization of mechanically activated channels produced by thromboxane A2 receptors (TxA2‐Rs) on those sensory endings; however, this has not been investigated. Accordingly, the purpose of this investigation was to determine the role played by TxA2‐Rs on the sensory endings of thin fiber muscle afferents in the exaggerated EPR in rats with HF‐rEF induced by coronary artery ligation. In decerebrate, unanesthetized rats, we found that injection of the TxA2‐R antagonist daltroban (80 μg) into the arterial supply of the hindlimb reduced the pressor response to 30 s of electrically induced 1 Hz dynamic hindlimb muscle contraction in HF‐rEF (n = 8, peak ∆MAP pre: 22 ± 3; post: 14 ± 2 mmHg; p = 0.01) but not sham (n = 10, peak ∆MAP pre: 13 ± 3; post: 11 ± 2 mmHg; p = 0.68) rats. In a separate group of HF‐rEF rats (n = 4), we found that the systemic (intravenous) injection of daltroban had no effect on the EPR (peak ΔMAP pre: 26 ± 7; post: 25 ± 7 mmHg; p = 0.50). Our data suggest that TxA2‐Rs on thin fiber muscle afferents contribute to the exaggerated EPR evoked in response to dynamic muscle contraction in HF‐rEF.

Voltage-gated potassium channel dysfunction in dorsal root ganglia contributes to the exaggerated exercise pressor reflex in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially IA, was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF.NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.

IL-6 signaling pathway contributes to exercise pressor reflex in rats with femoral artery occlusion in association with Kv4 activity in muscle afferent nerves.

Interleukin‐6 (IL‐6) via trans‐signaling pathway plays a role in modifying muscle sensory nerve‐exaggerated exercise pressor reflex in rats with ligated femoral arteries, but the underlying mechanisms are poorly understood. It is known that voltage‐gated potassium channel subfamily member Kv4 channels contribute to the excitabilities of sensory neurons and neuronal signaling transduction. Thus, in this study, we determined that 1) IL‐6 regulates the exaggerated exercise pressor reflex in rats with peripheral artery disease (PAD) induced by femoral artery ligation and 2) Kv4 channels in muscle dorsal root ganglion (DRG) neurons are engaged in the role played by IL‐6 trans‐signaling pathway. We found that the protein levels of IL‐6 and its receptor IL‐6R expression were increased in the DRGs of PAD rats with 3‐day of femoral artery occlusion. Inhibition of muscle afferents’ IL‐6 trans‐signaling pathway (gp130) by intra‐arterial administration of SC144, a gp130 inhibitor, into the hindlimb muscles of PAD rats alleviated blood pressure response to static muscle contraction. On the other hand, we found that 3‐day femoral occlusion decreased amplitude of Kv4 currents in rat muscle DRG neurons. The homo IL‐6/IL‐6Rα fusion protein (H. IL‐6/6Rα), but not IL‐6 alone significantly inhibited Kv4 currents in muscle DRG neurons; and the effect of H. IL‐6/6Rα was largely reverted by SC144. In conclusion, our data suggest that via trans‐signaling pathway upregulated IL‐6 in muscle afferent nerves by ischemic hindlimb muscles inhibits the activity of Kv4 channels and thus likely leads to adjustments of the exercise pressor reflex in PAD.

Effects of bradykinin on voltage-gated KV 4 channels in muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease.

During exercise, bradykinin (BK), a muscle metabolite in ischaemic muscles, exaggerates autonomic responses to activation of muscle afferent nerves in peripheral artery disease (PAD). We examined whether BK inhibits activity of KV 4 channels in muscle afferent neurons of PAD rats induced by femoral artery occlusion. We demonstrated that: 1) femoral occlusion attenuates KV 4 currents in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles and decreases the threshold of action potential firing; 2) BK has a greater inhibitory effect on KV 4 currents in muscle DRG neurons of PAD rats; and 3) expression of KV 4.3 is downregulated in DRGs of PAD rats and inhibition of KV 4.3 significantly decreases activity of KV 4 currents in muscle DRG neurons. Femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit activity of KV 4 channels in muscle afferent neurons and this is likely involved in the exaggerated exercise pressor reflex in PAD. Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in patients with peripheral artery diseases (PAD) and in PAD rats induced by femoral artery occlusion. However, the precise signalling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. A-type voltage-gated K+ (KV ) channels are quintessential regulators of cellular excitability in the various tissues. Among KV channels, KV 4 (i.e. KV 4.1 and KV 4.3) in primary sensory neurons mainly participate in physiological functions in regulation of mechanical and chemical sensation. However, little is known about the role of KV 4 in regulating neuronal activity in muscle afferent neurons of PAD. In addition, bradykinin (BK) is considered as a muscle metabolite contributing to the exaggerated exercise pressor reflex in PAD rats with femoral artery occlusion. Our data demonstrated that: 1) KV 4 currents are attenuated in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles of PAD rats, along with a decreasing threshold of action potential firing; 2) KV 4 currents are inhibited by application of BK onto muscle DRG neurons of PAD rats to a greater degree; and 3) expression of KV 4.3 is downregulated in the DRGs of PAD rats and KV 4.3 channel is a major contributor to the activity of KV 4 currents in muscle DRG neurons. In conclusion, data suggest that femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit the activity of KV 4 channels in muscle afferent neurons likely leading to the exaggerated exercise pressor reflex observed in PAD.

Exaggerated exercise pressor reflex in a mouse model of femoral artery ligation

The exercise pressor reflex (EPR) is a reflexive mechanism in which blood pressure and heart rate increase in response to skeletal muscle contraction. These reflexive increases are caused by both mechanical and metabolic stimulation of group III and IV afferent neurons innervating in contracting skeletal muscle. Patients with peripheral artery disease (PAD) have an exaggerated EPR which contributes to exercise intolerance and morbidity and mortality. Previous studies in our laboratory have demonstrated that the EPR is overactive in a rat model of heart failure similar to it is in human heart failure patients. We determined that this abnormality was initiated by a blunting of the metabolic component but maintained by the mechanosensitive component of the reflex. Recently, we developed a mouse model of EPR, and have validated that the mouse model is similar to our rat model. In this model, studied the EPR in a model of peripheral artery disease (PAD). Our current results demonstrated that the blood pressure increase induced by passive stretch was exaggerated in mice 72 hours following femoral artery ligation. This exaggerated rise in blood pressure was not affected by SB-705498 (a TRPv1 receptor inhibitor) but it was nearly abolished by Gadolinium (a mechanical sensitive group III afferent neurons inhibitor) and it was partially reduced by GsMTx-4 (a selective inhibitor of the Piezo2 receptor). These results indicate that the exaggerated EPR in a murine model of PAD is mediated by mechanically sensitive group III neurons, which is mediated, in part, by Piezo2 receptors.

Thromboxane A 2 Receptors Contribute to the Exaggerated Exercise Pressor Reflex in Rats with Heart Failure

Background Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fiber skeletal muscle afferents. During exercise, stimulation of these afferents initiates a reflex, termed the exercise pressor reflex (EPR), which contributes importantly to reflex increases in sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). In patients and animals with heart failure with reduced ejection fraction (HF-rEF), activation of the EPR contributes to exaggerated increases in SNA and is linked to decreased exercise tolerance and increased mortality. The exaggerated EPR in HF-rEF is attributed, at least in part, to an increased responsiveness of thin fiber skeletal muscle afferents produced by cyclooxygenase (COX) products of arachidonic metabolism. However, the COX metabolite receptors on the sensory endings that mediate the COX-induced afferent sensitization in HF-rEF are unknown. Purpose & Hypothesi The purpose of this investigation was to determine the role played by one of the major receptors for products of COX metabolism present on sensory neurons, namely thromboxane A2 receptors (TxA2-Rs), in the exaggerated EPR in HF-rEF rats. We tested the hypothesis that injection of the TxA2-R antagonist daltroban (80μg) into the arterial supply of the hindlimb would reduce the reflex increase in renal SNA (RSNA) and MAP evoked in response to 30 seconds of 1 Hz dynamic hindlimb muscle contraction in HF-rEF rats, but not control rats. Methods Experiments were performed on male Sprague-Dawley rats subjected to either a coronary artery ligation surgery to produce myocardial infarction and HF-rEF or a sham ligation. At least six weeks after the initial surgery, in decerebrate, unanesthetized rats we compared the EPR evoked by electrically induced hindlimb skeletal muscle contraction before and after injection of daltroban into the arterial supply of the hindlimb. Results Ejection fraction was significantly reduced in HF-rEF (47±3%) compared to sham (84±1%, p<0.01). We found that daltroban reduced the EPR response in HF-rEF (n=8; peak ΔMAP pre: 17±4; post: 12±2mmHg; P=0.04; peak ΔRSNA pre: 135±55; post: 46±13%; P=0.07), but not in SHAM rats (n=10; peak ΔMAP pre: 13±3; post: 11±2mmHg; P=0.48; peak ΔRSNA pre: 64±17; post: 63±15%; P=0.96;) rats. In a separate group of HF-rEF rats (n=4), we determined whether systemic circulation of daltroban accounted for the attenuating effects on EPR activation seen when daltroban was injected into the hindlimb arterial supply. We found that intravenous injection of daltroban had no effect on the EPR (peak ΔMAP pre: 26±7; post: 25±7mmHg; P=0.50). In these same four HF-rEF rats we also found that hindlimb arterial injection of the vehicle for daltroban (0.4mL saline containing 1% DMSO) had no effect on the EPR (peak ΔMAP pre: 23±8; post: 20±6mmHg; P=0.40). Conclusions These data suggest that that TxA2-R receptors on the sensory endings of muscle afferents contribute to the exaggerated EPR in HF-rEF. Our data enhance the understanding of the sympathetic and cardiovascular adjustments to exercise in the ~6 million Americans with HF-rEF.

Circulating NGF is correlated with indexes of diabetes progression and P2X 3 expression in UCD‐T2DM rats

Type 2 diabetes is associated with neuropathic symptoms, including development of peripheral neuropathy and an exaggerated exercise pressor reflex, both of which have been demonstrated in UC Davis Type 2 diabetes mellitus (UCD-T2DM) rats. This novel animal model more closely mimics the human disease state than traditional T2DM rodent models. Several studies have demonstrated changes in nerve growth factor (NGF) in diabetic patients, with some showing increases and others decreases, and this has been proposed as a potential driver of the development of peripheral neuropathy. Additionally, increased expression of purinergic 2X3 (P2X3) receptors has been suggested as a potential mechanism underlying peripheral nerve sensitization. However, interactions between these factors during diabetes progression are not fully understood. Therefore, the objective of this study was to investigate the relations between NGF, P2X3, and peripheral neuropathy during diabetes progression in UCD-T2DM rats. We hypothesized that NGF levels would be correlated with traditional indexes of diabetes (glucose and HbA1c) as well as P2X3 expression and mechanical allodynia as measured by paw withdrawal threshold. Methods Blood was collected monthly from the tail vein of male UCD-T2DM rats from 4 to 8 months of age. Serum levels of NGF, leptin, and insulin were measured by ELISA. Development of mechanical allodynia was tracked monthly using Von Frey filaments to measure paw withdrawal threshold. At 8 months of age, L4-5 dorsal root ganglia were harvested and P2X3 receptor protein levels were quantified using the Jess automated western blotting platform. Pearson correlations were calculated using Graphpad Prism 9. Results Consistent with wasting and loss of pancreatic beta cell function during diabetes progression, glucose and HbA1c increased significantly over time whereas body weight, insulin, and leptin levels decreased. Circulating NGF concentration was negatively correlated with glucose (r=-0.61, P<0.01) and HbA1c (r=-0.79, p<0.01) demonstrating that NGF decreased with the progression of diabetes. Circulating NGF concentration was positively correlated with body weight (r=0.61, p<0.01), insulin (r=0.77, p<0.01), and leptin (r=0.82, p<0.01) thereby showing the same decreasing trend with diabetes progression. Interestingly, NGF was negatively correlated with P2X3 receptor expression (r=-0.81, p=0.03) and neither NGF nor P2X3 were significantly associated with mechanical allodynia (NGF: r=0.14, p=0.45; P2X3: r= 0.15, p=0.75). Conclusion Circulating NGF is significantly associated with several indexes of diabetes progression in UCD-T2DM rats, including glucose, HbA1c, leptin, and insulin levels. Though decreasing concentrations of NGF are associated with increases in P2X3 receptor protein expression, neither NGF nor P2X3 appear to be associated with the development of mechanical allodynia.

Exaggerated Exercise Pressor Reflex During the Progression of Type 2 Diabetes Mellitus is Independent of Aging

We previously determined that the exercise pressor reflex and mechanoreflex are exaggerated during the progression of type 2 diabetes mellitus (T2DM) in UCD-T2DM rats. For this first study T2DM onset was determined using a non-fasting glucose threshold of 200 mg/dL. Importantly, aging, which may also be associated with autonomic modulation changes, was not investigated during this previous study. Therefore, the objective of the current study was to determine if these changes in the exercise pressor reflex during T2DM progression occur independent of aging. We hypothesized that the exercise pressor reflex and mechanoreflex would remain exaggerated during the progression of T2DM when compared against age-matched healthy controls. Methods Studies were conducted in fasted, unanesthetized, decerebrated UCD-T2DM rats, using a fasting glucose of 140 mg/dL as diagnostic criteria, at 4 different time points of disease progression (pre-onset: before developing diabetes; early-onset: 3-7 wks after onset; established: 23-28 wks after onset; and chronic: 39-44 wks after onset). Healthy, age-matched Sprague Dawley rats were used as controls (CTL). The exercise pressor reflex was evoked by statically and intermittently contracting the hindlimb for 30 s, and the mechanoreflex was evoked by stretching the Achilles tendon for 30 s. Peak changes in mean arterial pressure (MAP) and developed tension were measured and compared across all groups (one-way ANOVA followed by Holm Sidak's post hoc analysis). Results We found that fasting blood glucose was significantly higher after the onset of T2DM and remained above the diagnostic threshold (140 mg/dL) during the progression of the disease (p<0.05). Static hindlimb muscle contraction significantly increased peak pressor response in early-onset UCD-T2DM rats, but not in other stages of T2DM, compared to age-matched healthy controls (CTL: 10 ± 1 mmHg, n=7; T2DM: 24 ± 5 mmHg, n=6, p<0.05) with similar developed tensions, p>0.05. Likewise, intermittent hindlimb muscle contraction significantly increased peak pressor response in early-onset UCD-T2DM rats, but not later stages of T2DM, compared to their age-matched healthy controls (CTL: 9 ± 1 mmHg, n=6; T2DM: 36 ± 7 mmHg, n=4, p<0.05) with similar developed tensions, p>0.05. Similarly, passive tendon stretch significantly increased peak pressor response in early-onset UCD-T2DM rats, but not later stages of T2DM, compared to their age-matched healthy controls (CTL: 12 ± 5 mmHg, n=6; T2DM: 36 ± 8 mmHg, n=7, p<0.05) with similar developed tensions, p>0.05. Conclusion These findings suggest that the exercise pressor reflex and mechanoreflex are exaggerated at the onset of T2DM, as diagnosed by fasted glucose of 140mg/dL, but not later in the disease, and this change occurs independent of aging.

GsMTx-4 normalizes the exercise pressor reflex evoked by intermittent muscle contraction in early stage type 1 diabetic rats.

Emerging evidence suggests the exercise pressor reflex is exaggerated in early stage type 1 diabetes mellitus (T1DM). Piezo channels may play a role in this exaggeration, as blocking these channels attenuates the exaggerated pressor response to tendon stretch in T1DM rats. However, tendon stretch constitutes a different mechanical and physiological stimuli than that occurring during muscle contraction. Therefore, the purpose of this study was to determine the contribution of Piezo channels in evoking the pressor reflex during an intermittent muscle contraction in T1DM. In unanesthetized decerebrate rats, we compared the pressor and cardioaccelerator responses to intermittent muscle contraction before and after locally injecting grammostola spatulata mechanotoxin 4 (GsMTx-4, 0.25 µM) into the hindlimb vasculature. Although GsMTx-4 has a high potency for Piezo channels, it has also been suggested to block transient receptor potential cation (TRPC) channels. We, therefore, performed additional experiments to control for this possibility by also injecting SKF 96365 (10 µM), a TRPC channel blocker. We found that local injection of GsMTx-4, but not SKF 96365, attenuated the exaggerated peak pressor (ΔMAP before: 33 ± 3 mmHg, after: 22 ± 3 mmHg, P = 0.007) and pressor index (ΔBPi before: 668 ± 91 mmHg·s, after: 418 ± 81 mmHg·s, P = 0.021) response in streptozotocin (STZ) rats (n = 8). GsMTx-4 attenuated the exaggerated early onset pressor and the pressor response over time, which eliminated peak differences as well as those over time between T1DM and healthy controls. These data suggest that Piezo channels are an effective target to normalize the exercise pressor reflex in T1DM.NEW & NOTEWORTHY This is the first study to demonstrate that blocking Piezo channels is effective in ameliorating the exaggerated exercise pressor reflex evoked by intermittent muscle contraction, commonly occurring during physical activity, in T1DM. Thus, these findings suggest Piezo channels may serve as an effective therapeutic target to reduce the acute and prolonged cardiovascular strain that may occur during dynamic exercise in T1DM.

HIF-1α inhibition alleviates the exaggerated exercise pressor reflex in rats with peripheral artery disease induced by femoral artery occlusion.

Hypoxia‐inducible factor 1α (HIF‐1α) is a transcription factor mediating adaptive responses to hypoxia and ischemia. Our previous work showed that HIF‐1α is increased in muscle sensory nerves of rats with peripheral artery disease (PAD) induced by femoral artery occlusion. The present study was further to examine the role played by HIF‐1α in regulating the response of arterial blood pressure (BP) to the activation of muscle afferent nerve during static muscle contraction in rats with femoral artery occlusion. A rat model of femoral artery ligation was used to study PAD in this study. Western blot analysis was employed to examine the protein levels of HIF‐1α in the dorsal root ganglion (DRG) tissues. BAY87, a synthesized compound with the characteristics of highly potent and specific suppressive effects on expression and activity of HIF‐1α, was given into the arterial blood supply of the ischemic hindlimb muscles before the exercise pressor reflex was evoked by static muscle contraction. First, HIF‐1α was increased in the DRG of occluded limbs (optical density: 0.89 ± 0.13 in control versus 1.5 ± 0.05 in occlusion; p < 0.05, n = 6 in each group). Arterial injection of BAY87 (0.2 mg/kg) then inhibited expression of HIF‐1α in the DRG of occluded limbs 3 hr following its injection (optical density: 1.02 ± 0.09 in occluded limbs with BAY87 versus 1.06 ± 0.1 in control limbs; p > 0.05, n = 5 in each group). In addition, muscle contraction evoked a greater increase in BP in occluded rats. BAY87 attenuated the enhanced BP response in occluded rats to a greater degree than in control rats. Our data suggest that the inhibition of HIF‐1α alleviates the exaggeration of the exercise pressor reflex in rats under ischemic circumstances of the hindlimbs in PAD induced by femoral artery occlusion.