Protein Kinase C Epsilon Contributes to Chronic Mechanoreflex Sensitization in Rats with Heart Failure

Abstract

We investigated second-messenger signaling components linked to the stimulation of G_q protein-coupled receptors (e.g., thromboxane A₂, and bradykinin 2 receptors) on the sensory endings of 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 inositol 1,4,5-trisphosphate (IP₃) receptor antagonist Xestospongin C (5μg) or the PKCε translocation inhibitor PKCe141 (45μg) into the arterial supply of the hindlimb would reduce the increase in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) evoked during 30 s of 1 Hz dynamic hindlimb muscle stretch in decerebrate, unanesthetized HF-rEF rats but not sham-operated controls (SHAM). Ejection fraction was significantly reduced in HF-rEF (45 (19)%) compared to SHAM (80 (9)%; P < 0.001) rats. In HF-rEF rats (n=3M/2F), IP₃ receptor blockade had no effect on the peak ΔRSNA (pre: 99 (74), post: 133 (79) %, P=0.974) or peak ΔMAP response to stretch (peak ΔMAP; pre: 32 (14), post: 36 (21) mmHg, P=0.719). Conversely, in another group of HF-rEF rats, (n=4M/3F) the PKCε translocation inhibitor reduced the peak ΔRSNA (pre: 110 (77), post: 62 (58) %, P=0.029) and peak ΔMAP response to stretch (pre: 30 (20), post: 17 (16) mmHg, P=0.048). In SHAM counterparts, neither drug affected the mechanoreflex responses. Our findings highlight PKCε, but not IP₃ receptors, as a significant second-messenger in the chronic mechanoreflex sensitization in HF-rEF which may play a crucial role in the exaggerated sympathetic response to exercise in this patient population.

Graphical Abstract

Skeletal muscle contraction leads to exaggerated reflex increases in sympathetic nerve activity in heart failure patients with reduced ejection fraction (HF-rEF) which raises cardiovascular risk and impairs exercise tolerance. This is partly due to chronic sensitization of mechanically sensitive group III/IV muscle afferents via Gq protein-coupled thromboxane A₂ (TxA₂) and bradykinin B2 receptor signaling. The specific Gq protein-linked signaling mechanisms underlying this sensitization have not been fully explored, though indirect evidence strongly suggests the involvement of either inositol 1,4,5-trisphosphate (IP₃) receptors and/or protein kinase C epsilon (PKCε). Our findings demonstrate that PKCε, but not IP₃ receptors, within sensory endings of thin fiber muscle afferents plays a role in the mechanoreflex sensitization in HF-rEF rats.

Introduction

A withdrawal of parasympathetic nerve activity and an increase in sympathetic nerve activity (SNA) in response to exercise are essential for appropriate cardiovascular function and exercise performance. A feedforward mechanism, termed central command, and multiple feedback mechanisms, including the exercise pressor reflex and the carotid chemoreflex, underly those exercise-induced autonomic nervous system adjustments (Goodwin et al., 1972; Rowell & O’Leary, 1990; Stickland et al., 2007). In patients with heart failure with reduced ejection fraction (HF-rEF), however, activation of those mechanisms during exercise produces an exaggerated increase in SNA compared to that found in healthy counterparts (Sinoway & Li, 2005; Koba et al., 2006; Stickland et al., 2007) which is clinically significant because exercise plasma noradrenaline (NA) was found to be a signifincat prognostic marker of cardiac death in heart failure patients (Kinugawa et al., 2002). Morevoer, while exercise presor reflex activation in HF-rEF patients may increase cardiac output during small muscle mass exercise (Amann et al., 2014), it also constrains limb limb vascular conductance via augmented sympathetic outflow, thereby constraining convective O₂ delivery (Amann et al., 2014; Smith et al., 2020). Thus, understanding the mechanisms underlying the exaggerated exercise pressor reflex in HF-rEF carries clinical and functional relevance for the over 26 million people with HF-rEF worldwide (Ponikowski et al., 2014).

The exercise pressor reflex is activated when the sensory endings of group III and IV skeletal muscle afferents (collectively termed thin fiber muscle afferents) are stimulated by mechanical and/or metabolic stimuli associated with skeletal muscle contraction (McCloskey & Mitchell, 1972; Kaufman et al., 1982; Kaufman et al., 1983; Mense & Stahnke, 1983; Kaufman et al., 1984; Mense & Meyer, 1988; Rotto & Kaufman, 1988). The mechanically sensitive portion of the exercise pressor reflex (i.e., the mechanoreflex) contributes importantly to the overall reflex exaggeration in HF-rEF patients (Middlekauff et al., 2004; Antunes-Correa et al., 2014) and in a rat model of myocardial infarction-induced HF-rEF (Smith et al., 2003; Koba et al., 2008; Morales et al., 2012; Butenas et al., 2021c, b; Butenas et al., 2022). For example, hindlimb muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production) evokes a larger increase in group III muscle afferent firing rate in HF-rEF rats compared to the increase in firing rate in healthy control rats (Wang et al., 2010). We found recently G_q protein coupled thromboxane A₂ (TxA₂) receptors on the sensory endings of thin fiber muscle afferents contributed importantly to the exaggerated mechanoreflex (Butenas et al., 2021b) and exercise pressor reflex (Butenas et al., 2021c) control of renal SNA (RSNA) and mean arterial pressure (MAP) in HF-rEF rats. Similarly, Koba et al. (2010) found that the G_q protein coupled bradykinin B2 receptors on the sensory endings of thin fiber muscle afferents also contributed to the exaggerated mechanoreflex. However, neither TxA₂ or B2 receptors contributed to mechanoreflex or exercise pressor reflex generation in healthy rats (Koba et al., 2010; Butenas et al., 2021c, b). Thus, the development of HF-rEF results in a chronic, or persistent, sensitization of the thin fiber muscle afferents that underly mechanoreflex activation which is produced, at least in part, by G_q protein coupled TxA₂ and B2 receptors on the sensory endings of these afferents which does not require the accumulation of metabolites produced during muscle contractions.

The mechanisms underlying the G_q protein-mediated chronic mechanoreflex sensitization in HF-rEF have not been investigated. G_q protein stimulation results in the activation of phospholipase C which induces an increase in diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃) formation (Narumiya et al., 1999; Huang et al., 2004; Jang et al., 2020), both of which have been linked to sensory neuron sensitization (Borbiro & Rohacs, 2017). There is indirect experimental support for the possibility that both components of G_q coupled second-messenger systems are involved in sensitizing the mechanoreflex in HF-rEF. For example, when formed, IP₃ binds to its receptor on the endoplasmic reticulum which increases intracellular calcium concentrations (Borbiro & Rohacs, 2017). Elevated intracellular calcium has been shown to sensitize mechanically activated PIEZO channel-mediated currents in HEK293 cells (Eijkelkamp et al., 2013). Moreover, we found recently that IP₃ receptor signaling contributed importantly to mechanoreflex sensitization in rats with simulated peripheral artery disease (Rollins et al., 2021). Another G_q protein linked signaling component involved in mechanoreflex sensitization may involve DAG-induced translocation of protein kinase C (PKC) from the cytosol to the cell membrane within the sensory ending. For example, the PKCε isoform specifically has been identified as a key second messenger signaling component in mechanical hyperalgesia (Hucho et al., 2005). Together, these findings suggest that second-messenger signaling within sensory neurons involving IP₃ receptors and/or PKCε translocation contributes to the exaggerated mechanoreflex in HF-rEF, but these possibilities have not been investigated.

Based on the information above, we investigated the role played by signaling linked to IP₃ receptors and PKCε in evoking the exaggerated mechanoreflex in rats with HF-rEF. We tested the hypotheses that: 1) hindlimb arterial injection of the IP₃ receptor antagonist Xestospongin C (5 μg) would reduce the reflex increase RSNA and MAP evoked in response to 30 seconds of 1 Hz repetitive hindlimb muscle stretches in HF-rEF rats but not in healthy control rats, and 2) hindlimb arterial injection of the selective PKCε translocation inhibitor PKCe141 (45 μg) would reduce the reflex increase RSNA and MAP evoked in response to 30 seconds of 1 Hz hindlimb muscle stretches in HF-rEF rats but not in healthy control rats.

Methods & Materials

Ethical Approval.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Kansas State University (Protocol #4552) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011). Experiments were performed on ~14–19-week-old male and female Sprague-Dawley rats (n=59; Charles River Laboratories). Rats were housed two per cage in temperature (maintained at ~22°C) and light (12–12 hour light-dark cycle running from 7 AM to 7 PM)-controlled accredited facilities with standard rat chow and water provided ad libitum.

Myocardial infarction procedure.

Myocardial infarction (MI) was induced in 40 of the 59 rats by surgically ligating the left main coronary artery (Musch & Terrell, 1992). Briefly, rats were anesthetized initially with a 5% isoflurane-O₂ mixture (Butler Animal Health Supply, Elk Grove Village, IL, and Linweld, Dallas, TX) and maintained subsequently on 2.5% isoflurane-O₂ and then intubated and mechanically ventilated with a rodent respirator (Harvard model 680, Harvard Instruments, Holliston, MA) for the duration of the surgical procedure. After a single injection of the antiarrhythmic drug amiodarone (100 mg/kg ip), which was administered to improve survival rate following the ensuing coronary artery ligation surgery (Kolettis et al., 2007), a left thoracotomy was performed to expose the heart through the fifth intercostal space, and the left main coronary artery was ligated 1–2 mm distal to the edge of the left atrium with a 6–0 braided polyester suture. The thorax was then closed with 2–0 gut, and the skin was closed with 2–0 silk. Prior to termination of anesthesia, bupivacaine (1.5 mg/kg sc) and buprenorphine (0.03 mg/kg im) were administered to reduce pain associated with the surgery, along with ampicillin (50 mg/kg im) to reduce the risk of infection. After rats were removed from mechanical ventilation and anesthesia, they were monitored closely for ~6 h post-surgery. In the remaining 19 of 59 rats, a sham ligation of the coronary artery was performed in which 6–0 braided polyester suture was passed under the left main coronary artery, but not tied. These rats are referred to as “SHAM” rats from this point forward. Following completion of either MI or sham procedures, rats were housed one per cage for ten days to minimize risk of infection of the surgical site. During these ten days, the antibiotic Baytril (100 mg/mL) was administered in the drinking water. Following completion of the Baytril treatment, rats were housed two per cage as described above. All animals were monitored daily for 14 days following the MI or sham procedure for changes in behavior, gait/posture, breathing, appetite and body weight.

Echocardiograph Measurements.

Transthoracic echocardiograph measurements were performed with a commercially available system (Logiq S8; GE Health Care, Milwaukee, WI) one week before the final experimental protocol. Briefly, the rats were anaesthetized as described above. Once the rat was fully anesthetized, the isoflurane mixture was reduced to 2.5% isoflurane-O₂. Following 5 minutes at 2.5% isoflurane, echocardiograph measurements began. The transducer was positioned on the left anterior chest, and left ventricular dimensions were measured. The left ventricular fractional shortening (FS), ejection fraction (EF), end diastolic (LVEDV), end systolic volume (LVESV), and stroke volume (SV) were determined by echocardiographic measurements as previously described (Butenas et al., 2021a).

Surgical Procedures for Experimental Protocols.

In vivo experiments were performed between six and eight weeks following the MI or sham procedure. On the day of the experiment, rats were anesthetized as described above. Adequate depth of anesthesia 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) with a 2% isoflurane-balance O₂ gaseous mixture until the decerebration was completed (see below). The right jugular vein and both carotid arteries were cannulated with PE-50 catheters which were used for the injection of fluids, measurement of arterial blood pressure (physiological pressure transducer, AD Instruments), and sampling of arterial blood gasses (Radiometer). HR was calculated from the R-R interval measured by electrocardiogram (AD Instruments). The left superficial epigastric artery was cannulated with a PE-8 catheter whose tip was placed near the junction of the superficial epigastric and femoral arteries. A reversible snare was placed around the left iliac artery and vein (i.e., proximal to the location of the catheter placed in the superficial epigastric artery). The left calcaneal bone was severed and linked by string to a force transducer (Grass FT03), which, in turn, was attached to a rack and pinion.

Upon completion of the initial surgical procedures, rats were placed in a Kopf stereotaxic frame. After administering dexamethasone (0.2 mg i.v.) to minimize swelling of the brainstem, a pre-collicular decerebration was performed in which all brain tissue rostral to the superior colliculi was removed (Smith et al., 2001). The cranial cavity was filled with cotton balls and covered with glue (Kwik-Sil, World Precision Instruments). Following decerebration, anesthesia was reduced to 0.5%. A retroperitoneal approach was used to expose bundles of the left renal sympathetic nerve, which were then glued with Kwik-Sil onto a pair of thin stainless-steel recording electrodes connected to a high impedance probe (Grass Model HZP) and amplifier (Grass P511). Multiunit signals from the renal sympathetic nerve fibres were filtered at high and low frequencies (1 KHz and 100 Hz, respectively) for the measurement of RSNA.

Upon completion of all surgical procedures, anesthesia was terminated, and the rats’ lungs were ventilated with room air. Experimental protocols commenced at least one hour after reduction of isoflurane from 2% to 0.5%, and at least 30 minutes after reduction of isoflurane from 0.5% to 0%. Experiments were performed on decerebrate, unanesthetized rats because anesthesia has been shown to markedly blunt the exercise pressor reflex in the rat (Smith et al., 2001). Body core temperature was measured via a rectal probe and maintained at ~37–38°C by an automated heating system (Harvard Apparatus) and heat lamp. Arterial pH and blood gases were analyzed periodically throughout the experiment from arterial blood samples (~75 μL) and maintained within physiological ranges (pH: 7.35–7.45, PCO₂: ~38–40 mmHg, PO₂: ~100 mmHg) by administration of sodium bicarbonate and/or adjusting ventilation as necessary. At the end of all experiments in which RSNA was measured, postganglionic sympathetic nerve activity was abolished with administration of hexamethonium bromide (20 mg/kg iv) to allow for the quantification of background noise as described previously (Kempf et al., 2018). A pneumothorax was then performed, and the lungs were then excised and weighed. The heart was excised, and the atria and right ventricle (RV) were separated from the left ventricle (LV) and septum, and the RV, LV, and atria were weighed. In rats with HF-rEF, the LV infarction surface area was measured using planimetry and expressed as percent of LV endocardial surface area as described previously (Craig et al., 2019).

Isolated mechanoreflex protocols.

We first compared the RSNA, pressor, and cardioaccelerator responses evoked in response to 30 seconds of 1 Hz dynamic stretch of the triceps surae muscle before and after the injection of either the IP3 receptor antagonist Xestospongin C (SHAM n=8, 5M/3F; HF-rEF n=5, 3M/2F) or the PKCε inhibitor PKCe141 (SHAM n=6, 3M/3F; HF-rEF n=7, 4M/3F) into the arterial supply of the left hindlimb. Before initiating the protocol, we paralyzed the rats with pancuronium bromide (1 mg/kg iv) to avoid any spontaneous/reflex muscle contraction. In detail, at least 60 minutes following the decerebration procedure and termination of anesthesia, baseline muscle tension was set at ~80–100 g by manually turning the rack and pinion. Baseline blood pressure and heart rate were measured for ~30 seconds. An experienced investigator then initiated the stretch protocol in which the rack and pinion was turned back and forth manually at a 1 Hz frequency with the aid of a metronome for 30 seconds. The investigator aimed for a developed tension of ~0.6–0.8 kg on each stretch which is the tension generation typically developed during 1 Hz intermittent contraction protocols in decerebrate rats (Copp et al., 2016; Kempf et al., 2018). This stretch protocol was adapted from protocols described originally by Stebbins et al. (1988) and Daniels et al. (2000) in the cat. Approximately five minutes following the completion of the control stretch protocol, and after ensuring that blood pressure had returned to its pre-stretch baseline value, the appropriate solution (see below) was injected into the arterial supply of the left hindlimb via the left superficial epigastric artery catheter. After the appropriate time had passed the stretch protocol was repeated. At the end of each experiment, Evans blue dye was injected in the same manner as the experimental solution to confirm that the injectate had access to the triceps surae muscle circulation.

The following isolated mechanoreflex protocols were performed. Note that the specific injectate, whether an iliac artery and vein snare was pulled tight, the time the hindlimb was allowed to reperfuse if a snare was pulled tight, and the sample sizes are indicated.

Protocol 1:

Xestospongin C (IP₃ receptor antagonist, Cayman Chemical, Ann Arbor, MI, USA; 5 μg dissolved in 0.2 mL of 0.02% ethanol; established by Rollins et al., 2021); snare tight for 10 min, reperfuse for 30 min, n=8 (5M/3F) SHAM rats; n=5 (3M/2F) HF-rEF rats.

Protocol 2:

PKCe141 (PKCε translocation inhibitor, Sigma-Aldrich, St. Louis, MO, USA; 45 μg dissolved in 0.2 mL of 2.25% DMSO); no snare and infused with the aid of a constant infusion pump (Harvard Apparatus) at a flow rate of 20 μL/min for 10 min; n=6 (3M/3F) SHAM rats and n=7 (4M/3F) HF-rEF rats. The dose of 45 μg was established based on an estimation of the drug’s concentration within the rat hindlimb, aiming to achieve a concentration slightly higher than its IC₅₀ of 5.9 μM (Rechfeld et al., 2014).

Protocol 3:

0.2mL of 2.25% DMSO (vehicle for PKCe141), no snare and infused with the aid of a constant infusion pump (Kent Scientific) at a flow rate of 20 μL/min for 10 min, n=4 (1M/3F) HF-rEF rats.

Protocol 4:

PKCe141 (45 μg dissolved in 0.2mL of 2.25% DMSO) was injected into the right jugular vein with the aid of a constant infusion pump (Kent Scientific) at a flow rate of 20 μL/min for 10 min and therefore allowed to circulate systemically; n=4 (3M/1F) HF-rEF rats. Ten minutes elapsed between the intravenous injection of PKCe141 and the subsequent stretch maneuver exactly as aforementioned in Protocol 2 when PKCe141 was injected into the arterial supply of the hindlimb.

Drug injection protocols.

In three groups of HF-rEF rats, we compared the renal sympathetic, pressor, and cardioaccelerator response to injection of either lactic acid (24mMol in 0.2mL saline; n=5, 1M/4F), α,β-methylene ATP (20 μg in 0.2mL saline; n=4, 2M/2F), or bradykinin (5 μg in 0.2mL saline; n=4, 2M/2F) before and after the injection of the PKCε inhibitor PKCe141 (45 μg dissolved in 0.2mL of 2.25% DMSO) into the arterial supply of the left hindlimb. In detail, at least 60 minutes following the decerebration procedure and termination of anesthesia, baseline RSNA, blood pressure, and heart rate were measured for ~30 seconds. Lactic acid, α,β-methylene ATP, and bradykinin were all injected as a bolus over ~2 seconds into the arterial supply of the left hindlimb. For α,β-methylene ATP and bradykinin injections, an iliac artery and vein snare was pulled tight 30 seconds prior to injection and remained snared for 30 seconds after the injection was completed. Approximately five minutes following the completion of the control injection protocol, and after ensuring that blood pressure had returned to its pre-injection baseline value PKCe141 was infused as described above. After infusion of PKCe141, a second injection of either lactic acid, α,β-methylene ATP, or bradykinin was performed exactly as performed in the control injection. At the end of each experiment, Evans blue dye was injected in the same manner as the experimental solution to confirm that the injectate had access to the triceps surae muscle circulation.

Exercise pressor reflex protocols.

In SHAM (n=5, 3M/2F) and HF-rEF (n=6, 5M/1F) rats, we compared the renal sympathetic, pressor, and cardioaccelerator responses evoked in response to 30 seconds of 1 Hz dynamic hindlimb muscle contraction before and after the injection of PKCe141 (45 μg in 0.2mL saline containing 2.25% DMSO) into the arterial supply of the left hindlimb. Following recovery of isoflurane anesthesia, baseline muscle tension was set to ~100 g and baseline RSNA, blood pressure, and HR were measured for ~30 seconds. The sciatic nerve was then electrically stimulated using stainless steel electrodes for 30 seconds at a voltage of ~1.5x motor threshold (0.01 ms pulse duration, 500 ms train duration, 40 Hz frequency) which produced 1 Hz repetitive/dynamic contractions of the triceps surae muscles. Approximately 10 min following the control contraction maneuver, PKCe141 was infused as described above. At the end of each experiment, the rats were paralyzed with pancuronium bromide (1 mg/kg iv) and the sciatic nerve was stimulated for 30 s with the same parameters as those used to elicit contraction to ensure that the increase in RSNA, blood pressure and HR during contraction was not due to the electrical activation of the axons of the thin fiber muscle afferents in the sciatic nerve. No increase in RSNA, blood pressure or HR was observed during the stimulation period following the administration of pancuronium bromide. Evans blue dye was injected in the same manner as the experimental solution to confirm that the injectate had access to the triceps surae muscle circulation.

Tissue collection

In eight (4M/4F) SHAM and 10 (5M/5F) HF-rEF rats, the left and right L₄/L₅ DRG were harvested. Rats were first deeply anesthetized (5% isoflurane) and killed with an overdose of saturated KCl. Samples were isolated into 2 mL bead mill tubes containing ~0.5 g of 1.4 mm diameter ceramic beads, 300 μL of RP1 lysis buffer (Macherey-Nagel), and homogenized for 1 min at 5 m/s using Bead Mill 4 (Fisherbrand^™). Total protein and mRNA from tissues were prepared with the Nucleospin miRNA/Protein Kit (Macherey-Nagel, Dϋren, Germany) according to the manufacturer’s instructions. Total Protein and RNA concentrations were determined using the Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA) and stored in a −80°C freezer until further analysis.

Western blot experiments

Protein samples (20 μg) were separated on 4–12% Bis-Tris Protein Gels (Invitrogen^™) by gel electrophoresis in MES running buffer (Invitrogen^™) employing 200 V for 22 min. Gels were then transferred to mini-PVDF membranes using the iBlot 2 Dry Transfer Device (Invitrogen^™). Membranes were incubated for ~3 hours with the iBind device with iBind solution (Invitrogen^™) with a primary antibody for phosphorylated PKCε (Ser-729) diluted 1:100 (Millipore Sigma, Burlington, MA, USA; cat. no. 06-821-I) and a Goat anti-Rabbit IgG (H+L) secondary antibody conjugated with Horse Radish Peroxidase diluted 1:1,500 (Abcam; Cambridge, England; cat. no. ab205718). The membranes were then incubated for 5 min with 6mL of SuperSignal^™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and imaged with C-DiGit^® Blot Scanner (Li-Cor). The protein bands were quantified and analyzed using Image Studio software (Li-Cor), confirming the presence of phosphorylated PKCε (p-PKCε) at approximately 84 kDa, as specified by the manufacturer. The membranes were then stripped and re-incubated with a primary antibody for PKCε diluted 1:100 (ThermoFisher, Rockford, IL, USA; cat. no. MA5-14908) and a Goat anti-Rabbit IgG (H+L) secondary antibody conjugated with Horse Radish Peroxidase diluted 1:1,500 (Abcam; Cambridge, England; cat. no. ab205718). The membranes were then re-incubated with the chemiluminescent substrate and imaged confirming the presence of total PKCε (t-PKCε) at approximately 84 kDa, as specified by the manufacturer. Next, the membrane was stripped a final time and re-incubated with a primary antibody for GAPDH diluted 1:1,000 (Thermo Fisher Scientific; Rockford, IL, USA; cat. no. MA5-15738), along with a Goat anti-Mouse IgG (H+L), secondary antibody conjugated with Horse Radish Peroxidase diluted 1:1,000 (Thermo Fisher Scientific; Rockford, IL, USA; cat. no. 31430). The membranes were then re-incubated with the chemiluminescent substrate and imaged confirming the presence of GAPDH at approximately 38 kDa, as specified by the manufacturer.

Quantitative Reverse Transcriptase Polymerase Chain Reaction experiments

Complementary DNA (cDNA) was synthesized from RNA isolates (see above) using the High Capacity RNA-cDNA^™ kit (Thermo Fisher) according to the manufacturer’s instructions. Quantitative reverse transcriptase polymerase chain reaction experiments were then performed on the cDNA samples using TaqMan gene expression assays specific for: PKCε (Sequence proprietary; assay ID: Rn01785893_m1), along with GAPDH with (forward primer: 5′-ACCGCCTGTTGCGTGTTA-3′ and reverse primer: 5′-CAATCGCCAACGCCTCAA-3′; assay ID: Rn01775763_g1). All samples were run in duplicate for the gene of interest and the endogenous control (GAPDH). The results were analyzed with the comparative threshold (ΔΔCt) method.

Data analysis.

Muscle tension, blood pressure, HR and RSNA were measured and recorded in real time with a PowerLab and LabChart data acquisition system (AD Instruments). The original RSNA data were rectified and corrected for the background noise determined after the administration of hexamethonium bromide. RSNA was normalized by subtracting the baseline value and expressing the difference as a percent change from baseline for a given maneuver. Baselines for mean arterial pressure (MAP), RSNA and HR were determined from the 30-second baseline periods that preceded each maneuver. The peak increase in MAP (peak ΔMAP), RSNA (peak ΔRSNA), and HR (peak ΔHR) during a given maneuver was calculated as the difference between the peak values wherever they occurred during the maneuvers and their corresponding baseline value. The integrated RSNA for the first 5 seconds (∫ΔRSNA_{5 Sec}) was calculated by integrating the ΔRSNA (>0) during the first 5 seconds of the contraction maneuver. The change in tension-time indexes (ΔTTIs) and blood pressure indexes (BPIs) were calculated by integration of the area under curve during the stretch/contraction maneuver and subtracting the integrated area under the curve during the baseline period. Time courses of the increase in RSNA and MAP were plotted as their change from baseline. Baseline MAP, baseline HR, Peak ΔMAP, Peak ΔHR, Peak ΔRSNA, BPI, ∫ΔRSNA, and ΔTTIs were compared with multiple paired t-tests. Data for echocardiograph measurements, body and organ masses, heart morphometrics, protein and mRNA expression were analyzed with unpaired Student’s t-tests or Mann-Whitney U tests as appropriate. All data are expressed as mean (SD). Statistical significance was defined as P ≤ 0.05.

Results

Body mass, heart morphometrics

Body masses were not different between SHAM and HF-rEF rats (Table 1). The ratios of the RV, LV, and atria mass to body mass were greater in HF-rEF rats compared to SHAM rats. Additionally, LVEDV and LVESV were significantly greater, and ejection fraction and fractional shortening were significantly lower, in HF-rEF rats compared to SHAM rats. There was no difference in SV between groups.

Table 1.

Body and tissue weights and heart morphometrics in SHAM and HF-rEF rats

	SHAM	HF-rEF	P-value
Body weight (g)	460 (113)	444 (126)	0.650
Lung/body weight (mg/g)	3.22 (0.48)	3.55 (0.63)	0.049*
RV/body weight (mg/g)	0.51 (0.09)	0.57 (0.06)	0.032*
LV/body weight (mg/g)	1.99 (0.17)	2.15 (0.25)	0.015*
Atria/body weight (mg/g)	0.15 (0.04)	0.21 (0.06)	0.004*
LV EDV (mL)	0.96 (0.26)	1.94 (0.82)	<0.001*
LV ESV (mL)	0.19 (09)	1.20 (0.89)	<0.001*
Stroke volume (mL)	0.78 (0.26)	0.80 (0.32)	0.807
Fractional shortening (%)	43 (9)	20 (13)	<0.001*
Ejection fraction (%)	80 (9)	45 (19)	<0.001*
LV infarct size (%)	-	29 (6)	-

Values are mean (SD). LV, left ventricle; RV, right ventricle; EDV, end diastolicvolume; ESV, end systolic volume. Data were compared using student’s t-test. Asterisks indicate a statistically significantdifference between groups (P<0.05).

Effect of HF-rEF on the mechanoreflex and exercise pressor reflex

Pooled data from control conditions across all experiments in the present investigation in which hindlimb muscle stretch was performed (n=15 SHAM, n=21 HF-rEF rats) indicate that, compared to SHAM rats, HF-rEF rats had significantly larger pressor (peak ΔMAP, SHAM: 19 (13); HF-rEF: 31 (17) mmHg; P=0.016), and sympathetic (peak ΔRSNA, SHAM: 70 (70); HF-rEF: 112 (68) %; P=0.040) responses to the 30 s stretch maneuver. When comparing the control responses to 30 seconds of hindlimb contraction between SHAM and HF-rEF rats, no statistically significant differences were observed between the groups for either the pressor (peak ΔMAP: SHAM: 19 (7); HF-rEF: 16 (7) mmHg; P=0.473) or sympathetic response (peak ΔRSNA: SHAM: 56 (37); HF-rEF: 69 (50) %; P=0.096, see the experimental considerations paragraph in the discussion section for further comments). There was no difference between SHAM and HF-rEF rats for the tension developed during the stretch maneuver (ΔTTI, SHAM: 16 (3); HF-rEF 16 (3) kg⋅s; P=0.420) or the contraction maneuver (ΔTTI, SHAM: 12 (3); HF-rEF 17 (6) kg⋅s; P=0.129).

Effect of 1,4,5-trisphosphate (IP3) receptor blockade on isolated mechanoreflex activation

Injection of the IP₃ receptor antagonist Xestospongin C into the arterial supply of the hindlimb had no effect on the time course of the RSNA or pressor response to hindlimb skeletal muscle stretch in SHAM (n=8, 5M/3F) or HF-rEF rats (n=5, 3M/2F; Fig. 1). The ∫ΔRSNA and the ∫ΔRSNA_{5 Sec} as well as the Peak ΔMAP and BPI of the stretch maneuver before and after Xestospongin C injection in SHAM and HF-rEF rats are shown in Fig. 2. The tension developed during the stretch maneuver was not different before and after Xestospongin C (Table 2). An example of original recordings showing the RSNA and pressor response to stretch before and after Xestospongin C in a HF-rEF rat is shown in Fig. 5A. Baseline MAP, baseline HR, and Peak ΔHR before and after Xestospongin C in SHAM and HF-rEF rats are shown in Table 3.

Figure 1:

Effect of inositol 1,4,5-trisphosphate (IP₃) receptor blockade with Xestospongin C on the time course of isolated mechanoreflex activation. The Δ renal sympathetic nerve activity (Δ RSNA, A and B) and Δ mean arterial pressure (Δ MAP, C and D) response to 30 s of 1 Hz hindlimb skeletal muscle stretch before and after injection of the IP₃ receptor antagonist Xestospongin C (5 μg) into the arterial supply of the hindlimb of SHAM (left, n=5M/3F) and HF-rEF (right, n=3M/2F) rats. Data were analyzed with two-way repeated-measures ANOVA and Šidák multiple comparisons tests. Asterisks indicate statistically significant differences between conditions (P < 0.05).

Figure 2:

Effect of inositol 1,4,5-trisphosphate (IP₃) receptor blockade with Xestospongin C on isolated mechanoreflex activation. The integrated Δ in RSNA during the 1 Hz hindlimb skeletal muscle stretch maneuver for the full 30 s (∫ΔRSNA, A) and first 5 seconds (∫ΔRSNA_{5 Sec}, B), as well as the peak change in mean arterial pressure (Peak ΔMAP, C) and blood pressure index (BPI, D) response to the stretch maneuver before and after injection of the IP₃ receptor antagonist Xestospongin C (5 μg) into the arterial supply of the hindlimb of SHAM (n=5M/3F) and HF-rEF (n=3M/2F) rats. Data were analyzed with multiple t-tests and are expressed as mean values overlaid with individual responses (males in closed triangles, females in open circles). Asterisks indicate statistically significant differences between conditions (P < 0.05).

Table 2.

Baseline MAP, baseline RSNA, baseline HR, and Peak ΔHR in SHAM and HF-rEF rats.

	Pharmacological agent	n	Control	Postcondition	P value
Baseline MAP, mmHg
SHAM stretch	5μg Xestospongin C i.a.	5M/3F	88 (20)	79 (17)	0.116
HF-rEF stretch	5μg Xestospongin C i.a.	3M/2F	85 (7)	91 (16)	0.272
SHAM stretch	44μg PKCε i.a.	3M/3F	92 (29)	94 (25)	0.599
HF-rEF stretch	44μg PKCε i.a.	4M/3F	87 (11)	82 (25)	0.464
HF-rEF stretch	44μg PKCε i.v.	3M/1F	84 (10)	87 (14)	0.596
HF-rEF stretch	2.25% DMSO i.a.	1M/3F	79 (1)	87 (7)	0.307
HF-rEF lactic acid injection	44μg PKCε i.a.	1M/4F	81 (22)	81 (33)	0.966
HF-rEF α,β-methylene ATP injection	44μg PKCε i.a.	2M/2F	94 (26)	81 (15)	0.903
HF-rEF bradykinin injection	44μg PKCε i.a.	2M/2F	93 (4)	95 (4)	0.586
SHAM contraction	44μg PKCε i.a.	3M/2F	93 (13)	94 (20)	0.851
HF-rEF contraction	44μg PKCε i.a.	5M/1F	87 (15)	88 (12)	0.793
Baseline RSNA, V
SHAM stretch	5μg Xestospongin C i.a.	5M/3F	0.08 (0.09)	0.08 (0.08)	0.630
HF-rEF stretch	5μg Xestospongin C i.a.	3M/2F	0.02 (0.03)	0.03 (0.04)	0.233
SHAM stretch	44μg PKCε i.a.	3M/3F	0.04 (0.04)	0.05 (0.04)	0.047*
HF-rEF stretch	44μg PKCε i.a.	4M/3F	0.04 (0.03)	0.04 (0.03)	0.800
HF-rEF stretch	44μg PKCε i.v.	3M/1F	0.03 (0.04)	0.03 (0.04)	0.999
HF-rEF stretch	2.25% DMSO i.a.	1M/3F	0.04 (0.02)	0.05 (0.03)	0.161
HF-rEF lactic acid injection	44μg PKCε i.a.	1M/4F	0.05 (0.03)	0.05 (0.03)	0.152
HF-rEF α,β-methylene ATP injection	44μg PKCε i.a.	2M/2F	0.04 (0.03)	0.04 (0.03)	0.357
HF-rEF bradykinin injection	44μg PKCε i.a.	2M/2F	0.05 (0.03)	0.05 (0.04)	0.765
SHAM contraction	44μg PKCε i.a.	3M/2F	0.06 (0.02)	0.05 (0.02)	0.327
HF-rEF contraction	44μg PKCε i.a.	5M/1F	0.03 (0.03)	0.04 (0.03)	0.330
Baseline HR, bpm
SHAM stretch	5μg Xestospongin C i.a.	5M/3F	459 (31)	474 (23)	0.261
HF-rEF stretch	5μg Xestospongin C i.a.	3M/2F	445 (36)	468 (38)	0.325
SHAM stretch	44μg PKCε i.a.	3M/3F	476 (41)	481 (34)	0.542
HF-rEF stretch	44μg PKCε i.a.	4M/3F	463 (34)	459 (48)	0.730
HF-rEF stretch	44μg PKCε i.v.	3M/1F	467 (24)	476 (22)	0.013*
HF-rEF stretch	2.25% DMSO i.a.	1M/3F	469 (15)	469 (20)	0.847
HF-rEF lactic acid injection	44μg PKCε i.a.	1M/4F	485 (66)	482 (63)	0.898
HF-rEF α,β-methylene ATP injection	44μg PKCε i.a.	2M/2F	507 (14)	512 (18)	0.166
HF-rEF bradykinin injection	44μg PKCε i.a.	2M/2F	455 (50)	449 (52)	0.414
SHAM contraction	44μg PKCε i.a.	3M/2F	409 (49)	408 (40)	0.967
HF-rEF contraction	44μg PKCε i.a.	5M/1F	446 (24)	442 (22)	0.425
Peak ΔHR, bpm
SHAM stretch	5μg Xestospongin C i.a.	5M/3F	8 (6)	9 (11)	0.924
HF-rEF stretch	5μg Xestospongin C i.a.	3M/2F	10 (4)	12 (11)	0.728
SHAM stretch	44μg PKCε i.a.	3M/3F	9 (10)	11 (10)	0.533
HF-rEF stretch	44μg PKCε i.a.	4M/3F	24 (24)	9 (11)	0.221
HF-rEF stretch	44μg PKCε i.v.	3M/1F	14 (10)	9 (10)	0.085
HF-rEF stretch	2.25% DMSO i.a.	1M/3F	14 (8)	14 (12)	0.812
HF-rEF lactic acid injection	44μg PKCε i.a.	1M/4F	3 (2)	2 (2)	0.895
HF-rEF α,β-methylene ATP injection	44μg PKCε i.a.	2M/2F	1 (2)	2 (2)	0.391
HF-rEF bradykinin injection	44μg PKCε i.a.	2M/2F	6 (6)	10 (10)	0.414
SHAM contraction	44μg PKCε i.a.	3M/2F	22 (11)	36 (25)	0.158
HF-rEF contraction	44μg PKCε i.a.	5M/1F	11 (10)	11 (7)	0.956

Values are mean (SD). MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity; HR, heart rate. Data were compared with Student’s t-tests. Asterisks indicate statistically significant differences between conditions (P < 0.05).

Figure 5:

Original tracings from two HF-rEF rats showing the renal sympathetic and blood pressure response to 30 s of 1 Hz hindlimb skeletal muscle stretch before (left) and after (right) injecting either the inositol 1,4,5-trisphosphate (IP₃) receptor inhibitor Xestospongin C (5μg; Panel A) or the PKCε inhibitor PKCe141 (45 μg; Panel B) into the arterial supply of the hindlimb.

Table 3.

Tension-time index in SHAM and HF-rEF rats.

	Pharmacological agent	n	Control	Postcondition	P value
ΔTTI, kg⋅s
SHAM stretch	5μg Xestospongin C i.a.	5M/3F	17 (6)	16 (3)	0.563
HF-rEF stretch	5μg Xestospongin C i.a.	3M/2F	17 (4)	17 (4)	0.631
SHAM stretch	44μg PKCε i.a.	3M/3F	16 (3)	16 (3)	0.203
HF-rEF stretch	44μg PKCε i.a.	4M/3F	18 (3)	19 (3)	0.520
HF-rEF stretch	44μg PKCε i.v.	3M/1F	15 (2)	16 (2)	0.189
HF-rEF stretch	2.25% DMSO i.a.	1M/3F	14 (2)	14 (2)	0.167
SHAM contraction	44μg PKCε i.a.	3M/2F	12 (4)	12 (4)	0.986
HF-rEF contraction	44μg PKCε i.a.	5M/1F	17 (5)	16 (5)	0.206

Values are mean (SD). ΔTTI, tension-time index. Data were compared with Student’s t-tests.

Effect of protein kinase C epsilon (PKCε) inhibition on isolated mechanoreflex activation

Injection of the PKCε inhibitor PKCe141 into the arterial supply of the hindlimb significantly reduced the time course of the RSNA response to hindlimb skeletal muscle stretch in HF-rEF (n=7, 4M/3F) and SHAM (n=6, 3M/3F; Fig. 3) rats. However, the time course of the MAP response was only reduced in HF-rEF rats. The ∫ΔRSNA and the ∫ΔRSNA_{5 Sec} of the stretch maneuver was significantly reduced after PKCe141 in HF-rEF rats but not SHAM rats (Fig. 4A & B). The ∫ΔRSNA and the ∫ΔRSNA_{5 Sec} as well as the Peak ΔMAP and BPI of the stretch maneuvers before and after PKCe141 injection in SHAM and HF-rEF rats are shown in Fig. 4. The tension developed during the stretch maneuver was not different before and after injection of PKCe141 (Table 2). An example of original recordings from a HF-rEF rat showing the RSNA and pressor response to stretch before and after PKCe141 is shown in Fig. 5B. Baseline MAP, baseline HR, and Peak ΔHR before and after PKCe141 injection in SHAM and HF-rEF rats are shown in Table 3.

Figure 3:

Effect of protein kinase C epsilon (PKCε) receptor inhibition with PKCe141 on the time course of isolated mechanoreflex activation. The Δ renal sympathetic nerve activity (Δ RSNA, A and B) and Δ mean arterial pressure (Δ MAP, C and D) response to 30 s of 1 Hz hindlimb skeletal muscle stretch before and after injection of the PKCε inhibitor PKCe141 (45 μg) into the arterial supply of the hindlimb of SHAM (left, n=4M/3F) and HF-rEF (right, n=4M/3F) rats. Data were analyzed with two-way repeated-measures ANOVA and Šidák multiple comparisons tests. Asterisks indicate statistically significant differences between conditions (P < 0.05).

Figure 4:

Effect of protein kinase C epsilon (PKCε) inhibition with PKCe141 on isolated mechanoreflex activation. The integrated Δ in RSNA during the 1 Hz hindlimb skeletal muscle stretch maneuver for the full 30 s (∫ΔRSNA, A) and first 5 seconds (∫ΔRSNA_{5 Sec}, B), as well as the peak change in mean arterial pressure (Peak ΔMAP, C) and blood pressure index (BPI, D) response to the stretch maneuver before and after injection of the PKCε inhibitor PKCe141 (45 μg) into the arterial supply of the hindlimb of SHAM (n=4M/3F) and HF-rEF (n=4M/3F) rats. Data were analyzed with multiple t-tests and are expressed as mean values overlaid with individual responses (males in closed triangles, females in open circles). Asterisks indicate statistically significant differences between conditions (P < 0.05).

In systemic control experiments in HF-rEF rats, injection of PKCe141 into the jugular vein had no effect on the time course of the RSNA or pressor response to 1 Hz dynamic hindlimb skeletal muscle stretch (n=4, 3M/1F; Fig. 6A & C). In a different group of HF-rEF rats (n=4, 1M/3F), injection of 0.2mL saline containing 2.25% DMSO into the arterial supply of the hindlimb had no effect on the time course of the RSNA or pressor response to 1 Hz dynamic hindlimb skeletal muscle stretch (Fig. 6B & D). Tension developed during the stretch maneuver was not different before and after i.v. injection of PKCe141 or i.a. injection of the vehicle for PKCe141 (Table 2). Baseline MAP, baseline HR, and Peak ΔHR are shown in Table 3.

Figure 6:

Systemic and vehicle controls for the protein kinase C epsilon (PKCε) inhibitor PKCe141 on isolated mechanoreflex activation. The Δ renal sympathetic nerve activity (Δ RSNA, A and B) and Δ mean arterial pressure (Δ MAP, C and D) response to 30 s of 1 Hz hindlimb skeletal muscle stretch in HF-rEF rats before and after intravenous injection (jugular vein) of the PKCε receptor antagonist PKCe141 (45 μg; n=3M/2F) as well as before and after injection of the vehicle for the PKCε inhibitor PKCe141 into the arterial supply of the hindlimb (i.e., 0.2mL saline containing 2.25% DMSO; n=1M/3F). Data were analyzed with two-way repeated-measures ANOVA and Šidák multiple comparisons tests. Asterisks indicate statistically significant differences between conditions (P < 0.05).

Effect of PKCε inhibition on RSNA and pressor responses to lactic acid, α,β-methylene ATP and bradykinin injection

Injection of the PKCε inhibitor PKCe141 into the arterial supply of the hindlimb had no effect on the Peak ΔRSNA or Peak ΔMAP response to hindlimb arterial injection of 24 mMol lactic acid (n=5, 1M/4F), 20 μg of α,β-methylene ATP (n=4, 2M/2F), or 5 μg of bradykinin (n=4, 2M/2F)(Fig. 7). Baseline MAP, baseline HR, and Peak ΔHR before and after PKCe141 injection in HF-rEF rats are shown in Table 3.

Figure 7:

Effect of protein kinase C epsilon (PKCε) inhibition with PKCe141 on isolated metaboreflex activation in HF-rEF rats. The peak Δ in mean arterial pressure (Peak ΔMAP, A) and peak Δ in renal sympathetic nerve activity (Peak ΔRSNA, B) response to hindlimb arterial injection of lactic acid (24mMol; n=1M/4F), α,β-methylene ATP (20 μg; n=2M/2F), and bradykinin (5 μg; n=2M/2F) before and after injection of the PKCε inhibitor PKCe141 (45 μg) into the arterial supply of the hindlimb. Data were analyzed with multiple t-tests and are expressed as mean values overlaid with individual responses (males in closed triangles, females in open circles). Asterisks indicate statistically significant differences between conditions (P < 0.05).

Effect of PKCε inhibition on the exercise pressor reflex

Injection of PKCe141 into the arterial supply of the hindlimb significantly reduced the time course of the RSNA and pressor response to muscle contraction in HF-rEF rats (n=5M/1F), but not SHAM rats (n=3M/2F; Fig. 8). The ∫ΔRSNA and the ∫ΔRSNA_{5 Sec} as well as the Peak ΔMAP and BPI of the contraction maneuvers before and after PKCe141 injection in SHAM and HF-rEF rats are shown in Fig. 9. The tension developed during the contraction maneuvers was not different before and after injection of PKCe141 (Table 3). Baseline MAP, baseline HR, and Peak ΔHR before and after PKCe141 injection in SHAM and HF-rEF rats are shown in Table 2.

Figure 8:

Effect of protein kinase C epsilon (PKCε) receptor blockade with PKCe141 on the time course of exercise pressor reflex activation. The Δ renal sympathetic nerve activity (Δ RSNA, A and B) and Δ mean arterial pressure (Δ MAP, C and D) response to 30 s of 1 Hz hindlimb skeletal muscle contraction before and after injection of the PKCε inhibitor PKCe141 (45 μg) into the arterial supply of the hindlimb of SHAM (left, n=3M/2F) and HF-rEF (right, n=5M/1F) rats. Data were analyzed with two-way repeated-measures ANOVA and Šidák multiple comparisons tests. Asterisks indicate statistically significant differences between conditions (P < 0.05).

Figure 9:

Effect of protein kinase C epsilon (PKCε) inhibition with PKCe141 on exercise pressor reflex activation. The integrated Δ in RSNA during the 1 Hz hindlimb skeletal muscle stretch maneuver for the full 30 s (∫ΔRSNA, A) and first 5 seconds (∫ΔRSNA_{5 Sec}, B), as well as the peak change in mean arterial pressure (Peak ΔMAP, C) and blood pressure index (BPI, D) response to the contraction maneuver before and after injection of the PKCε inhibitor PKCe141 (45 μg) into the arterial supply of the hindlimb of SHAM (n=3M/2F) and HF-rEF (n=5M/1F) rats. Data were analyzed with multiple t-tests and are expressed as mean values overlaid with individual responses (males in closed triangles, females in open circles). Asterisks indicate statistically significant differences between conditions (P < 0.05).

PKCε protein and mRNA expression in L₄/L₅ DRG

There was no difference between SHAM (n=4M/4F) and HF-rEF (n=5M/5F) rats in total PKCε (t-PKCε) protein or the ratio of phosphorylated PKCε (p-PKCε) protein to t-PKCε protein expression in within L₄/L₅ DRG tissue (Fig. 10A). Similarly, in those same rats, we found no difference between SHAM and HF-rEF rats in PKCε mRNA expression within L₄/L₅ DRG tissue (Fig. 10B).

Figure 10:

Total PKCε (t-PKCε), phosphorylated PKCε (p-PKCε), and the ratio of p-PKCε to t-PKCε protein expression (A) and PKCε mRNA expression within L₄/L₅ DRG tissue from SHAM (n=4M/4F) and HF-rEF (n=5M/5F) rats. GAPDH was used as protein loading control and as a reference gene sample. Top: original western blot images showing staining with anti-PKCε, anti-p-PKCε (Ser-729) and anti-GAPDH of L₄/L₅ DRG tissue SHAM and HF-rEF rats. Data were analyzed with Student’s t test and are expressed as mean overlaid with individual values.

Discussion

This investigation built upon prior research demonstrating the involvement of TxA₂ (Butenas et al., 2021c, b) and B2 receptors (Koba et al., 2010) on thin fiber muscle afferents in the exaggerated mechanoreflex and exercise pressor reflex in HF-rEF rats. Specifically, we investigated the contribution of IP₃ receptors and PKCε, key components of second-messenger signaling associated with Gq protein-coupled TxA₂ and B2 receptors, to the chronic sensitization of mechanically activated channels/afferents underlying mechanoreflex activation in HF-rEF rats. We found that hindlimb arterial injection of the IP₃ receptor antagonist Xestospongin C did not affect the sympathetic and pressor response to hindlimb muscle stretch in HF-rEF rats, whereas hindlimb arterial injection of the PKCε translocation inhibitor PKCe141 attenuated those responses. In SHAM rats, neither Xestospongin C nor PKCe141 injection substantially impacted the RSNA or pressor response to hindlimb muscle stretch. These results suggest that in HF-rEF, PKCε signaling, but not IP3 receptor signaling, within thin fiber muscle afferents contributes to chronic mechanoreflex sensitization.

Xestospongin C, derived from a marine sponge species (Xestospongia; (Gafni et al., 1997)), inhibits IP₃ receptors and prevents the release of intracellular calcium stores into the cytosol across various species (Nakagawa et al., 1984; Ruiz et al., 2009; Sylantyev et al., 2013; Azab et al., 2015). In the SHAM cohort presented herein, similar to our prior findings in healthy rats (Rollins et al., 2021), hindlimb arterial injection of Xestospongin C (5 μg) did not reduce the sympathetic or pressor response to dynamic stretch. Moreover, Xestospongin C (5 μg) injection had no effect on the exaggerated sympathetic or pressor response to dynamic hindlimb muscle stretch in rats with HF-rEF. This lack of effect of Xestospongin C on the mechanoreflex in HF-rEF rats contrasts with our previous finding that Xestospongin C reduced the exaggerated pressor response to dynamic stretch in a rat model of simulated peripheral artery disease (Rollins et al., 2021). Thus, there appear to be differences in the second-messenger signaling pathway(s) associated with G_q protein coupled receptors that mediate chronic mechanoreflex sensitization across rat models of different cardiovascular diseases. Specifically, IP₃ receptor signaling appears necessary for the chronic mechanoreflex sensitization in rats with simulated peripheral artery disease but not in rats with HF-rEF.

PKCe141 is a thienoquinoline that interferes with the binding of PKCε and its anchoring protein, RACK2 (Rechfeld et al., 2014). We found in HF-rEF rats that hindlimb arterial injection of the PKCε translocation inhibitor PKCe141, but not the vehicle for PKCe141, attenuated the sympathetic and pressor response to hindlimb skeletal muscle stretch. This identifies PKCε translocation as a second-messenger signaling component linked to G_q protein coupled TxA₂ and B2 receptors on thin fiber muscle afferents which have been found to contribute to isolated mechanoreflex activation sensitization in this model (Koba et al., 2010; Butenas et al., 2021b). We also extended the isolated mechanoreflex findings to experiments in which the mechanoreflex was activated in a more physiological manner during rhythmic 1 Hz hindlimb skeletal muscle contraction. Similar to the stretch experiments, in the contraction experiments inhibition of PKCε translocation in HF-rEF rats attenuated the sympathetic and pressor response to hindlimb muscle contraction. In contrast, in SHAM rats there was little to no effect of PKCe141 on the sympathetic or pressor response to hindlimb muscle stretch or contraction. Thus, collectively, the present results suggest that PKCε translocation contributes to the chronic sensitization of mechanically activated channels/afferents that underly mechanoreflex activation in HF-rEF. Additionally, we found no difference between SHAM and HF-rEF rats in total PKCε expression or phosphorylated PKCε expression within L₄/L₅ DRG tissue. This suggests that the PKCε translocation/activity induced mechanoreflex sensitization in HF-rEF is not accompanied by elevated PKCε transcription, translation, or phosphorylation.

The present finding that injection of PKCe141 into the jugular vein had no effect on the sympathetic and pressor response to hindlimb muscle stretch in HF-rEF rats suggests that the effect of PKCe141 when injected into the arterial supply of the hindlimb is attributable to PKCε inhibition within the sensory endings of thin fiber muscle afferents and not effects elsewhere in the mechanoreflex arc such as the spinal cord and/or brainstem. We should note, however, that the systemic control experiments were not designed to investigate a possible physiological role for PKCε within the spinal cord or brainstem in HF-rEF. Rather, they were designed only to inform the conclusions of the experiments in which PKCe141 is injected into the arterial supply of the hindlimb. The present finding that PKCe141 had no effect on the sympathetic and pressor response to injection of lactic acid, α,β-methylene ATP, or bradykinin into the arterial supply of the hindlimb suggests that the effect of PKCe141 on the sympathetic and pressor response to stretch and contraction in HF-rEF rats is not attributable to an “off-target” effect that may have produced a generalized reduction in muscle afferent responsiveness.

Several experimental considerations warrant discussion. First, we did not specifically investigate potential sex differences in the magnitude of PKCe-mediated mechanoreflex sensitization. Nonetheless, our study indicates that the effect was observed in both sexes, suggesting it was not driven solely by one sex. Second, the hindlimb muscle stretch model of isolated mechanoreflex activation stimulates mechanically activated channels by passively lengthening the muscle. During the muscle contractions associated with most common forms of exercise, mechanically-sensitive channels on the sensory endings of thin fiber muscle afferents are stimulated when muscle length shortens and intramuscular pressure increases (Gallagher et al., 2001). Although the nature of the stimulus is different, hindlimb muscle stretch in the rat has been shown to stimulate 87% of the same group III muscle afferents as does hindlimb muscle contraction in the healthy rat (Stone et al., 2015). Third, analysis of PKCε mRNA and protein expression in whole DRG tissue may have masked effects of HF-rEF on receptor expression that were limited to subpopulations of group III/IV muscle afferents. Lastly, our study was specifically powered to investigate the effect of pharmacological manipulations within the SHAM and HF-rEF groups. While we detected significantly larger sympathetic and pressor responses to stretch in HF-rEF rats compared to SHAM rats, we did not observe a difference in those responses to contraction. We believe this is due to having a sufficient sample size for the stretch comparison (n=15 SHAM and 21 HF-rEF), but an insufficient sample size to detect a significant difference in the contraction comparison (n=5 SHAM and 6 HF-rEF). Notably, the existing literature consistently demonstrates that reflex sympathetic and/or pressor responses to hindlimb muscle contraction are exaggerated in the rat myocardial infarction-induced model of HF-rEF compared to SHAM counterparts (Smith et al., 2003; Smith et al., 2005; Koba et al., 2008; Butenas et al., 2021c, b).

In summary, we investigated second-messenger signaling components associated with G_q protein coupled receptors which underly the chronic mechanoreflex sensitization in male and female rats with HF-rEF. Our findings described above suggest that intracellular signaling within the sensory ending of thin fiber muscle afferents involving PKCε, but not IP₃ receptors, contribute importantly to the chronic mechanoreflex sensitization and overall exaggerated exercise pressor reflex in male and female rats with HF-rEF. Importantly, excessive activation of thin fiber muscle afferents during exercise in HF-rEF patients (i.e., exaggerated exercise pressor reflex) contributes to impaired exercise tolerance and increases cardiovascular risk. Thus, our findings reveal important mechanisms within the sensory endings of these thin fiber muscle afferents which may carry important functional and clinical implications for HF-rEF patients.

Key Points:

• Skeletal muscle contraction results in exaggerated reflex increases in sympathetic nerve activity in heart failure patients with reduced ejection fraction (HF-rEF) compared to healthy individuals, contributing to increased cardiovascular risk and impaired tolerance for mild exercise.

• The exaggerated reflex sympathetic responses in HF-rEF may be attributed to a chronic sensitization of mechanically sensitive thin fiber muscle afferents mediated, at least in part, by stimulation of Gq protein-coupled thromboxane A₂ and bradykinin B2 receptors on muscle afferent sensory endings.

• The specific Gq protein-linked signaling mechanisms that produce the chronic mechanoreflex sensitization in HF-rEF have not been investigated but may involve inositol 1,4,5-trisphosphate (IP₃) receptors and/or protein kinase C epsilon (PKCε).

• Here we demonstrate that PKCε, but not IP₃ receptors, within the sensory endings of thin fiber muscle afferents plays a role in the sensitization of mechanically sensitive thin fiber muscle afferents in rats with HF-rEF.

First Author Profile

Alec L.E. Butenas earned his PhD from the Department of Kinesiology at Kansas State University in 2023 under the mentorship of Dr. Steven W. Copp. He is currently a postdoctoral research fellow at the Breathing Research and Therapeutics (BREATHE) Center, mentored by Dr. Gordon S. Mitchell. Dr. Butenas’ research interests focus on understanding the mechanisms regulating spinal neural plasticity following acute intermittent hypoxia and harnessing this plasticity to treat clinical disorders that compromise breathing, such as cervical spinal cord injury.

Data Availability Statement:

The data that support the findings of this study are available from the corresponding author upon reasonable request.