Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Nov 21;174(24):4826–4835. doi: 10.1111/bph.14064

Transient receptor potential vanilloid 1 inhibitors block laparotomy‐ and opioid‐induced infarct size reduction in rats

Helen M Heymann 1,†, Yun Wu 1,2,†, Yao Lu 1, Nir Qvit 3, Garrett J Gross 4, Eric R Gross 1,✉
PMCID: PMC5727239  PMID: 28982207

Abstract

Background and Purpose

In light of the opioid epidemic, physicians are increasingly prescribing non‐opioid analgesics to surgical patients. Transient receptor potential vanilloid 1 (TRPV1) inhibitors are potentially alternative pain therapeutics for surgery. Here, we examined in rodents whether the cardioprotection conferred by two common procedures during surgery, a laparotomy or morphine delivery, is mediated by the TRPV1 channel. We further tested whether an experimental analgesic peptide (known as P5) targeted against the TRPV1 C‐terminus region interferes with laparotomy‐ or morphine‐induced cardioprotection.

Experimental Approach

Male Sprague–Dawley rats were subjected to 30 min coronary occlusion followed by 120 min reperfusion. Before ischaemia, a laparotomy with or without capsaicin application (0.1% cream, a TRPV1 activator) was performed. Additional rats were given morphine (0.3 mg·kg−1) with or without capsaicin. In addition, capsazepine (3 mg·kg−1, a classical TRPV1 inhibitor), or P5 (3 mg·kg−1, a peptide analgesic and TRPV1 inhibitor), was given either alone or prior to a laparotomy or morphine administration. Myocardial infarct size was determined.

Key Results

A laparotomy, in addition to combining a laparotomy with capsaicin cream, reduced infarct size versus control. Morphine, in addition to combining morphine administration with capsaicin cream, also reduced infarct size versus control. When TRPV1 inhibitors capsazepine or P5 were given, either TRPV1 inhibitor abolished the infarct size reduction mediated by a laparotomy or morphine.

Conclusions and Implications

Inhibiting the TRPV1 channel blocks laparotomy‐ or morphine‐induced cardioprotection. Impaired organ protection may be a potential pitfall of using TRPV1 inhibitors for pain control.

Abbreviations

AAR/LV

area at risk as a percentage of left ventricle size

CAP

capsaicin

CON

control

CPZ

capsazepine

IS/AAR

infarct size as a percentage of area at risk

LAP

laparotomy

MOR

morphine

TRPV1

transient receptor potential vanilloid 1

Introduction

Opioids are the mainstay of analgesia in surgical patients. However, the associated social and economic impact of opioid abuse, addiction and overdoses are shifting how physicians approach pain control in the operating room. Opioid misuse is a leading public health concern in the United States (Kolodny et al., 2015; Rudd et al., 2016), and trends of increasing opioid abuse and overdoses are developing in the European Union (Novak et al., 2016). In the United Kingdom, opioid prescriptions rose 58% between 2000 and 2010 (Zin et al., 2014) and within this time frame, an increase in opioid‐related deaths was also identified (Giraudon et al., 2013). In response to this epidemic, utilizing non‐opioid analgesics or adjuvants for surgery is becoming a favoured option (Savarese and Tabler, 2017). In addition, finding non‐opioid receptor targets and developing therapeutics to use in synergy with or to replace opioids for pain control remain an active focus for researchers.

The transient receptor potential vanilloid 1 (TRPV1) channel is a novel non‐opioid target that has potential as a treatment for pain in surgical and non‐surgical patients. TRPV1 is a nonspecific cation channel mediating responses to cellular stress including pain by gating calcium (Caterina et al., 1997). Although initially discovered only in neurons, TRPV1 is broadly expressed in non‐neuronal tissues including those found in the kidney, lung, heart and brain. Furthermore, TRPV1 activation reduces ischaemia‐reperfusion injury for these organs (Ueda et al., 2008; Muzzi et al., 2012; Wang et al., 2012; Hurt et al., 2016). Therefore, since TRPV1 is widely expressed and when activated limits ischaemia‐reperfusion injury, it is critical to identify whether inhibiting TRPV1 for pain relief may interfere with the agents or interventions physicians administer in the operating room which can decrease organ injury.

Commonly, in the operating room, patients receive opioids, and during surgery, an incision is performed by a surgeon. Previous work suggests that a type of incision to the abdomen (known as a laparotomy) reduces infarct size in rodent and canine models of myocardial ischaemia‐reperfusion injury (Jones et al., 2009; Gross et al., 2011). Here, we hypothesized that myocardial protection conferred by a laparotomy or morphine delivery is mediated by TRPV1. We used a rodent model of myocardial ischaemia‐reperfusion injury to determine whether TRPV1 is important in mediating myocardial protection provided by either a laparotomy or opioid administration. We further investigated whether TRPV1 inhibitors, including the peptide P5, previously shown as an effective pain reliever experimentally (Valente et al., 2011), and a classical TRPV1 inhibitor capsazepine may limit the cardiac protection afforded by a laparotomy or opioid.

Methods

Animals

Eight‐ to 10‐week‐old male Sprague–Dawley rats (250–300 g; Charles River, USA) were used in these studies. Rats were housed in the facility 1 week prior to the start of experiments to acclimatize them. All rats were housed in a temperature‐, humidity‐ and light‐controlled (12 h cycle) room under standard pathogen‐free housing conditions. Up to three rats were housed in individually‐ventilated cages with at least 2 cm of wood shavings as bedding and free access to food pellets and water. The study protocol was approved by the Animal Care and Use Committee at the Medical College of Wisconsin, Milwuakee, Wisconsin and Stanford University, Stanford, California (AAPLAC 22220). All studies conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015).

Pharmacological agents

Morphine (0.3 mg·kg−1 i.v. bolus; Sigma, St. Louis, MO, USA) was dissolved in saline. Capsazepine (3 mg·kg−1 i.v. bolus; Sigma), the classical TRPV1 inhibitor, was dissolved in DMSO. Capsaicin (CAP) cream (0.1%; CVS Pharmacy, Woonsocket, Rhode Island, USA) was administered on the abdomen. The doses of morphine and capsazepine were determined from previous studies using our rodent myocardial ischaemia‐reperfusion model (Gross et al., 2009; Small et al., 2015; Hurt et al., 2016).

P5 (3 mg·kg−1 i.v. bolus) was synthesized by our laboratory using a Liberty peptide synthesizer. Purity was determined at greater than 95% by HPLC. The P5 sequence, discovered and previously published by another research group, is part of the TRP domain, a highly conserved region of the C terminus adjacent to the inner pore (Figure 1A; Valente et al., 2011). To allow for intracellular entry, the sequence was conjugated to the cell‐penetrating peptide TAT47–57 (Figure 1B). The peptide was dissolved in saline.

Figure 1.

Figure 1

Open in a new tab

Peptide P5, a TRPV1 inhibitor. (A) Crystal structure of the TRPV1 subunit. P5 is a 12 amino acid sequence of the TRP domain, a highly conserved region in the receptor C terminus next to the TRPV1 inner pore‐forming unit. (B) The P5 peptide was synthesized and conjugated to a partial TAT sequence consisting of amino acids 47–57 to allow for intracellular entry (Sweitzer et al., 2004).

Surgical preparation

The protocol for rodent preparation and cardiac ischaemia‐reperfusion experiments has been previously described in detail (Gross et al., 2013b; Small et al., 2015). Surgical procedures were performed between 9:00 and 11:00 h during weekdays. Briefly, rats were anaesthetized with inactin (thiobutabarbital, 100 mg·kg−1 i.p.; Sigma), placed on a heating pad, and a tracheotomy was performed. Rats were ventilated (30 to 40 breaths·min−1; tidal volume, 8 mL·kg−1), and the ventilator was adjusted to maintain a normal pH (7.35 to 7.45) and end‐tidal carbon dioxide (35 to 45 mmHg) by using a blood gas machine (Radiometer ABL‐80; Radiometer America, Brea, CA, USA). Body temperature was monitored with a rectal thermometer (Thermalert TH‐5; Physitemp Instruments, Clifton, NJ, USA) and maintained at 36 to 38°C by using heating pads and heat lamps. Catheters were placed in the carotid artery and jugular vein for measurement of systemic blood pressure, heart rate and blood gases and for administration of drugs or vehicle (Figure 2A).

Figure 2.

Figure 2

Open in a new tab

Experimental protocol: (A) graphical description of the rat myocardial infarction protocol. (B) Representative heart staining for infarct size determination. First, the left anterior descending artery was again occluded and the area at risk was negatively stained by patent blue dye (left). After the left ventricle was sliced into equal cross sections, the tissue was stained by triphenyltetrazolium chloride where viable tissue turned red and nonviable infarcted tissue remained white (right). (C) Experimental protocol for laparotomy studies. After 30 min at baseline, all rats were subjected to 30 min of coronary artery occlusion followed by 2 h of reperfusion. In the laparotomy groups, rats were treated with LAP, CAP or LAP + CAP respectively 15 min prior to 30 min of ischaemia, labelled with a blue arrow in the figure. In a subset of groups, the TRPV1 inhibitor CPZ or P5 was administered 10 min prior to laparotomy or alone 25 min prior to ischaemia, labelled with a grey arrow. (D) Experimental protocol for morphine studies. MOR or MOR + CAP was administered 5 min prior to ischaemia, labelled with a red arrow in the figure. In a subset of groups, the TRPV1 inhibitor capsazepine or P5 was administered 10 min prior to morphine or alone 15 min prior to ischaemia, labelled with a grey arrow. BL, baseline; Isc, ischaemia; Rep, reperfusion.

The heart was exposed by a left thoracotomy in the fourth intercostal space. The left anterior descending coronary artery was isolated, and a suture (6–0 prolene; Ethicon, Somerville, USA) was placed around it to induce ischaemia‐reperfusion. After surgical manipulation and adjustment of the ventilator settings based on blood gas analysis, rodents were allowed to stabilize for 30 min before initiation of the experimental protocol. The hearts were subjected to 30 min of left anterior descending coronary artery occlusion followed by 2 h of reperfusion. After reperfusion, the left anterior descending coronary artery was again occluded, and the heart was negatively stained for the area at risk by injection of patent blue dye (Sigma) through the internal jugular vein. The heart was then excised, both atria and the right ventricle were removed and the left ventricle was cut into five equal slices to create cross sections from apex to base. The slices were separated into normal zone and area at risk, both followed by incubation in 1% triphenyltetrazolium chloride (Sigma) to measure the viability of myocardial tissue. Viable tissue was stained red, while nonviable tissue remained unstained or white (Figure 2B). Infarct size as a percentage of area at risk (IS/AAR) and area at risk as a percentage of left ventricle size (AAR/LV) were determined gravimetrically. Heart rate, blood pressure and rate pressure product were monitored and calculated throughout the experimental protocol using a PowerLab monitoring system (MLS060/8 PowerLab 4/35; ADInstruments, Colorado Springs, CO, USA).

Experimental design

After surgical preparation and stabilization, rats were randomly assigned to different treatment groups involving either laparotomy studies (Figure 2C) or morphine studies (Figure 2D). In all groups, rats were subjected to 30 min of left anterior descending coronary artery occlusion followed by 2 h of reperfusion. Blinding was undertaken when possible in experiments and data analysis; however, it was not feasible to blind the operator with regard to performing or not performing a laparotomy.

In our first series, a laparotomy was performed. This was conducted by performing a 4 cm transverse skin incision via the abdominal midline of the rats with a scalpel similar to previously described protocols (Gross et al., 2013a,b). Additionally, we applied topical capsaicin cream on the abdomen while performing a laparotomy or gave capsaicin cream alone. For subsets of these groups, the TRPV1 inhibitor capsazepine or the TRPV1 inhibitor P5 was given 10 min prior to an abdominal incision or alone 25 min prior to ischaemia.

For the morphine studies, morphine was administered 5 min prior to ischaemia. We also gave capsaicin cream and morphine together; with the capsaicin cream applied on the abdomen immediately followed by morphine administration. TRPV1 inhibitors capsazepine or P5 were also administered 10 min prior to morphine or alone 15 min prior to ischaemia.

Statistical analysis

Based on our previous studies and by using a power analysis with α = 0.05 and 80% power, a minimum of six experiments are required to detect at least a 15% difference in myocardial infarct size between groups (Gross et al., 2009). All data are shown as mean ± SEM. Differences between groups in IS/AAR, AAR/LV and haemodynamic parameters were compared by a one‐way ANOVA followed by Bonferroni correction for multiplicity. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). A P < 0.01 was considered statistically significant and denoted by * or # throughout the manuscript. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015).

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b).

Results

A total of 95 rats were used for 90 successful experiments. Three rats were excluded from the capsazepine alone group in the morphine studies secondary to intractable ventricular fibrillation during ischaemia. One rat in the P5 alone group of the laparotomy studies was excluded secondary to complications with the surgical preparation. One rat was excluded from the capsazepine plus morphine group secondary to inadequate release of the suture during reperfusion. For the completed studies, no statistical differences in haemodynamics including heart rate, blood pressure and rate pressure product occurred in any of the treatment groups (Table 1).

Table 1.

Haemodynamics

Group n Baseline 15 min ischaemia 2 h reperfusion
HR MAP RPP HR MAP RPP HR MAP RPP
Laparotomy studies
CON 6 387 ± 12 126 ± 8 57 ± 4 400 ± 11 110 ± 5 51 ± 3 378 ± 9 80 ± 5 40 ± 3
LAP 6 388 ± 12 128 ± 4 59 ± 2 388 ± 11 110 ± 7 50 ± 3 358 ± 12 86 ± 5 39 ± 2
CAP 6 380 ± 17 121 ± 6 55 ± 4 392 ± 22 107 ± 6 49 ± 4 372 ± 22 91 ± 8 41 ± 5
LAP + CAP 6 377 ± 8 124 ± 4 55 ± 2 388 ± 6 115 ± 5 51 ± 3 370 ± 7 94 ± 4 42 ± 2
CPZ + LAP 6 382 ± 9 123 ± 5 55 ± 2 363 ± 14 107 ± 8 44 ± 4 355 ± 13 80 ± 5 36 ± 2
P5 + LAP 6 375 ± 4 133 ± 4 60 ± 2 370 ± 7 111 ± 4 49 ± 2 365 ± 6 95 ± 4 43 ± 1
CPZ 6 394 ± 10 121 ± 4 55 ± 2 406 ± 13 117 ± 8 54 ± 5 380 ± 7 82 ± 2 41 ± 2
P5 6 390 ± 19 112 ± 7 52 ± 4 393 ± 21 103 ± 7 49 ± 5 373 ± 13 85 ± 5 41 ± 3
Morphine studies
CON 6 406 ± 7 100 ± 8 46 ± 4 403 ± 5 100 ± 7 45 ± 3 375 ± 10 83 ± 2 38 ± 2
MOR 6 388 ± 17 98 ± 5 45 ± 2 398 ± 11 96 ± 4 44 ± 2 366 ± 16 70 ± 3 33 ± 1
MOR + CAP 6 399 ± 16 101 ± 6 52 ± 3 405 ± 15 96 ± 8 49 ± 3 374 ± 11 82 ± 5 44 ± 4
CPZ + MOR 6 390 ± 9 113 ± 3 50 ± 2 377 ± 16 96 ± 7 41 ± 2 371 ± 20 69 ± 2 34 ± 3
P5 + MOR 6 421 ± 7 99 ± 6 50 ± 2 401 ± 11 96 ± 11 45 ± 5 366 ± 10 67 ± 1 33 ± 1
CPZ 6 402 ± 7 105 ± 5 48 ± 3 405 ± 5 105 ± 8 47 ± 3 385 ± 11 80 ± 5 37 ± 3
P5 6 417 ± 13 99 ± 6 50 ± 2 406 ± 6 85 ± 12 41 ± 5 382 ± 8 74 ± 7 35 ± 2
Open in a new tab

Groups, number of animals per group (n) and haemodynamics acquired for the studies. HR, MAP and RPP (defined as the product of HR and systolic blood pressure) were assessed at baseline, during ischaemia and at 2 h of reperfusion. Data are presented as mean ± SEM (n = 6). No significant differences were found comparing each group to the respective control group. HR, heart rate; MAP, mean arterial pressure; n, number of animals per group; RPP, rate pressure product.

A laparotomy performed prior to cardiac ischaemia‐reperfusion reduced myocardial infarct size versus untreated rodents [LAP, 44 ± 2%* vs. control (CON), 66 ± 1%; Figure 3A]. Interestingly, the infarct size reduction afforded by a laparotomy could be mimicked by applying capsaicin cream to the abdomen (CAP, 49 ± 1%* vs. CON, 66 ± 1%; Figure 3A). When given together, the combination of an incision and capsaicin was not statistically different (LAP + CAP, 40 ± 2% vs. LAP, 44 ± 2%; Figure 3A). No statistically significant differences in AAR/LV were noted for these treatment groups (Figure 3B).

Figure 3.

Figure 3

Open in a new tab

Laparotomy studies: laparotomy‐induced reduction of myocardial infarct size is mediated by TRPV1. (A) IS/AAR for rats receiving a laparotomy, the TRPV1 activator capsaicin or a combination of both. Laparotomy or capsaicin reduces infarct size, and the combination of laparotomy and capsaicin induce no further reduction. (B) AAR/LV for corresponding experimental groups showed no statistically significant differences. n = 6 per group, *P < 0.01 versus CON.

Importantly, the administration of the TRPV1 inhibitor capsazepine or P5 blocked the protective effect of a laparotomy (LAP, 44 ± 2% vs. CPZ + LAP, 58 ± 1%#; P5 + LAP, 65 ± 2%#; Figure 4A). Compared to control groups, no significant change in IS/AAR occurred when capsazepine or P5 was given alone. In addition, no statistically significant differences were noted in AAR/LV for the majority of these treatment groups when compared to control (Figure 4B). For the group receiving P5 plus laparotomy, the AAR/LV was significantly less when compared to the laparotomy group alone (LAP, 43 ± 2% vs. P5 + LAP, 34 ± 2%#; Figure 4B).

Figure 4.

Figure 4

Open in a new tab

Laparotomy studies: the administration of either TRPV1 inhibitor capsazepine (CPZ) or P5 blocked cardiac protection afforded by a laparotomy (LAP). (A) IS/AAR for rats receiving a laparotomy, a laparotomy combined with either capsazepine or P5, or capsazepine or P5 given alone. The administration of capsazepine or P5 eliminated cardiac protection generated by a laparotomy. No effect occurred when capsazepine or P5 were given alone. (D) AAR/LV for each corresponding experimental group. n = 6 per group, *P < 0.01 versus CON; #P < 0.01 versus LAP.

We next questioned whether morphine decreases myocardial injury through TRPV1 activation. morphine given prior to ischaemia reduced myocardial infarct size when compared to control (MOR, 37 ± 3%*, vs. CON 61 ± 2%; Figure 5A). When morphine and capsaicin were given together, no further reduction in infarct size was seen compared to giving morphine alone (MOR + CAP, 43 ± 3%, vs. MOR, 37 ± 3%; Figure 5A). No differences in AAR/LV were noted among these groups (Figure 5B).

Figure 5.

Figure 5

Open in a new tab

Morphine studies: morphine‐induced reduction of myocardial infarct size is mediated by TRPV1. (A) IS/AAR for rats receiving morphine (MOR) or a combination of morphine and the TRPV1 activator capsaicin (CAP). Both morphine and the combination of morphine and capsaicin reduced infarct size. (B) AAR/LV for corresponding groups. n = 6 per group; *P < 0.01 versus CON.

When TRPV1 inhibitors capsazepine or P5 were given before morphine, the ability of morphine to decrease myocardial injury was blocked (MOR, 37 ± 3% vs. CPZ + MOR, 62 ± 3%#; P5 + MOR, 58 ± 2%#; Figure 6A). No effect on myocardial infarct size was noted when either TRPV1 inhibitor was given alone. No significant differences were noted in AAR/LV for these treatment groups (Figure 6B).

Figure 6.

Figure 6

Open in a new tab

Morphine studies: (A) IS/AAR for rats receiving morphine (MOR), morphine combined with either TRPV1 inhibitor capsazepine (CPZ) or P5, or capsazepine or P5 alone. Administration of TRPV1 inhibitors capsazepine and P5 eliminated cardiac protection generated by morphine. No effect occurred when capsazepine or P5 were given alone. (B) AAR/LV for corresponding experimental groups. n = 6 per group; *P < 0.01versus CON; #P < 0.01 versus morphine.

Discussion

The importance of this study is that TRPV1 inhibitors may block organ‐protecting mechanisms activated by common operating room procedures and drugs administered intraoperatively. For this study, we describe how TRPV1 activation is essential for laparotomy‐ or morphine‐induced cardioprotection. Topical capsaicin (exclusively activating TRPV1 (Caterina et al., 1997)) or an abdominal incision significantly reduced myocardial infarct size. The combinations of abdominal incision with topical capsaicin as well as intravenous morphine with topical capsaicin did not further decrease infarct size, respectively, suggesting both interventions mediate cardioprotection specifically through TRPV1. Importantly, inhibiting TRPV1 via capsazepine or the peptide P5 abolished protection afforded by a laparotomy or morphine delivery.

Previously, COX inhibitors emerged as promising analgesic alternatives. However, acetylsalicylic acid and COX‐2 inhibitors were shown to inhibit ischaemic preconditioning‐induced and opioid‐induced benefits of the myocardium in experimental models (Shinmura et al., 2000, 2002, 2003). Analysis of the celecoxib Long‐term Arthritis Safety Study (CLASS) and vioxx Gastrointestinal Outcomes Research (VIGOR) clinical trials further demonstrated an increased myocardial infarction rate in patients taking COX‐2 inhibitors when compared to a placebo group from a separate primary prevention trial (Mukherjee et al., 2001). Since crosstalk exists between pain signalling pathways and cardioprotection, as shown in this example, the contribution of TRPV1 in mediating cardiac protection specifically and organ protection in general must be considered prior to developing non‐narcotic analgesics for clinical use that directly block TRPV1 channels.

Here, we describe how the peptide P5, which mitigates pain behaviour in rodents (Valente et al., 2011), inhibits cardioprotection by either a laparotomy or intravenous opioid administration. Protein–protein interactions are involved in the coupling of the stimulus causing TRPV1 channel gating and these interactions can be blocked by peptides mimicking a specific binding epitope. For P5, a sequence of the TRPV1 domain located near the inner pore forming unit, blocking this region inhibits TRPV1 activation by capsaicin and pH in rat dorsal root ganglion neurons grown in vitro and capsacin‐induced knee joint nociceptive fibre activation in mice (Valente et al., 2011). Therefore, the ability of P5 to inhibit cardioprotection by a laparotomy or morphine raises concerns regarding the utility of TRPV1 inhibitors as pain relievers, particularly in people at risk for organ injury. Many TRPV1 inhibitors have not been tested to determine how they may affect organ protection. As general pathways of pain signalling and organ protection are interconnected, impairment of organ protection may be a pitfall of using these drugs as analgesics.

A laparotomy and opioid administration probably share common signalling pathways leading to cardioprotection, and TRPV1 is a major mechanism for both of these cardioprotective modalities. TRPV1 was previously identified in cardiac afferent nerves (Zahner et al., 2003). In TRPV1 knockout mice using an isolated heart protocol, ischaemic preconditioning‐induced protection is abolished compared to wild‐type mice (Zhong and Wang, 2007). These data suggest that the cardioprotective role mediated by TRPV1 is within the heart itself. If cardiac protection was neuron mediated, the ability for ischaemic preconditioning to reduce myocardial infarct size should not be abolished in an isolated heart model. We and others recently identified that TRPV1 is present and functional within the cardiac myocyte (Andrei et al., 2016; Hurt et al., 2016). TRPV1 also modulates myocardial ischaemia‐reperfusion injury through the regulation of mitochondrial membrane potential (Hurt et al., 2016). These findings indicate that TRPV1 within the cardiac myocyte acts as an end‐effector and mediator of myocardial protection from ischaemia‐reperfusion injury.

Although the mechanism of remote conditioning is complex, our previous study suggests that PKCγ and PKCε mediate laparotomy‐induced cardioprotection (Gross et al., 2013b). Further, an abdominal incision leads to translocation of PKCε from the cytosol to the membrane in the myocardium which is blocked in bradykinin receptor knockout mice (Jones et al, 2009). In particular, the triggering of epoxyeicosatrienoic acids (EETs) plays an important role in mediating laparotomy‐induced cardioprotection as part of the bradykinin pathway (Gross et al., 2013a). The neuronal trigger and end effector for remote conditioning in addition to the possible interaction between TRPV1, EETs and the PKC isozymes required for cardioprotection need further exploration.

Besides laparotomy, remote conditioning can be accomplished by a blood pressure cuff, femoral nerve stimulation or an abdominal incision (Heusch et al., 2015). Remote preconditioning by a blood pressure cuff can be easily applied and is not harmful to a person. Although initial smaller studies examining remote preconditioning by a blood pressure cuff showed promising results in regard to cardioprotection (Hoole et al., 2009; Thielmann et al., 2013), two larger clinical trials described no difference in outcomes between remote conditioning versus sham treatment in patients who underwent cardiac surgery (Hausenloy et al., 2015; Meybohm et al., 2015). Among the rationale for these findings that remote conditioning may not be an effective cardioprotective strategy is the possibility that propofol blocks the remote conditioning signal. Further, multiple other cardioprotective agents including opioids and volatile anaesthetics are administered to patients which may have to be considered (Zaugg and Lucchinetti, 2015; Wagner et al., 2016). It is also interesting to note that in patients who underwent percutaneous coronary intervention, morphine produced an additive effect with remote conditioning by a blood pressure cuff which reduced peak troponin I levels and achieved a greater percentage of ST‐segment resolution compared to untreated patients or those who received remote conditioning (Rentoukas et al., 2010). Further, remote conditioning significantly reduced major adverse kidney events at 90 days after cardiac surgery in patients at high risk for acute kidney injury (Zarbock et al., 2017). Taken together, the clinical benefits of remote conditioning are relatively promising, and further research is needed on whether the mechanism of remote conditioning involves TRPV1.

In addition to the heart, the tissue‐protective effects of remote conditioning exist in the brain, lung, kidney, intestine and skeletal muscle (Tapuria et al., 2008; Jensen et al., 2011; Er et al., 2012). Therefore, inhibition of TRPV1 would likely affect endogenous protection in other organs. In the kidney, activation of TRPV1 ameliorates ischaemia‐reperfusion induced acute kidney injury (Chen et al., 2014). Perivascular sensory nerve‐mediated vasodilation was impaired in the mesenteric arteries of TRPV1 knockout mice (Wang et al., 2006). Compared to wild‐type mice, TRPV1 knockout mice also show enhanced local inflammation and acceleration of lipopolysaccharide‐induced sepsis, indirectly causing organ damage (Fernandes et al., 2012). Our findings we present here for the heart may have larger implications and maybe a mechanism in general for organ protection from ischaemia‐reperfusion injury.

Several potential limitations exist within our study. For the rat group that received both P5 and a laparotomy, the AAR/LV was significantly less when compared to the laparotomy group alone. However, a smaller AAR/LV tends to be associated with less infarct size, which likely underestimated rather than overestimated the effect of P5 blocking the laparotomy. Interspecies differences between rats and humans may lead to variability in cardioprotection by a laparotomy or morphine delivery. However, laparotomy‐mediated cardiac protection is also effective in canines (Gross et al., 2011). In addition, opioid‐induced cardioprotection is reported in humans (Murphy et al., 2006; Wong et al., 2010). Additionally, our group size was not powered to differentiate whether a combination of laparotomy with capsaicin may have had subtle additive effects. We speculate that with a larger cohort, these combinations of treatment strategies may perhaps gain significance when compared to the single treatment strategies tested. Further, although infarct size is significantly reduced in rodents receiving a laparotomy or morphine, we did not examine cardiac function for these studies. However, our model used does allow us to study cellular mechanisms involved during myocardial ischaemia‐reperfusion injury and clearly suggests that infarct size reduction by morphine or laparotomy is mediated by a TRPV1‐dependent mechanism. Even with these potential limitations, our study likely has important implications for surgical patients.

It is also important to recognize that although low dose capsaicin (0.1%) applied to the abdomen reduces myocardial injury, a higher dose of capsaicin (such as the 8% capsaicin patch) causes cell death probably secondary to TRPV1‐dependent calcium overload. Intravenous capsaicin administration also has a narrow therapeutic window to induce cardioprotection (Hurt et al., 2016). In this respect, and when considering that TRPV1 inhibitors block organ protection, an alternative strategy for developing drugs against TRPV1 is to indirectly modulate protein interactions with TRPV1 instead of directly modifying TRPV1 itself. This is supported by recent evidence that a novel synthesized peptide, V1‐cal, which inhibits the interaction of calcineurin with TRPV1, reduces pain in experimental pain models (McAllister et al., 2016) and reduces myocardial infarct size during ischaemia‐reperfusion injury (Hurt et al., 2016).

In conclusion, a laparotomy or intravenous morphine reduces myocardial ischaemia‐reperfusion injury via the TRPV1 channel. Blocking TRPV1 channels limits laparotomy‐ or morphine‐induced cardioprotection. A schematic for the suggested signalling process leading to cardioprotection is shown in Figure 7. This intriguing subject needs further study particularly with the increasing use of non‐opioid analgesics during surgery and the current investment in developing TRPV1 inhibitors as pain therapeutics.

Figure 7.

Figure 7

Open in a new tab

Summary figure: a laparotomy or morphine administration activates TRPV1 channels, which subsequently leads to a reduction in myocardial infarct size. The TRPV1 inhibitors capsazepine (CPZ) and P5 abolish cardioprotection induced by these two common perioperative procedures.

Author contributions

G.J.G., E.R.G. and H.M.H. contributed to experimental design. Y.W., Y.L. and N.Q. performed experiments for the study. Y.W. and H.M.H. contributed to data analysis. H.M.H., Y.W. and E.R.G. contributed to writing the manuscript. H.M.H., Y.W., G.J.G. and E.R.G. contributed to revising the paper.

Conflict of interest

E.R.G. holds a patent on peptide modulators of specific calcineurin protein–protein interactions.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

This study was supported by the NIH (NHLBI HL109212 and NIGMS GM119522 to E.R.G.), funding from the Priority Department of the Second Affiliated Hospital of Anhui Medical University (to Y.W.) and a FAER medical student anaesthesia research fellowship (to H.M.H.).

Heymann, H. M. , Wu, Y. , Lu, Y. , Qvit, N. , Gross, G. J. , and Gross, E. R. (2017) Transient receptor potential vanilloid 1 inhibitors block laparotomy‐ and opioid‐induced infarct size reduction in rats. British Journal of Pharmacology, 174: 4826–4835. doi: 10.1111/bph.14064.

References

  1. Alexander SPH, Catterall WA, Kelly E, Marrion N, Peters JA, Benson HE et al (2015a). The Concise Guide to PHARMACOLOGY 2015/16: Voltage‐gated ion channels. Br J Pharmacol 172: 5904–5941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander SPH, Davenport AP, Kelly E, Marrion N, Peters JA, Benson HE et al (2015b). The Concise Guide to PHARMACOLOGY 2015/16: G protein‐coupled receptors. Br J Pharmacol 172: 5744–5869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andrei SR, Sinharoy P, Bratz IN, Damron DS (2016). TRPA1 is functionally co‐expressed with TRPV1 in cardiac muscle: co‐localization at z‐discs, costameres and intercalated discs. Channels (Austin) 10: 395–409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997). The capsaicin receptor: a heat‐activated ion channel in the pain pathway. Nature 389: 816–824. [DOI] [PubMed] [Google Scholar]
  5. Chen L, Marko L, Kassmann M, Zhu Y, Wu K, Gollasch M (2014). Role of TRPV1 channels in ischemia/reperfusion‐induced acute kidney injury. PLoS One 9: e109842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA et al (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Er F, Nia AM, Dopp H, Hellmich M, Dahlem KM, Caglayan E et al (2012). Ischemic preconditioning for prevention of contrast medium‐induced nephropathy: randomized pilot RenPro Trial (Renal Protection Trial). Circulation 126: 296–303. [DOI] [PubMed] [Google Scholar]
  8. Fernandes ES, Liang L, Smillie SJ, Kaiser F, Purcell R, Rivett DW et al (2012). TRPV1 deletion enhances local inflammation and accelerates the onset of systemic inflammatory response syndrome. J Immunol 188: 5741–5751. [DOI] [PubMed] [Google Scholar]
  9. Giraudon I, Lowitz K, Dargan PI, Wood DM, Dart RC (2013). Prescription opioid abuse in the UK. Br J Clin Pharmacol 76: 823–824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gross ER, Hsu AK, Gross GJ (2009). Acute methadone treatment reduces myocardial infarct size via the delta‐opioid receptor in rats during reperfusion. Anesth Analg 109: 1395–1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gross GJ, Baker JE, Moore J, Falck JR, Nithipatikom K (2011). Abdominal surgical incision induces remote preconditioning of trauma (RPCT) via activation of bradykinin receptors (BK2R) and the cytochrome P450 epoxygenase pathway in canine hearts. Cardiovasc Drugs Ther 25: 517–522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gross GJ, Hsu A, Gross ER, Falck JR, Nithipatikom K (2013a). Factors mediating remote preconditioning of trauma in the rat heart: central role of the cytochrome p450 epoxygenase pathway in mediating infarct size reduction. J Cardiovasc Pharmacol Ther 18: 38–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gross ER, Hsu AK, Urban TJ, Mochly‐Rosen D, Gross GJ (2013b). Nociceptive‐induced myocardial remote conditioning is mediated by neuronal gamma protein kinase C. Basic Res Cardiol 108: 381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hausenloy DJ, Candilio L, Evans R, Ariti C, Jenkins DP, Kolvekar S et al (2015). Remote Ischemic Preconditioning and Outcomes of Cardiac Surgery. N Engl J Med 373: 1408–1417. [DOI] [PubMed] [Google Scholar]
  15. Heusch G, Botker HE, Przyklenk K, Redington A, Yellon D (2015). Remote ischemic conditioning. J Am Coll Cardiol 65: 177–195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoole SP, Heck PM, Sharples L, Khan SN, Duehmke R, Densem CG et al (2009). Cardiac Remote Ischemic Preconditioning in Coronary Stenting (CRISP Stent) Study: a prospective, randomized control trial. Circulation 119: 820–827. [DOI] [PubMed] [Google Scholar]
  17. Hurt CM, Lu Y, Stary CM, Piplani H, Small BA, Urban TJ et al (2016). Transient receptor potential vanilloid 1 regulates mitochondrial membrane potential and myocardial reperfusion injury. J Am Heart Assoc 5 pii: e003774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jensen HA, Loukogeorgakis S, Yannopoulos F, Rimpilainen E, Petzold A, Tuominen H et al (2011). Remote ischemic preconditioning protects the brain against injury after hypothermic circulatory arrest. Circulation 123: 714–721. [DOI] [PubMed] [Google Scholar]
  19. Jones WK, Fan GC, Liao S, Zhang JM, Wang Y, Weintraub NL et al (2009). Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase C signaling. Circulation 120: S1–S9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kolodny A, Courtwright DT, Hwang CS, Kreiner P, Eadie JL, Clark TW. (2015). The prescription opioid and heroin crisis: a public health approach to an epidemic of addiction. Annu Rev Public Health 36: 559–574. [DOI] [PubMed] [Google Scholar]
  22. McAllister S, Zambelli V, Gross ER. (2016). Breaking the TRPV1‐Calcineurin Interaction as a Novel Means for Pain Control. FASEB J 30: Supplement 927: 2. [Google Scholar]
  23. McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Meybohm P, Bein B, Brosteanu O, Cremer J, Gruenewald M, Stoppe C et al (2015). A Multicenter Trial of Remote Ischemic Preconditioning for Heart Surgery. N Engl J Med 373: 1397–1407. [DOI] [PubMed] [Google Scholar]
  25. Mukherjee D, Nissen SE, Topol EJ. (2001). Risk of cardiovascular events associated with selective COX‐2 inhibitors. JAMA 286: 954–959. [DOI] [PubMed] [Google Scholar]
  26. Murphy GS, Szokol JW, Marymont JH, Avram MJ, Vender JS (2006). Opioids and cardioprotection: the impact of morphine and fentanyl on recovery of ventricular function after cardiopulmonary bypass. J Cardiothorac Vasc Anesth 20: 493–502. [DOI] [PubMed] [Google Scholar]
  27. Muzzi M, Felici R, Cavone L, Gerace E, Minassi A, Appendino G et al (2012). Ischemic neuroprotection by TRPV1 receptor‐induced hypothermia. J Cereb Blood Flow Metab 32: 978–982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Novak SP, Håkansson A, Martinez‐Raga J, Reimer J, Krotki K, Varughese S (2016). Nonmedical use of prescription drugs in the European Union. BMC Psychiatry 16: 274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rentoukas I, Giannopoulos G, Kaoukis A, Kossyvakis C, Raisakis K, Driva M et al (2010). Cardioprotective role of remote ischemic periconditioning in primary percutaneous coronary intervention: enhancement by opioid action. JACC Cardiovasc Interv 3: 49–55. [DOI] [PubMed] [Google Scholar]
  30. Rudd RA, Aleshire N, Zibbell JE, Gladden RM (2016). Increases in drug and opioid overdose deaths – United States, 2000–2014. MMWR Morb Mortal Wkly Rep 64: 1378–1382. [DOI] [PubMed] [Google Scholar]
  31. Savarese JJ, Tabler NG Jr. (2017). Multimodal analgesia as an alternative to the risks of opioid monotherapy in surgical pain management. J Healthc Risk Manag 37: 24–30. [DOI] [PubMed] [Google Scholar]
  32. Shinmura K, Tang XL, Wang Y, Xuan YT, Liu SQ, Takano H et al (2000). Cyclooxygenase‐2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits. Proc Natl Acad Sci U S A 97: 10197–10202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shinmura K, Nagai M, Tamaki K, Tani M, Bolli R. (2002). COX‐2-derived prostacyclin mediates opioid‐induced late phase of preconditioning in isolated rat hearts. Am J Physiol Heart Circ Physiol 283: H2534–2543. [DOI] [PubMed] [Google Scholar]
  34. Shinmura K, Kodani E, Xuan YT, Dawn B, Tang XL, Bolli R. (2003). Effect of aspirin on late preconditioning against myocardial stunning in conscious rabbits. J Am Coll Cardiol 41: 1183–1194. [DOI] [PubMed] [Google Scholar]
  35. Small BA, Lu Y, Hsu AK, Gross GJ, Gross ER (2015). Morphine reduces myocardial infarct size via heat shock protein 90 in rodents. Biomed Res Int 2015: 129612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SPH et al (2016). The IUPHAR/BPS guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucleic Acids Res 44: D1054–D1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sweitzer SM, Wong SM, Peters MC, Mochly‐Rosen D, Yeomans DC, Kendig JJ (2004). Protein kinase C epsilon and gamma: involvement in formalin‐induced nociception in neonatal rats. J Pharmacol Exp Ther 309: 616–625. [DOI] [PubMed] [Google Scholar]
  38. Tapuria N, Kumar Y, Habib MM, Abu Amara M, Seifalian AM, Davidson BR (2008). Remote ischemic preconditioning: a novel protective method from ischemia reperfusion injury – a review. J Surg Res 150: 304–330. [DOI] [PubMed] [Google Scholar]
  39. Thielmann M, Kottenberg E, Kleinbongard P, Wendt D, Gedik N, Pasa S et al (2013). Cardioprotective and prognostic effects of remote ischaemic preconditioning in patients undergoing coronary artery bypass surgery: a single‐centre randomised, double‐blind, controlled trial. Lancet 382: 597–604. [DOI] [PubMed] [Google Scholar]
  40. Ueda K, Tsuji F, Hirata T, Takaoka M, Matsumura Y. (2008). Preventive effect of TRPV1 agonists capsaicin and resiniferatoxin on ischemia/reperfusion‐induced renal injury in rats. J Cardiovasc Pharmacol 51: 513–520. [DOI] [PubMed] [Google Scholar]
  41. Valente P, Fernandez‐Carvajal A, Camprubi‐Robles M, Gomis A, Quirce S, Viana F et al (2011). Membrane‐tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity. FASEB J 25: 1628–1640. [DOI] [PubMed] [Google Scholar]
  42. Wagner NM, Lu Y, Gross ER. (2016). Remote Ischemic Preconditioning and Cardiac Surgery. N Engl J Med 374: 490. [DOI] [PubMed] [Google Scholar]
  43. Wang LH, Luo M, Wang Y, Galligan JJ, Wang DH (2006). Impaired vasodilation in response to perivascular nerve stimulation in mesenteric arteries of TRPV1‐null mutant mice. J Hypertens 24: 2399–2408. [DOI] [PubMed] [Google Scholar]
  44. Wang M, Ji P, Wang R, Zhao L, Xia Z (2012). TRPV1 agonist capsaicin attenuates lung ischemia‐reperfusion injury in rabbits. J Surg Res 173: 153–160. [DOI] [PubMed] [Google Scholar]
  45. Wong GT, Huang Z, Ji S, Irwin MG (2010). Remifentanil reduces the release of biochemical markers of myocardial damage after coronary artery bypass surgery: a randomized trial. J Cardiothorac Vasc Anesth 24: 790–796. [DOI] [PubMed] [Google Scholar]
  46. Zahner MR, Li DP, Chen SR, Pan HL. (2003). Cardiac vanilloid receptor 1‐expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats. J Physiol 551: 515–523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zarbock A, Kellum JA, Van Aken H, Schmidt C, Küllmar M, Rosenberger P et al (2017). Long‐term Effects of Remote Ischemic Preconditioning on Kidney Function in High‐risk Cardiac Surgery Patients: Follow‐up Results from the RenalRIP Trial. Anesthesiology 126: 787–798. [DOI] [PubMed] [Google Scholar]
  48. Zaugg M, Lucchinetti E. (2015). Remote Ischemic Preconditioning in Cardiac Surgery–Ineffective and Risky? N Engl J Med 373: 1470–1472. [DOI] [PubMed] [Google Scholar]
  49. Zhong B, Wang DH. (2007). TRPV1 gene knockout impairs preconditioning protection against myocardial injury in isolated perfused hearts in mice. Am J Physiol Heart Circ Physiol 293: H1791–1798. [DOI] [PubMed] [Google Scholar]
  50. Zin CS, Chen LC, Knaggs RD (2014). Changes in trends and pattern of strong opioid prescribing in primary care. Eur J Pain 18: 1343–1351. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES