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1Department of Medicine, 2Neuroscience Program, and 3Cell and Molecular Biology Program, Michigan State University, East Lansing, Michigan; and 4Department of Pharmacology, Guangzhou Medical College, Guangzhou, China
Submitted 9 February 2007 ; accepted in final form 18 June 2007
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ABSTRACT |
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transient receptor potential vanilloid type 1; ischemia-reperfusion; substance P; calcitonin gene-related peptide
Capsaicin-sensitive sensory nerves are widely distributed in the cardiovascular system and can be activated by a variety of physical and chemical stimuli. The myocardium and the coronary vascular system possess dense capsaicin-sensitive sensory nerve innervation (14, 48). Activation of capsaicin-sensitive sensory nerves can be brought about by a range of diverse stimuli binding to the capsaicin receptor known as the transient receptor potential vanilloid 1 (TRPV1) channels. The TRPV1 is a nonselective cation channel mainly expressed in primary sensory neurons and sensory C and A
fibers (13), which is now believed to be a molecular integrator of noxious stimuli. The TRPV1 may be activated by physical and chemical mediators including noxious heat, protons, vanilloid compounds (4, 7) such as capsaicin, and lipid metabolites including lipoxygenase products (17, 44). Moreover, immunofluorescence labeling revealed that TRPV1-containing afferent nerve fibers are widely distributed on the epicardial surface of the ventricle (48). During myocardial ischemia, TRPV1-positive sensory nerves integrate and respond to multiple ischemic metabolites and cause chest pain (30). The responses of these sensory nerves include not only transmitting signals to the central nervous system but also releasing sensory neurotransmitters such as substance P (SP) and calcitonin gene-related peptide (CGRP), which coexpress in TRPV1-positive sensory neurons (15, 16).
CGRP is a 37 amino acid neuropeptide, which is found in primary afferent A and C fibers, including those innervating the heart (1, 26). Indeed, CGRP has been recognized as the most potent vasodilatory peptide, which may play an important role in protecting the heart from ischemic injury and damage (1, 26). It has been shown that the protective effects of endogenous CGRP rely on the intact function of capsaicin-sensitive sensory nerves and that capsaicin pretreatment abolishes CGRP protective effects (22). These results indicate that TRPV1-mediated CGRP release may play a key role in regulating CGRP action.
SP colocalizes with CGRP in cardiac C-fiber endings innervating coronary vessels and is released in the heart during ischemia to act as a potent coronary and peripheral vasodilator (2). Moreover, pretreatment of isolated hearts with a SP-receptor antagonist or depletion of sensory neuropeptides impairs postischemic recovery, suggesting a role for endogenously released SP in cardioprotection (43). Also, studies using hearts from TRPV1 gene knockout mice show that TRPV1-dependent SP release contributes to cardiac protection during I/R (45).
Although a previous study from our laboratory showed that TRPV1 gene deletion impairs cardiac recovery after I/R injury (45), it is unknown whether TRPV1 plays a role in PC-induced protection of hearts, given that the molecular mechanisms underlying this process are poorly defined. To test the hypothesis that TRPV1 expressed in sensory nerves innervating the heart plays a key role in PC-induced protection against myocardial injury and the lack of TRPV1 impairs such protection, hearts of gene-targeted TRPV1 null-mutant (TRPV1–/–) or wild-type (WT) mice were used to determine whether ischemic PC activates the TRPV1, resulting in the release of SP and CGRP to convey the beneficial effects of PC against I/R.
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MATERIALS AND METHODS |
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Male TRPV1–/– strain B6.129S4-TRPV1tm1Jul and matching control WT strain C57BL/6J mice were used (Jackson, Bar Harbor, ME). Mice were heparinized (500 U/kg ip) and anesthetized with pentobarbital sodium (50 mg/kg ip). Hearts from TRPV1–/– and WT mice were cannulated and retrogradely perfused at 37°C and 80 mmHg with Krebs-Henseleit buffer [containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25 NaHCO3, 0.5 Na-EDTA, and 11 glucose, saturated with 95% O2-5% CO2, pH 7.4] through the aorta in a noncirculating Langendorff apparatus as described previously (47). A water-filled balloon was inserted into the left ventricle and adjusted to a left ventricular (LV) end-diastolic pressure (LVEDP) of 5 to 8 mmHg. The distal end of the catheter was connected to a Digi-Med heart performance analyzer via a pressure transducer. Coronary flow (CF) was continuously measured using an ultrasonic flow probe placed in the aortic perfusion line. Hearts were paced at 400 beats/min, except during ischemic PC and sustained global ischemia to avoid inducing excessive ventricular tachyarrhythmia during reperfusion, and pacing was reinitiated 2 min after reperfusion. LV developed pressure (LVDP; peak systolic minus LVEDP) and peak-positive maximum rate of rise of LV pressure (+dP/dt) during isovolumic contraction were used as indexes of LV systolic function; LVEDP was used as an index of LV diastolic function. The experiments were approved by the Michigan State University Animal Care and Use Committee.
Experimental Protocols
All hearts were allowed to stabilize for 25 min before three cycles of 5 min of ischemia followed by 5 min of reperfusion. All nonpreconditioned hearts were time-matched perfused with the same duration as that of preconditioned hearts.
Group 1: controls. As normal controls (nonischemic), WT and TRPV1–/– hearts were perfused for 130 min. For I/R controls, WT and TRPV1–/– hearts were first perfused with Krebs-Henseleit buffer for 55 min (time-matched perfusion of the equilibration period plus the PC period) and subsequently subjected to 30 min of no-flow normothermic global ischemia followed by 40 min of reperfusion.
Group 2: PC. WT and TRPV1–/– hearts were perfused with Krebs-Henseleit buffer for 25 min as the equilibration period and subjected to PC with three cycles of 5 min of ischemia followed by 5 min of reperfusion. The vehicle solution was added to the perfusate (at 1% of the CF rate) 5 min before PC and continuously perfused for another 5 min during the first circle of PC. The hearts were subjected to 30 min of no-flow normothermic global ischemia followed by 40 min of reperfusion as that of the I/R control groups.
Group 3: PC plus CGRP8-37. To determine the role of endogenous CGRP during PC, WT and TRPV1–/– hearts were treated with the standard PC protocol as that of group 2, except that CGRP8-37 (10–6 M), a selective CGRP receptor antagonist, instead of vehicle was perfused. Two additional concentrations of CGRP8-37, 10–5 and 10–7 M, were used in WT hearts. An additional drug control of CGRP8-37 was added. After a 20-min equilibration period, WT hearts were perfused with CGRP8-37 (10–6 M) for 15 min without PC, followed by perfusion with Krebs-Henseleit buffer for 20 min (time-matched perfusion of the PC period) and then subjected to I/R.
Group 4: PC plus RP-67580. To determine the role of endogenous SP during PC, hearts from WT and TRPV1–/– mice were perfused as that of CGRP8-37 with RP-67580 (10–7 M), a selective neurokinin-1 (NK1) receptor antagonist. Two additional concentrations of RP-67580, 10–6 and 10–8 M, were used in WT hearts. An additional drug control of RP-67580 (10–7 M) was added as that of the CGRP8-37 group.
Lactate Dehydrogenase Release
In addition to the measurement of cardiac function, cardiac injury was assessed by measuring lactate dehydrogenase (LDH) release. Perfusion effluent was collected during the first 10 to 20 min of I/R and stored at –80°C until analyzed. Total LDH levels were determined with the use of a CytoTox 96 assay (Promega). The data were expressed as absorbance units released per milliliter per minute per gram of heart wet tissues (42).
Measurement of Myocardial Infarct Size
Risk area and infarct size were measured 30 min after postischemia reperfusion. Hearts were perfused for 10 min at a flow rate of 2 ml/min with a 1% 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in Krebs buffer. TTC stained all living tissue brick red and left the infarct area unstained (white). Hearts were then removed from the perfusion system and sliced perpendicularly along the long axis from the apex to base in about 2 mm per section. Sections were incubated for another 10 min at 37°C in 1% TTC. Once the staining was stable, the slices were fixed in 10% formalin for 48 h and weighed. Both sides of each slice were photographed, and the infarct areas in the photos were quantified with the use of the National Institutes of Health software of ImageJ version 1.37v. Given the hearts were subjected to global ischemia, total cross-sectional areas of hearts were defined as total risk areas. The ratio of the infarct area to total risk area (%infarct size) of two sides of each slice was calculated and multiplied by the percent weight of the slice.
Measurement of SP and CGRP
WT and TRPV1–/– hearts were cut into pieces and put into tubes containing 1.5 ml Krebs-Henseleit buffer that was saturated with 95% O2-5% CO2 at 37°C continuously for 30 min (the stabilization period) (45). In the PC group, hearts were subjected to three cyclic episodes of incubation with Krebs-Henseleit buffer [free of gas and D-glucose, using L-(–)-glucose as a substitution] in an anaerobic chamber and aerated with 100% N2 to remove residual oxygen for 10 min (an insult that did not induce myocardial cell death as measured by LDH release), followed by normoxic culture in normal glucose Krebs-Henseleit buffer for 10 min. Additional WT and TRPV1–/– hearts were treated the same as that of the PC group, except that capsazepine (CAPZ; 10–6 M) was added to the solution. In the normal control group, WT and TRPV1–/– hearts were treated the same as that of the PC group, except with no deprivation of oxygen and glucose. The samples were purified and analyzed by radioimmunoassay. The assay was performed as recommended by the supplier. Commercially available rat CGRP and SP radioimmunoassay kits (Peninsula) were used for determination of SP and CGRP release, which was normalized by the heart weight.
Statistical Analysis
All values are expressed as means ± SE. Differences among groups were determined by one-way ANOVA followed by the Tukey-Kramer multiple comparison test. The results were considered statistically significant at P < 0.05.
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RESULTS |
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There were no statistically significant differences in the hemodynamics between groups under baseline and during PC periods (data not shown). PC before I/R prevented the increase in LVEDP in WT hearts, although PC also suppressed further elevation of LVEDP in TRPV1–/– hearts. As a result, PC protected against elevation of LVEDP much more effectively in WT than in TRPV1–/– hearts (Fig. 1). PC enhanced recovery of LVDP, CF, and +dP/dt in WT hearts but not in TRPV1–/– hearts (Fig. 1). Thus hearts from WT hearts showed significantly better preservation of postischemic function by PC, whereas PC had less protective effect on hearts from TRPV1 –/– mice.
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To determine whether endogenous CGRP plays a role in PC-induced cardiac protection, the selective CGRP receptor antagonist CGRP8-37 (10–6 M) was given before and during PC. CGRP8-37 blocked PC-induced cardioprotective effects in WT mice by increasing LVEDP and inhibiting recovery of LVDP, CF, and +dP/dt in WT hearts, but it had no effect on these parameters in TRVR1–/– hearts (Fig. 2). Lower (10–7 M) or higher (10–5 M) concentrations of CGRP8-37 had similar effects on cardiac recovery in WT hearts as that evoked by CGRP8-37 at 10–6 M (data not shown). CGRP8-37 (10–6 M) had no effect on cardiac function in WT hearts without I/R, and CGRP8-37 given 20 min before I/R had no effect on cardiac recovery after I/R (data not shown).
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The effect of endogenous SP on PC-inducing cardiac protection was assessed by pretreatment of the hearts with the NK1 receptor antagonist RP-67580 (10–7 M). The protective effects of PC were abolished in the presence of RP-67580 by increasing LVEDP and decreasing LVDP and +dP/dt, but it had no effect on these parameters in TRVR1–/– hearts (Fig. 3). RP-67580 (10–7 M) had no significant effect on CF in WT or TRVR1–/– hearts. Lower (10–8 M) or higher (10–6 M) concentrations of RP-67580 had similar effects on postischemic recovery in WT hearts as that evoked by RP-67580 at 10–7 M (data not shown). RP-67580 (10–7 M) had no effect on cardiac function in WT hearts without I/R, and RP-67580 given 25 min before I/R had no effect on cardiac recovery after I/R (data not shown).
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LDH levels and infarct areas after I/R were significantly lower in WT hearts with PC compared with WT hearts without PC and TRPV1–/– hearts with or without PC, and these parameters were lower in TRPV1–/– hearts with PC than in TRPV1–/– hearts without PC (Figs. 4 and 5). These results indicate that PC protects hearts against I/R-induced injury in WT hearts, but its protection is impaired in TRPV1–/– hearts, given that the protective effects of PC in WT hearts were much more effective than in TRPV1–/– hearts.
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The release of SP and CGRP at baseline (normal control) was not different between WT and TRPV1–/– hearts. SP release in both WT and TRVR1–/– hearts subjected to PC increased remarkably compared with the baseline (P < 0.05), but the magnitude of the increase was smaller in TRPV1–/– hearts than in WT hearts (Fig. 6). Furthermore, a blockade of the TRPV1 receptor with CAPZ attenuated SP release in WT but not TRPV1–/– hearts subjected to PC, indicating that SP release is partially mediated by the TRPV1 receptor. CGRP release in WT but not TRPV1–/– hearts subjected to PC increased remarkably compared with the baseline (P < 0.05). Furthermore, blockade of the TRPV1 with CAPZ attenuated CGRP release in WT but not TRPV1–/– hearts subjected to PC treatment (Fig. 6).
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DISCUSSION |
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Clinical studies have shown that patients with angina have a lower in-hospital death rate and a smaller infarct size than patients without angina, suggesting that PC by preinfarction angina might render the myocardium more resistant to infarction from the subsequent prolonged ischemic episode (41). TRPV1-positive afferent nerves innervating the heart have been found to be widely distributed on the epicardial surface of the ventricle (48), which can be activated in the setting of myocardial ischemia to mediate the sensation of angina (30). Recent studies also show that TRPV1 directly modulates endothelial function in the brain (12) and nitric oxide (NO) release from endothelial cells in mesenteric arteries (31), suggesting the possibility of TRPV1 action on endothelial cells of coronary arteries. Indeed, TRPV1 has been shown to integrate and respond to multiple ischemic metabolites, serving as a polymodal detector of pain-producing chemical and physical stimuli (30).
It has been shown that PC caused by brief periods of acute myocardial ischemia produces various metabolites, including bradykinin (BK), reactive oxygen species, protons, and arachidonate metabolites, some of which cause CGRP release from TRPV1-positive sensory nerve fibers (11). BK, acting on B2 bradykinin receptors, has been shown to improve LV function by reducing the incidence of arrhythmias, attenuating myocardial necrosis, and preventing apoptosis after myocardial I/R (9). Importantly, BK may excite sensory nerve endings by activating TRPV1 via production of 12-lipoxygenase metabolites of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE). 12-HPETE, structurally similar to capsaicin, is the most potent endogenous TRPV1 agonist (37) that has been reported to protect against I/R injury through activation of TRPV1 (36). The production of 12(S)-hydroeicosatetraenoic acid, an immediate metabolic product of 12-HPETE, is increased during PC to protect against I/R injury in the heart (28). Reactive oxygen species, one of the known PC triggers, may activate TRPV1 via the cyclooxygenase pathway of prostaglandin ethanolamides (33). The intracellular mechanism may involve protein kinase C (PKC), given it has been shown that PKC plays a pivotal role during PC (47), possibility via mediating BK- or ATP-induced sensitization of the TRPV1 (27, 39). Thus metabolites produced by PC may stimulate cardiac afferent nerves through direct or indirect TRPV1 activation, leading to depolarization and the release of sensory neurotransmitters such as CGRP and SP.
Evidence suggests that an ischemic insult capable of inducing TRPV1 activation leads to a release of sensory neuropeptides such as SP, CGRP, and other neurokinins from sensory nerve terminals (10, 25). These neuropeptides produce coronary vasodilation and negative inotropic and chronotropic effects, which would be expected to limit the deleterious consequences of ischemia on the myocardium (35). Our previous study (45) has shown that I/R injury caused more profound impairment in terms of LVEDP, LVDP, and CF in TRPV1–/– than in WT hearts, indicating that TRPV1 protects against I/R injury. Moreover, exogenous CGRR and SP added to the perfusion solution before ischemia improved recovery not only in WT but also in TRPV1–/– hearts (45). These results indicate that the substitution of SP and CGRP before I/R is capable of inducing PC-like protection, observations also supported by others showing that exogenous or endogenous CGRP and SP have cardiac protection (5, 22, 23). Moreover, the facts that PC caused higher SP and CGRP release in WT than in TRPV1–/– hearts and that acute blockade of the TRPV1 decreased SP and CGRP release in WT hearts indicate that these neuropeptides may account for, at least in part, TRPV1-mediated PC protection.
CGRP is one of the most potent vasodilators identified to date in many species (1). In addition to vasodilation, CGRP has been suggested to play a protective role after myocardial infarction and vascular damage (5, 22, 23). A wealth of pharmacological data indicates that depletion of CGRP from sensory nerves by prior pretreatment with capsaicin greatly inhibits or abolishes the protective effect of ischemic PC (22). However, capsaicin induces quite nonspecific depletion of neuropeptides from sensory nerve terminals. To avoid this problem, TRPV1–/– hearts were used in the present study. The fact that basal CGRP and SP release is similar in the WT and TRPV1–/– hearts indicates that sensory neuropeptide synthesis and release in TRPV1–/– hearts are not impaired under the resting condition. In contrast, ischemic PC causes increased release of CGRP in WT but not in TRPV1–/– hearts. Likewise, the blockade of the CGRP receptor impairs the PC protective effects in WT but not in TRPV1–/– hearts. Our data suggest that endogenous CGRP released from isolated mouse hearts by activation of TRPV1 by PC contributes, at least in part, to PC-induced cardiac protection. These results are consistent with reports by others showing that TVPV1, but not acid-sensing channels (ASIC3) or B2 receptors, mediates acid-evoked CGRP release from mouse hearts (38). A cautionary note: Given the fact that sensory nerves also possess afferent function in vivo via sending signal to the central nervous system, the data obtained from the Langendorff in vitro model may merely reflect efferent function (neuropeptide release) of sensory nerves activated by TRPV1. Future studies of in vivo animal models may provide additional insight into TRPV1 function in PC-induced cardiac protection.
The mechanisms underlying CGRP-induced PC protection are unclear. However, several possibilities exist. The cardiac protective effects afforded by CGRP-mediated ischemic PC have been suggested to be related to an inhibition of cardiac TNF-
production (21) to induce an anti-inflammatory effect to prevent postischemic leukocyte rolling and adhesion (18) and to reduce the oxidative stress damages via inhibition of apoptosis due to the I/R sequence (34). CGRP may also offer a protection from ischemia by causing microvascular vasodilator by activation of an NO- and endothelium-independent or -dependent vasodilator pathway (1).
It has been shown that SP is colocalized with other sensory neuropeptides, especially CGRP and neurokinin A in sensory nerve terminals (24), and is released from cardiac afferent fibers during myocardial ischemia to protect the heart from I/R injury (16, 43). In the present study, we found that PC increased SP release in both WT and TRVR1–/– hearts, although the magnitude of the increase was smaller in TRPV1–/– hearts than in WT hearts. Also, a blockade of the NK1 receptor impaired PC-induced protection in WT but not in TRPV1–/– hearts. These data extend our previous finding (45) by showing that not only harmful I/R but also short-term and nonlethal PC increases SP release and that TRPV1 activation is responsible for, at lease in part, PC-induced SP release. The cardiac protective effect of SP is possibly mediated by NO release leading to vasodilatation of coronary arteries (3, 6). However, the mechanisms underlying the protective effect of SP on the myocardium cannot be fully explained by coronary vasodilatation. The data in the present study showed that RP-67580 increased LVEDP and decreased LVDP and +dP/dt but had no effect on CF in WT hearts. These results indicate that the protective effects of SP may not depend solely on improved total perfusion of the heart; rather, it may affect the distribution of regional myocardial flow and thus produces its beneficial effect without altering total cardiac flow. It has been shown that SP increases NO synthesis (3), which acts as a second messenger, resulting in the activation of PKC and tyrosine kinase phosphatidylinositol 3-kinase as well as an opening of the ATP-sensitive potassium channel (8). Moreover, NK1 activated by SP stimulates cyclooxygenase-2 and prostaglandin E2 expression (20). All of these may mediate SP-induced PC protection of the heart.
In summary, the experiments presented in the present study provide direct evidence that TRPV1 plays a role in mediating ischemia PC via, at least in part, increasing endogenous CGRP and SP. Given that sensory nerve function is impaired in many pathological conditions, such as diabetes and aging in which ischemic PC has been shown to be attenuated, our findings may provide a new molecular basic for the treatment of these conditions.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 vs. PC-TRPV1–/–; 
P < 0.05 vs. WT hearts.
