TY - JOUR
AU - Schwenke, Daryl, O
AB - Abstract Acute myocardial infarction (MI) triggers an adverse increase in cardiac sympathetic nerve activity (SNA). Whereas β-adrenergic receptor (β-AR) blockers are routinely used for the management of MI, they may also counter β-AR–mediated vasodilation of coronary vessels. We have reported that ghrelin prevents sympathetic activation following MI. Whether ghrelin modulates coronary vascular tone following MI, either through the modulation of SNA or directly as a vasoactive mediator, has never been addressed. We used synchrotron microangiography to image coronary perfusion and vessel internal diameter (ID) in anesthetized Sprague-Dawley rats, before and then again 30 minutes after induction of an MI (left coronary artery ligation). Rats were injected with either saline or ghrelin (150 µg/kg, subcutaneously), immediately following the MI or sham surgery. Coronary angiograms were also recorded following β-AR blockade (propranolol, 2 mg/kg, intravenously). Finally, wire myography was used to assess the effect of ghrelin on vascular tone in isolated human internal mammary arteries (IMAs). Acute MI enhanced coronary perfusion to nonischemicregions through dilation of small arterioles (ID 50 to 250 µm) and microvessel recruitment, irrespective of ghrelin treatment. In ghrelin-treated rats, β-AR blockade did not alter the ischemia-induced vasodilation, yet in saline-treated rats, β-AR blockade abolished the vasodilation of small arterioles. Finally, ghrelin caused a dose-dependent vasodilation of IMA rings (preconstricted with phenylephrine). In summary, this study highlights ghrelin as a promising adjunct therapy that can be used in combination with routine β-AR blockade treatment for preserving coronary blood flow and cardiac performance in patients who suffer an acute MI. An acute myocardial infarction (MI) is associated with severe damage to the myocardium that impairs cardiac function but is also exacerbated by sustained overstimulation of the nerves that control heart function; i.e., sympathetic nerve activity (SNA) (1). Indeed, it is the initial increase in cardiac SNA within the first hours following MI that triggers the generation of ventricular arrhythmias (1, 2), which is ultimately responsible for sudden heart failure and death (3). The peptide hormone ghrelin has a diverse range of physiological effects. As a growth hormone-releasing hormone, ghrelin has been shown to improve cardiac growth and function in patients with chronic heart failure (4). Moreover, the early administration of ghrelin, immediately following acute MI, acts centrally to prevent cardiac sympathetic activation (5, 6), which ultimately reduces the incidence of arrhythmias and attenuates the severity of cardiac dysfunction and remodeling (7). The functional capacity of the heart is also highly dependent on adequate coronary blood flow. Coronary vascular tone and hence, blood flow are tightly controlled by SNA through regional β-adrenergic receptor (β-AR)–mediated vasodilation and α-AR–mediated vasoconstriction. Although ghrelin prevents sympathetic activation, it remains unclear how sympathoinhibition impacts the modulation of coronary perfusion and thus, cardiac function following acute MI. Hence, the well-documented cardioprotective properties of ghrelin following acute MI may be linked to preservation of coronary perfusion through sympathetic modulation of coronary vascular tone. Ghrelin may also directly modulate coronary blood flow, independent of neurohormonal input, as in vitro and ex vivo studies have reported ghrelin to have potent vasoactive properties. However, subsequent reports that directly investigate vasomotor responses have been controversial, suggesting that ghrelin can act systemically as either a vasodilator (8–10) or a vasoconstrictor (11–13). The discrepancy among studies may be attributed to, at least in part, differences between species (rodent vs human) and differences in the source and size of vessel (e.g., coronary vs mesenteric, conduit vs resistance), methodological approach, and experimental protocol. Thus, it is critical to understand the direct vascular response of ghrelin if it is to be considered a therapeutic for the treatment of acute MI in a clinical setting, as the optimization of coronary flow to the ischemic myocardium is critical for improving the outcome in the subacute phase. Accordingly, the first aim of this study was to determine the impact that ghrelin has on coronary vascular tone in vivo in the early stages following an acute MI in an anesthetized rat. Second, with the use of nonspecific beta-blockade in vivo, we aimed to identify whether the effects of ghrelin on coronary vascular tone are mediated indirectly via sympathoinhibition. Moreover, we aimed to assess the direct vasoactive properties of ghrelin, void of any neurohormonal input, using isolated human internal mammary arteries (IMAs). The use of the IMAs is pertinent, as it is the gold standard vessel for surgical coronary artery bypass grafting (CABG) and thus, represents the kind of vessel that is important for maintaining coronary perfusion in patients with ischemic cardiac disease who would potentially be eligible for receiving ghrelin therapy. Materials and Methods In vivo imaging of the rat coronary circulation using synchrotron microangiography The coronary circulation was visualized in vivo, using synchrotron radiation (SR) microangiography at the Super Photon Ring-8 GeV (SPring-8) Facility BL28B2 Beam Line (Hyogo, Japan). The use of SR for high-resolution imaging of both pulmonary and coronary vessels in vivo in anesthetized rats has previously been described in detail (14, 15). Animals All animal experiments were approved by the Animal Ethics Committee of SPring-8 and conducted in accordance with the guidelines of the Physiological Society of Japan and the Animal Research: Reporting In Vivo Experiments guidelines. Experiments were conducted on 32 male Sprague-Dawley rats (∼10 weeks old; body weight ∼300 to 340 g). All rats were on a 12-hour light/dark cycle at 25 ± 1°C and provided with food and water ad libitum. Anesthesia and surgical preparation Rats were anesthetized with pentobarbital sodium [60 mg/kg, intraperitoneally (IP), somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan] and analgesic agent butorphanol tartrate (0.5 mg/kg, IP, vetorphale; Meiji Seika, Tokyo, Japan). Supplemental doses of pentobarbital (∼15 mg/kg/h, IP) and butorphanol tartrate (0.025 mg/kg/h, IP) were periodically administered to maintain a surgical level of anesthesia, evident by the complete absence of the limb-withdrawal reflex. Throughout the experimental protocol, body temperature was maintained at 37°C, using a rectal thermistor coupled with a thermostatically controlled heating pad. The trachea was cannulated and the lungs ventilated with a rodent ventilator (model 680; Harvard Apparatus, Holliston, MA). The inspirate gas was enriched with oxygen (∼50% O2), and the ventilator settings were adjusted (tidal volume ∼3.5 mL; breathing rate ∼80/minutes) to maintain arterial partial pressure of carbon dioxide normocapnic. The femoral artery and vein were cannulated for measurement of systemic arterial blood pressure (ABP) and fluid administration [3 mL/h, intravenously (IV), 0.9% sodium chloride saline; Ohtsuka, Tokyo, Japan], respectively. The right common carotid artery was cannulated with a 20-gauge angiocath catheter (Argyle, Nihon Covidien, Tokyo) that was advanced to close proximity of the aortic valve so that iodinated contrast medium (Iomeron 350; Bracco-Eisai, Tokyo, Japan) directly entered and perfused the coronary vessels using a high-speed injector. A left thoracotomy was performed between the first and second rib to access the anterior surface of the left ventricle. A 7.0 Prolene suture (Ethicon, Somerville, NJ) was loosely placed around the left anterior descending (LAD) coronary artery, which was located between the appendage of the left atrium and the base of the pulmonary artery. A fine polyethylene catheter was inserted through a pinhole made in the apex of the left ventricle for the continuous measurement of left ventricular pressure (LVP), from which we could measure left ventricular end–systolic pressure (LVESP) and calculate the maximum rate of LVP development (dP/dtmax), which is an indicator of ventricular contractility. Microangiography experimental protocol The surgically prepared rat was strategically positioned supine in front of and perpendicular to the Saticon X-ray detector (Hitachi Denshi Techno-System, Tokyo, Japan, and Hamamatsu Photonics, Shizuoka, Japan) so that the thorax was in alignment with a 9.5 × 9.5-mm imaging field (i.e., between the first and third ribs; Fig. 1). Figure 1. Open in new tabDownload slide A schematic diagram showing the experimental setup for coronary microangiography in an anesthetized rat model using monochromatic SR at the SPring-8 facilities in Hyogo, Japan. A typical angiogram of the supine rat is recorded from a lateral view of the heart. Figure 1. Open in new tabDownload slide A schematic diagram showing the experimental setup for coronary microangiography in an anesthetized rat model using monochromatic SR at the SPring-8 facilities in Hyogo, Japan. A typical angiogram of the supine rat is recorded from a lateral view of the heart. During each brief imaging scan, monochromatic SR at 33.2 keV and a flux of ∼1010 photons/mm2/second passed through the chest of the anesthetized rats. Iodinated contrast medium (Iomeron 350 with 160 U/mL heparin sulfate) was injected intra-arterially as a bolus (0.2 to 0.4 mL at 0.4 mL/s) into the aorta and cine-radiograms were obtained over 2 seconds. For each 2-second period of scanning (a single exposure sequence), 100 frames were recorded (10-bit resolution) with a shutter open time of ∼1 ms. Rats were given at least 10 minutes to recover from each bolus injection of a contrast agent. Following baseline imaging of the coronary circulation, in two groups of rats, MI was induced by complete ligation of the LAD coronary artery, and then, rats were immediately injected [subcutaneously (SC)] with either (1) saline [MI and saline group (MI + Sal), n = 8, Group 3] or (2) ghrelin [MI and ghrelin group (MI + Ghr), 150 µg/kg, n = 8, Group 4]. Coronary angiograms were then again recorded 30 minutes after injection. In separate groups of sham-operated rats (control = suture placed around the LAD but no occlusion), angiogram images were also recorded at baseline and 30 minutes after injection of either (3) saline [sham and saline group (Sham + Sal), n = 8, Group 1] or (4) ghrelin [sham and ghrelin group (Sham + Ghr), n = 8, Group 2]. An additional small cohort of sham animals (n = 4) was administered the growth hormone secretagogue receptor 1a (GHS-R1a) antagonist, JMV2959 (6 mg/kg, IP; EMD Millipore, Burlington, MA), 2 minutes before ghrelin, so as to determine whether the vasoactive effects of ghrelin are mediated through the ghrelin receptor (Ghr + JMV2959, Group 5). Following the 30-minute angiogram, all rats in the four groups received an IV bolus of the nonselective β-AR blocker, propranolol (2 mg/kg; M5391; Sigma-Aldrich, St. Louis, MO), and 10 minutes later, coronary angiograms were once again recorded. Acylated ghrelin (rat) was used in this study and was sourced from Tocris Bioscience (catalog no. 1465; Bristol, UK). Measurement of infarct size At the completion of each experiment, the rat was euthanized (100 mg/kg pentobarbital sodium) and the heart excised and sectioned into 2-mm horizontal slices down the vertical plane. The sections were then stained with 2,3,5-triphenyltetrazolium solution (Sigma-Aldrich) at 37°C and subsequently fixed in 10% formalin for 20 minutes. Slices were mounted and photographed. Total infarct size was determined by the measurement of the area of the infarction for each slice, the multiplication of the area by the slice thickness, and the summation of the area of all slices. Infarct size was presented as a percentage of the total left ventricular wall. Data analysis ABP and LVP were continuously recorded and data sampled at 400 Hz using a PowerLab data acquisition system (model 8/S; ADInstruments, Dunedin, New Zealand). Heart rate (HR) was derived from the arterial systolic peaks, and dP/dtmax was derived from the LVP signal using LabChart (v. 8). The imaging analysis program, Image Proplus (version 7.0.1; Media Cybernetics, Rockville, MD) was used to enhance contrast and the clarity of angiogram images, as previously described (16). All imaged vessel branches were counted manually. The diameters of two to four vessels of each branching generation were measured in each rat to ensure that a wide variety of vessel sizes was selected from each frame. Vessels were categorized according to internal diameter (ID; in micrometers): 50 to 150, 150 to 250, 250 to 350, and >350. A 50-µm-thick tungsten filament, placed directly across the corner of the detector’s window, appearing in all recorded images, was subsequently used as a reference for calculating vessel ID. IMA wire myography Vessel preparation The collection of human vessels was approved by the local Human Ethics Committee (approval no.: LRS12-01-001), and informed consent was obtained. Discarded IMA sections from 12 patients (ages 51 to 83 years; 1 female, 11 male) who underwent CABG were retrieved. Each of the retrieved sections of IMA were immediately placed in ice-cold oxygenated physiological salt solution, maintained and transferred to the laboratory within 10 minutes. Normalization A 2 mm-wide ring of the vessel was sectioned from the segment of IMA and mounted onto a wire myograph (Model 120CW; Danish Myo Technology A/S, Aarhus, Denmark) containing Krebs solutions (pH 7.4 at 37°C), which was aerated with carbogen (95% O2/5% carbon dioxide) and left for 1 hour to equilibrate. The IMA ring was normalized in accordance with the Danish Myo Technology A/S Normalization Module for LabChart, which required the IMA ring to be progressively stretched in a step-wise manner so as to determine the length-tension curve and optimal basal tension. The vessels were left for a further 1 hour to equilibrate before commencing the experimental protocol. Ghrelin protocol The viability of the IMA vascular smooth muscle and endothelial integrity was confirmed based on the development of tension in response to potassium chloride (0.1 mol ⋅ L−1) and relaxation in response to acetylcholine (ACh; 10 µmol ⋅ L−1), respectively. All IMA rings were preconstricted with phenylephrine (PE; 0.1 mol ⋅ L−1), before a cumulative concentration response curve to ghrelin (1, 3, 10, 100, and 300 nmol ⋅ L−1) was established. The magnitude of the dilator response to each dose of ghrelin was expressed as a percentage of the maximum constriction to PE (assigned to 100%). At the end of each experiment, responses to potassium chloride and ACh further confirmed that all vessels were still viable and intact. Statistical analysis All statistical analyses were conducted using Prism (v6.0a; GraphPad Software, La Jolla, CA). All results are presented as means ± standard error of the mean (SEM). Given that the variance of angiography data was similar between groups, two-way analysis of variance (repeated-measures) were used to test whether vessel caliber (ID), vessel number, and hemodynamic responses were substantially influenced by the effects of MI, beta-blockade, and their interaction. One-way analysis of variance (factorial) was used to test for differences (1) within groups, between baseline and treatment periods and (2) between groups for each treatment period. Where statistical significance was reached, post hoc analyses were incorporated using the paired or unpaired t test with the Dunnet correction (temporal effects) or Sidak correction (between groups) for multiple comparisons. P ≤ 0.05 was predetermined as the level of significance for all statistical analyses. Results Coronary perfusion and hemodynamic responses to acute MI The microangiograms illustrated in Fig. 2a highlight the changes in coronary perfusion following 30 minutes of myocardial ischemia (i.e., LAD occlusion) in a MI + Sal rat (Group 3). Interestingly, MI caused an increase in coronary perfusion to nonischemic regions of the heart, evident by an increase in the number of opaque third-order vessel branches, visualized by angiography (Fig. 2a and 2b), as well as extensive dilation of those arterioles, previously visualized at baseline with an ID of 50 to 250 µm (Fig. 3b). As expected, the functional capacity of the heart was substantially impaired following acute MI, evident by a 39% reduction in contractility (dP/dtmax) and consequently, a 23% reduction in both LVESP and mean ABP (MABP) for MI + Sal rats (Table 1). Figure 2. Open in new tabDownload slide (a) Representative microangiogram images showing the contrast-enhanced coronary arterial branching network down to the third order in an anesthetized rat (MI + Sal) at (i) baseline and (ii) following acute MI, achieved by ligation of the LAD artery. A small wire clip was used to mark the ligation site. The recruitment of additional third-order branches was evident 30 minutes after induction of MI (red arrows). The tungsten wire in the bottom right corner of both angiogram frames is a reference of 50 µm diameter. (b) The number of opaque vessels (means ± SEM) at each of the first three branching generations of the coronary circulation in anesthetized rats, treated with saline or ghrelin (150 µg/kg, SC), 30 minutes after sham surgery (Sham + Sal, n = 8; Sham + Ghr, n = 8), or induction of an MI (MI + Sal, n = 8; MI + Ghr, n = 8). †Significantly different from sham (P < 0.05). Figure 2. Open in new tabDownload slide (a) Representative microangiogram images showing the contrast-enhanced coronary arterial branching network down to the third order in an anesthetized rat (MI + Sal) at (i) baseline and (ii) following acute MI, achieved by ligation of the LAD artery. A small wire clip was used to mark the ligation site. The recruitment of additional third-order branches was evident 30 minutes after induction of MI (red arrows). The tungsten wire in the bottom right corner of both angiogram frames is a reference of 50 µm diameter. (b) The number of opaque vessels (means ± SEM) at each of the first three branching generations of the coronary circulation in anesthetized rats, treated with saline or ghrelin (150 µg/kg, SC), 30 minutes after sham surgery (Sham + Sal, n = 8; Sham + Ghr, n = 8), or induction of an MI (MI + Sal, n = 8; MI + Ghr, n = 8). †Significantly different from sham (P < 0.05). Figure 3. Open in new tabDownload slide The relationship between coronary vessel size and the magnitude (percent) of change in vessel diameter (ID), 30 minutes after (a) sham surgery (Groups 1 and 2 and Group 5) or (b) MI (Groups 3 and 4), in rats treated with saline or ghrelin (150 µg/kg, SC). Ghrelin/saline was administered at the time of MI induction (or sham procedure). For sham Group 5 (n = 4), the GHS-R1a blocker (JMV2959, 6 mg/kg IP) was administered 2 minutes before ghrelin injection. Following the 30-minute coronary angiography imaging, all rats (except Group 5) received a bolus dose of propranolol (2 mg/kg, IV; a nonspecific β-AR blockade), and any further change in coronary caliber (relative to baseline ID) is presented for (c) sham rats and (d) MI rats. *Significant difference between saline vs ghrelin (P < 0.05). †Significant difference between sham vs MI (P < 0.05). ‡Significant reduction/increase in vessel caliber compared with baseline (pre-MI/sham procedure; P < 0.05). Figure 3. Open in new tabDownload slide The relationship between coronary vessel size and the magnitude (percent) of change in vessel diameter (ID), 30 minutes after (a) sham surgery (Groups 1 and 2 and Group 5) or (b) MI (Groups 3 and 4), in rats treated with saline or ghrelin (150 µg/kg, SC). Ghrelin/saline was administered at the time of MI induction (or sham procedure). For sham Group 5 (n = 4), the GHS-R1a blocker (JMV2959, 6 mg/kg IP) was administered 2 minutes before ghrelin injection. Following the 30-minute coronary angiography imaging, all rats (except Group 5) received a bolus dose of propranolol (2 mg/kg, IV; a nonspecific β-AR blockade), and any further change in coronary caliber (relative to baseline ID) is presented for (c) sham rats and (d) MI rats. *Significant difference between saline vs ghrelin (P < 0.05). †Significant difference between sham vs MI (P < 0.05). ‡Significant reduction/increase in vessel caliber compared with baseline (pre-MI/sham procedure; P < 0.05). Table 1. MABP, HR, LVESP, and Myocardial Contractility (dP/dtmax) of Anesthetized Rats Before and Then Again After 30 Minutes of Receiving an MI or Sham Surgery and Treated with Saline or Ghrelin (150 µg/kg, SC) and Then Again 10 Minutes After β-AR Blockade (Propranolol, 2 mg/kg, IV) Baseline T30 Min Propranolol Sham + Sal (n = 8) MABP (mmHg) 109 ± 5 110 ± 7 102 ± 8 HR (⋅ min−1) 407 ± 8 369 ± 15 320 ± 16a LVESP (mmHg) 129 ± 5 131 ± 5 121 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.04 ± 0.62 4.62 ± 0.68 4.06 ± 0.63 Sham + Ghr (150 µg/kg; n = 8) MABP (mmHg) 108 ± 6 110 ± 8 95 ± 8 HR (⋅ min−1) 385 ± 12 356 ± 8 302 ± 9a LVESP (mmHg) 124 ± 7 121 ± 6 107 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.25 ± 0.34 4.74 ± 0.50 4.08 ± 0.33 MI + Sal (n = 8) MABP (mmHg) 118 ± 4 91 ± 5b 71 ± 6a HR (⋅ min−1) 395 ± 13 342 ± 17c 276 ± 13a LVESP (mmHg) 132 ± 5 102 ± 6b 87 ± 6a dP/dtmax (mmHg ⋅ ms−1) 4.67 ± 0.71 2.83 ± 0.49b 2.12 ± 0.26 MI + Ghr (150 µg/kg; n = 8) MABP (mmHg) 116 ± 5 107 ± 9 94 ± 8d,e HR (⋅ min−1) 406 ± 11 367 ± 17 304 ± 12a LVESP (mmHg) 134 ± 5 121 ± 4c,e 111 ± 6e dP/dtmax (mmHg ⋅ ms−1) 4.90 ± 0.59 3.92 ± 0.39c 3.55 ± 0.45e Baseline T30 Min Propranolol Sham + Sal (n = 8) MABP (mmHg) 109 ± 5 110 ± 7 102 ± 8 HR (⋅ min−1) 407 ± 8 369 ± 15 320 ± 16a LVESP (mmHg) 129 ± 5 131 ± 5 121 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.04 ± 0.62 4.62 ± 0.68 4.06 ± 0.63 Sham + Ghr (150 µg/kg; n = 8) MABP (mmHg) 108 ± 6 110 ± 8 95 ± 8 HR (⋅ min−1) 385 ± 12 356 ± 8 302 ± 9a LVESP (mmHg) 124 ± 7 121 ± 6 107 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.25 ± 0.34 4.74 ± 0.50 4.08 ± 0.33 MI + Sal (n = 8) MABP (mmHg) 118 ± 4 91 ± 5b 71 ± 6a HR (⋅ min−1) 395 ± 13 342 ± 17c 276 ± 13a LVESP (mmHg) 132 ± 5 102 ± 6b 87 ± 6a dP/dtmax (mmHg ⋅ ms−1) 4.67 ± 0.71 2.83 ± 0.49b 2.12 ± 0.26 MI + Ghr (150 µg/kg; n = 8) MABP (mmHg) 116 ± 5 107 ± 9 94 ± 8d,e HR (⋅ min−1) 406 ± 11 367 ± 17 304 ± 12a LVESP (mmHg) 134 ± 5 121 ± 4c,e 111 ± 6e dP/dtmax (mmHg ⋅ ms−1) 4.90 ± 0.59 3.92 ± 0.39c 3.55 ± 0.45e Ghrelin was administered at the time of inducing the infarct. The MI was induced by LAD occlusion. Data are presented as means ± SEM. Abbreviation: T30 Min, 30 minutes after surgery (MI or sham) and treatment (saline or ghrelin). a Significant difference between T30 Min and Propranolol (P < 0.01). b Significant difference between baseline and T30 Min (P < 0.01). c Significant difference between baseline and T30 Min (P < 0.05). d Significant difference between T30 Min and Propranolol (P < 0.05). e Significant difference between MI rats treated with saline vs ghrelin (P < 0.05). Open in new tab Table 1. MABP, HR, LVESP, and Myocardial Contractility (dP/dtmax) of Anesthetized Rats Before and Then Again After 30 Minutes of Receiving an MI or Sham Surgery and Treated with Saline or Ghrelin (150 µg/kg, SC) and Then Again 10 Minutes After β-AR Blockade (Propranolol, 2 mg/kg, IV) Baseline T30 Min Propranolol Sham + Sal (n = 8) MABP (mmHg) 109 ± 5 110 ± 7 102 ± 8 HR (⋅ min−1) 407 ± 8 369 ± 15 320 ± 16a LVESP (mmHg) 129 ± 5 131 ± 5 121 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.04 ± 0.62 4.62 ± 0.68 4.06 ± 0.63 Sham + Ghr (150 µg/kg; n = 8) MABP (mmHg) 108 ± 6 110 ± 8 95 ± 8 HR (⋅ min−1) 385 ± 12 356 ± 8 302 ± 9a LVESP (mmHg) 124 ± 7 121 ± 6 107 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.25 ± 0.34 4.74 ± 0.50 4.08 ± 0.33 MI + Sal (n = 8) MABP (mmHg) 118 ± 4 91 ± 5b 71 ± 6a HR (⋅ min−1) 395 ± 13 342 ± 17c 276 ± 13a LVESP (mmHg) 132 ± 5 102 ± 6b 87 ± 6a dP/dtmax (mmHg ⋅ ms−1) 4.67 ± 0.71 2.83 ± 0.49b 2.12 ± 0.26 MI + Ghr (150 µg/kg; n = 8) MABP (mmHg) 116 ± 5 107 ± 9 94 ± 8d,e HR (⋅ min−1) 406 ± 11 367 ± 17 304 ± 12a LVESP (mmHg) 134 ± 5 121 ± 4c,e 111 ± 6e dP/dtmax (mmHg ⋅ ms−1) 4.90 ± 0.59 3.92 ± 0.39c 3.55 ± 0.45e Baseline T30 Min Propranolol Sham + Sal (n = 8) MABP (mmHg) 109 ± 5 110 ± 7 102 ± 8 HR (⋅ min−1) 407 ± 8 369 ± 15 320 ± 16a LVESP (mmHg) 129 ± 5 131 ± 5 121 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.04 ± 0.62 4.62 ± 0.68 4.06 ± 0.63 Sham + Ghr (150 µg/kg; n = 8) MABP (mmHg) 108 ± 6 110 ± 8 95 ± 8 HR (⋅ min−1) 385 ± 12 356 ± 8 302 ± 9a LVESP (mmHg) 124 ± 7 121 ± 6 107 ± 7 dP/dtmax (mmHg ⋅ ms−1) 5.25 ± 0.34 4.74 ± 0.50 4.08 ± 0.33 MI + Sal (n = 8) MABP (mmHg) 118 ± 4 91 ± 5b 71 ± 6a HR (⋅ min−1) 395 ± 13 342 ± 17c 276 ± 13a LVESP (mmHg) 132 ± 5 102 ± 6b 87 ± 6a dP/dtmax (mmHg ⋅ ms−1) 4.67 ± 0.71 2.83 ± 0.49b 2.12 ± 0.26 MI + Ghr (150 µg/kg; n = 8) MABP (mmHg) 116 ± 5 107 ± 9 94 ± 8d,e HR (⋅ min−1) 406 ± 11 367 ± 17 304 ± 12a LVESP (mmHg) 134 ± 5 121 ± 4c,e 111 ± 6e dP/dtmax (mmHg ⋅ ms−1) 4.90 ± 0.59 3.92 ± 0.39c 3.55 ± 0.45e Ghrelin was administered at the time of inducing the infarct. The MI was induced by LAD occlusion. Data are presented as means ± SEM. Abbreviation: T30 Min, 30 minutes after surgery (MI or sham) and treatment (saline or ghrelin). a Significant difference between T30 Min and Propranolol (P < 0.01). b Significant difference between baseline and T30 Min (P < 0.01). c Significant difference between baseline and T30 Min (P < 0.05). d Significant difference between T30 Min and Propranolol (P < 0.05). e Significant difference between MI rats treated with saline vs ghrelin (P < 0.05). Open in new tab Ghrelin effects on coronary perfusion in vivo In sham-treated animals (Group 2), ghrelin appeared to cause both vasodilation of the small coronary arterioles (ID 50 to 150 µm) and vasoconstriction of the larger conduit arteries (ID >350 µm; Fig. 3a) but did not substantially alter cardiac function (Table 1). The vasoactive effect of ghrelin in sham animals was abolished when the GHS-R1a antagonist, JMV2959, was administered before ghrelin injection (Fig. 3a). As MABP was not substantially altered by ghrelin treatment after 30 minutes, these changes in coronary vessels are unlikely to be a result of changes in coronary perfusion pressure. Following an acute MI, the administration of ghrelin (MI + Ghr) attenuated the severity of cardiac dysfunction, based on a significantly smaller MI-induced reduction in LVESP (10% decrease; P < 0.05 vs saline-treated MI), although the magnitude of reduction in contractility was not statistically different between MI + Ghr rats (20% decrease in dP/dtmax relative to baseline) and MI + Sal rats (∼40% decrease; Table 1). Infarct size was similar for MI + Sal rats (30.1 ± 3.4%) and MI + Ghr rats (28.6% ± 2.9%). Despite the clear differences in the severity of cardiac dysfunction between groups, the MI-induced changes in coronary perfusion were similar for both MI + Sal and MI + Ghr rats (Groups 3 and 4); i.e., ischemia enhanced coronary perfusion by microvessel recruitment (third-order branches) and extensive dilation of 50 to 250 µm-sized vessels, visualized previously at baseline, regardless of whether animals received ghrelin or not following the MI (Figs. 2b and 3b). Responses to β-AR blockade (propranolol) following sham and MI Next, this study aimed to determine whether β-AR blockade altered coronary perfusion in the presence of ghrelin, as (1) SNA modulates cardiac function and coronary vascular tone, (2) ghrelin is known to prevent the adverse increase in SNA following acute MI, and (3) beta-blockers are routinely administered for the treatment of MI. In sham rats, treated either without or with ghrelin (Groups 1 and 2, respectively), propranolol caused a 14% decrease in HR (P < 0.01) but did not significantly reduce contractility (dP/dtmax), LVESP, or MABP relative to the 30-minute period postsham and -MI (Table 1). In ghrelin-treated sham rats, propranolol did not cause any additional changes to vessel caliber compared with that induced by ghrelin alone (Fig. 3c). However, in saline-treated sham rats (Group 1), propranolol caused a small, albeit significant, 15% constriction (P < 0.05) of the large conduit vessels (ID >350 µm), which interestingly, was similar in magnitude to that caused by ghrelin in sham-treated rats (Fig. 3a vs 3c). All other smaller vessel sizes (i.e., ID <350 µm) in sham rats were not significantly altered by propranolol. In MI rats, treated either with or without ghrelin (Groups 4 and 3, respectively), β-AR blockade caused a further significant decrease in HR, MABP, and LVESP (significant for MI + Sal rats only), even though contractility was largely preserved (Table 1). Interestingly, propranolol did not further modify the ID of any coronary vessels in MI + Ghr rats. In contrast, in the untreated MI + Sal rats, β-AR blockade abolished the dilatory effect that was observed under ischemia alone for the 50 to 150 µm-sized arterioles, such that the vessel ID had essentially returned to pre-MI size (Fig. 3d). Human IMA myography Of the 12 IMAs retrieved, four did not respond to either potassium and/or ACh and were, therefore, not used for experimentation. Basal force development in IMA rings following normalization was 18.4 ± 2.0 mN (n = 8). IMA rings were preconstricted with PE (− 0.1 mmol ⋅ L−1), and the cumulative concentration response curve to ghrelin was assessed (Fig. 4a). Ghrelin caused a dose-dependent decrease in vessel-wall tension (i.e., vasodilation), reaching a minimal tension that was 30.7% ± 7% of the PE-preconstricted tension in response to 300 nmol ⋅ L−1 ghrelin (Fig. 4b). The half maximal effective concentration for ghrelin (effective concentration that caused 50% relaxation) was 43.4 ± 9.3 nmol ⋅ L−1, based on the fitted curve equation Y = 100/[1 + (X0.54)/(43.440.54)]. Figure 4. Open in new tabDownload slide (a) A representative, developed tension tracing recorded from a wire myography system for an isolated human IMA ring. Trace illustrates the vascular-wall tension developed during preconstriction with PE (0.1 mmol ⋅ L−1) and, subsequently, the progressive decrease in tension (vasodilation) in response to cumulative doses of ghrelin (0 to 300 nmol ⋅ L−1). (b) Mean concentration-relaxation curve for PE-preconstricted IMAs in response to ghrelin. Values are expressed as means ± SEM (arteries from n = 8 subjects). The response for each ghrelin dose is expressed as a percentage of the maximum tension to PE. EC50, half maximal effective concentration (effective concentration that caused 50% relaxation). Figure 4. Open in new tabDownload slide (a) A representative, developed tension tracing recorded from a wire myography system for an isolated human IMA ring. Trace illustrates the vascular-wall tension developed during preconstriction with PE (0.1 mmol ⋅ L−1) and, subsequently, the progressive decrease in tension (vasodilation) in response to cumulative doses of ghrelin (0 to 300 nmol ⋅ L−1). (b) Mean concentration-relaxation curve for PE-preconstricted IMAs in response to ghrelin. Values are expressed as means ± SEM (arteries from n = 8 subjects). The response for each ghrelin dose is expressed as a percentage of the maximum tension to PE. EC50, half maximal effective concentration (effective concentration that caused 50% relaxation). Discussion The primary findings of this study show that in an animal model, (1) ghrelin has the potential to both dilate and constrict coronary vessels in vivo, depending on the size and anatomical position (function) of the vessel; (2) the vasoactive properties of ghrelin, however, appear to be masked following MI, presumably as a result of the overwhelming dilatory effects of myocardial ischemia; (3) the ischemia-induced vasodilation of small arteries–arterioles is abolished by β-AR blockade but is preserved with the adjunct administration of ghrelin, and (4) ghrelin has potent, dose-dependent vasodilatory properties in human mammary arteries. Ever since its discovery in 1999 (17), ghrelin has emerged as an important therapeutic modulator of cardiac function in health and disease (18). Whereas the exact mechanisms that underpin the cardioprotective properties of ghrelin remain to be fully elucidated, various groups have shown that ghrelin prevents MI-induced sympathetic activation (6, 7, 19, 20), inhibits myocardial apoptosis (21), promotes angiogenesis (22, 23), reduces cardiac cachexia and dysfunction (24), preserves cardiac contractility (25), and attenuates cardiac remodeling (26). Although ghrelin is known to modulate coronary vasculature tone directly, at least in animal models, the few reports, to date, do not agree as to whether ghrelin dilates or constricts coronary vessels (10–13). In this study, one advantage of using SR microangiography was the ability to visualize the extensive coronary vascular network, from the first- to third-order arteriole branching in an in vivo animal model, revealing that ghrelin has the potential to both dilate and constrict coronary vessels, depending on the size and thus, physiological function of the vessel. We showed that ghrelin dilated resistance small arteries and arterioles (ID 50 to 150 µm) but constricted conduit vessels (ID >350 µm). The reason for the apparent dual vasoactive effects of ghrelin remains unclear, but several factors can be considered. Coronary vascular tone is meticulously modulated by neuronal (SNA) and hormonal factors (e.g., adrenaline), as well as local autoregulation (adenosine, O2 content), to ensure optimal blood flow to the highly aerobic-dependent myocardium. In this study, we used an in vivo animal model to assess directly the effect of ghrelin on the intact coronary circulation as a whole. Therefore, we cannot entirely exclude the possibility that vasoactive effects of acute ghrelin administration were influenced by indirect nonvascular effects through changes in cardiac work or cardiac SNA, which are important modulators of coronary vascular tone. However, all of the measured parameters of cardiac work (HR, LVESP, contractility, and ABP) for sham rats in this study were unaltered by ghrelin. Moreover, we have consistently reported that modulation of SNA by ghrelin is not evident in sham animals, rather only in those animals with an MI where SNA adversely increases (6, 7, 27). Two circulating isoforms of ghrelin exist: acyl and des-acyl ghrelin, the latter arising from both the degradation of acyl ghrelin and the direct release from ghrelin cells into the circulation. In general, the biological activity of ghrelin had been attributed to the acyl isoform, as it had long been accepted that des-acyl ghrelin was void of any biological activity (10, 28). Interestingly, Grossini et al. (12) had earlier reported that intracoronary infusion of acyl ghrelin reduced coronary perfusion (i.e., constriction) in anesthetized pigs, whereas des-acyl ghrelin increased coronary perfusion in the same model (10). Nonetheless, others have also shown that (acyl) ghrelin constricts canine coronary arteries (outer diameter >200 µm) (11), as well as rat coronary arterioles (outer diameter ∼100 µm) (13). In contrast, several studies advocate acyl ghrelin as a systemic vasodilator. Okumura et al. (29) originally reported that ghrelin caused a direct vasodilation-mediated increase in forearm blood flow, and Virdis et al. (30) reported that ghrelin augmented the vasodilatory response to ACh in hypertensive subjects. Moreover, acyl ghrelin has been shown to dilate IMAs (preconstricted with endothelin-1) potently from human patients (9, 28), which is consistent with the IMA results observed in this study. The physiological responses to acyl ghrelin are mediated via GHS-R1a. The receptor for des-acyl ghrelin remains unknown. Sax et al. (11) identified the GHS-R1a within canine coronary arterioles and showed that acyl ghrelin constricted isolated coronary arterioles. However, the vasoconstriction was not mediated through the GHS-R1a but rather, a yet-unidentified receptor. In comparison, in our study, we noted that GHS-R1a blockade appeared to abolish the ghrelin-mediated changes in rat coronary vascular tone, suggesting that ghrelin likely mediates its vascular effects, at least in part, via the GHS-R1a. Ultimately, given our observations that first, large conduit arteries can either constrict (rat coronary) or dilate (IMA) in response to ghrelin and second, different vessels, even from within the same vascular bed (e.g., coronary network), respond differently to ghrelin indicates that vascular responses to ghrelin are determined by multiple factors, including the following: (1) the presence, location, distribution, and type of ghrelin receptors present, (2) the type of vessel (e.g., coronary vs IMA; conduit vs resistance), (3) species (e.g., rat vs human), (4) experimental setup (in vitro vs in vivo), and thus, (5) whether vessels are exposed to acyl ghrelin alone (isolated vessels) or in conjunction with des-acyl ghrelin (in vivo, via the breakdown of acyl ghrelin). Hence, further research is required to elucidate the vasoactive properties of ghrelin. Coronary perfusion following myocardial ischemia It is well known that myocardial autoregulation is a predominant regulator of coronary blood flow; that is, O2 delivery to the myocardium is tightly matched to O2 demands. In the absence of sufficient O2, as is the case for myocardial ischemia, the accumulation of metabolic metabolites (e.g., hydrogen, potassium, and adenosine) within the interstitial fluid acts to dilate coronary vessels potently to increase blood flow and thus, O2 delivery (31). In this study, coronary microangiographic imaging confirmed that LAD occlusion provoked extensive dilation of all coronary resistance arterioles (ID <250 µm) in the nonischemic regions of the heart. However, one limitation of this study is that we were unable to quantify net coronary blood flow. Hence, we cannot comment on potential changes in flow per se, as even though local vascular resistance would have decreased in these dilated vessels, perfusion pressure (i.e., ABP) was reduced, and ventricular contractility (dP/dtmax) was also significantly impaired following acute MI. One key observation in this study was that the vasoactive properties of ghrelin, which were evident in the nonischemic heart, were absent or perhaps more accurately masked in the MI heart as a result of the dilatory effects ischemia. We have previously reported that the maximal capacity of coronary vessels to dilate (i.e., increase vessel caliber) appears to be ∼20%, based on the dilatory responses to the potent nitric oxide donor, nitroprusside, combined either with or without ACh (14, 32). Therefore, the inability of ghrelin to dilate the coronary vessels further may simply have been a result of the fact that maximal dilation was already attained with ischemia alone (i.e., 20% increase in vessel caliber). Importantly, we noted that ghrelin reduced the severity of cardiac dysfunction (LVESP) and preserved ABP in MI + Ghr rats, which is consistent with previous reports that ghrelin preserves cardiac inotropy (24). Hence, given that flow is the product of vascular resistance and perfusion pressure, it is plausible that ghrelin augmented the increase in coronary blood flow to nonischemic regions of the heart following acute MI. Clinical implications Patients who experience an acute MI are commonly prescribed β-AR blockers as part of standard clinical practice (33). Furthermore, some patients will be eligible for CABG. Accordingly, before the advocation of ghrelin as an adjunct therapy for MI, we aimed first to identify the effect of ghrelin on coronary perfusion in conjunction with a nonspecific β-AR blocker (propranolol). As our results clearly indicated that ghrelin causes moderate constriction of the large conduit coronary vessels, at least in the rat coronary circulation, we next aimed to reaffirm that in our hands, ghrelin did not constrict human mammary arteries, which are large conduit vessels, generally used as the gold standard vessel for surgical CABG. Previous studies have reported that nonselective β-AR stimulation facilitates a vasodilation-mediated increase in coronary blood flow (34, 35). The research team of Muller et al. (36) previously reported that β-AR inhibition using propranolol in humans reduced coronary blood flow (37) and that the overall β-AR–mediated dilatory response was the product of both β1-AR–mediated metabolic and β2-AR–mediated neurogenic vasodilation (38). In agreement, we also noted that propranolol constricted the coronary conduit vessels, specifically the >300-µm vessels, in saline-treated sham animals, which was likely matched by reduced cardiac work (HR, ABP, LVESP, and contractility) and thus, myocardial O2 consumption. More importantly, the use of beta-blockade following acute MI in this study abolished the potent vasodilatory effect of ischemia alone on the 50- to 150-µm resistance vessels, thereby increasing vascular resistance to blood flow. Interestingly, the secretion of ghrelin is mediated, in part, through activation of β1-AR on the membrane of ghrelin cells, and moreover, β1-AR-deficient mice have impaired ghrelin secretion and a lower plasma concentration of ghrelin (39). Therefore, the coronary constrictor response to β-AR blockade in the MI + Sal animals of this study may have been exacerbated, at least in part, as a result of suppression of endogenous ghrelin-mediated dilation of coronary vascular tone. This is an area of research that warrants further investigation, especially with the consideration that beta-blockers are a routine therapy used in clinics for cardiac and vascular diseases. In ghrelin-treated animals, both sham and MI, β-AR blockade had no effect on coronary perfusion, although it did reduce cardiac work (MI + Ghr rats), indicating that the adjunct use of ghrelin with the beta-blockade may have an additive beneficial effect of preserving the enhanced coronary flow (O2 delivery), while reducing cardiac work (O2 delivery). The apparent lack of an effect of the β-AR blockade on the coronary circulation of ghrelin-treated animals is likely linked to the sympathoinhibitory properties of ghrelin, as we have consistently confirmed that ghrelin effectively prevents adverse sympathetic activation following acute MI (5–7, 40). It is plausible, therefore, that the effect of the β-AR blockade in ghrelin-treated MI animals was negligible, as the β-ARs were not activated in the first place. One further limitation of this study is that we did not differentiate between the role of β1-AR and β2-AR receptors in the modulation of coronary perfusion following acute MI, primarily as our aim was to assess global β-AR blockade on coronary perfusion. Importantly, however, in the clinical setting, selective β1-AR blockade using metoprolol is preferentially used, as it reduces coronary vascular resistance, presumably via vasodilation (41). Collectively, these experimental data, in combination with reports in the literature, advocate ghrelin as a potential complementary therapy (with β-AR blockers) for the treatment of myocardial ischemia, as ghrelin has the potential to treat the origin of the increase in SNA following acute MI (6) rather than the symptom of an increase in SNA. The findings of this and other studies also suggest that ghrelin treatment is likely to increase flow in bypass graft segments that use IMAs. Conclusion We have used SR microangiography to demonstrate that ghrelin dilates small coronary arterioles and importantly, preserves an ischemia-mediated increase in coronary perfusion, even in the presence of β-AR blockers that have the potential to impair perfusion. We further used wire myography to confirm that ghrelin directly and potently dilates human conduit IMAs, which is important, considering these vessels are routinely used for CABG surgery. Ultimately, this study further expands our understanding concerning the diverse cardioprotective properties of ghrelin, and in doing so, we advocate ghrelin as a promising adjunct therapy that can be used in combination with routine β-AR blockade treatment for preserving cardiac performance in patients who suffer an acute MI. Abbreviations: ABP arterial blood pressure ACh acetylcholine CABG coronary artery bypass grafting dP/dtmax maximum rate of left ventricular pressure development GHS-R1a growth hormone secretagogue receptor 1a HR heart rate ID internal diameter IMA internal mammary artery IP intraperitoneally IV intravenously LAD left anterior descending LVESP left ventricular end–systolic pressure LVP left ventricular pressure MABP mean arterial blood pressure MI myocardial infarction MI + Ghr myocardial infarction and ghrelin group MI + Sal myocardial infarction and saline group PE phenylephrine SC subcutaneously SEM standard error of the mean Sham + Ghr sham and ghrelin group Sham + Sal sham and saline group SNA sympathetic nerve activity SPring-8 Super Photon Ring-8 GeV SR synchrotron radiation β-AR beta-adrenergic receptor. 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Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2018 Endocrine Society
TI - Ghrelin Preserves Ischemia-Induced Vasodilation of Male Rat Coronary Vessels Following β-Adrenergic Receptor Blockade
JF - Endocrinology
DO - 10.1210/en.2017-03070
DA - 2018-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ghrelin-preserves-ischemia-induced-vasodilation-of-male-rat-coronary-Tb0izVT0i6
SP - 1763
VL - 159
IS - 4
DP - DeepDyve
ER -