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The cardioprotective effects of carvedilol on ischemia and reperfusion injury by AMPK signaling pathway
Biomedicine & Pharmacotherapy 117 (2019) 109106
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
The cardioprotective effects of carvedilol on ischemia and reperfusion injury by AMPK signaling pathway
T
Haiyan Hua,b,c, Xuan Lib, Di Renb, Yi Tand,e, Jimei Chenc, Lei Yangc, Ruiping Chenc, Ji Lib, ⁎ Ping Zhua,c, a Department of Cardiac surgery, Affiliated of South China Hospital, Southern Medical University (Guangdong Provincial People's Hospital), Southern Medical University/ The Second School of Clinical Medicine, Guangzhou 510515, China b Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, MS, United States c Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China d Pediatric Research Institute, Department of Pediatrics, University of Louisville, Louisville, KY, United states e Wendy L. Novak Diabetes Care Center, Louisville, KY, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: AMPK Carvedilol Cardioprotection Ischemia/reperfusion
Carvedilol, a third generation beta blocker, is in clinical use for heart failure patients. However, besides adrenergic receptor blockade, the pharmacological effects of carvedilol on cardiomyocytes remain unknown. AMPactivated protein kinase (AMPK) is an emerging target recognized for heart failure treatment. The mechanical properties and intracellular Ca2+ properties were measured in isolated cardiomyocyte contractile functions in response to ischemic stress. Treatment of cardiomyocytes with carvedilol augmented phosphorylation of AMPK and downstream acetyl CoA carboxylase (ACC), and ameliorated hypoxia-induced impairment in maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt), and the impaired peak height and peak shortening (PS) amplitude caused by hypoxia. Carvedilol treatment improved calcium homeostasis with rescuing the peak Ca2+ signal, the maximum rate of Ca2+ change during contraction (+dF/dt) and the maximum rate of Ca2+ change during relaxation (-dF/dt) under hypoxia conditions. In mouse hearts perfused ex vivo with carvedilol, the function of post-ischemia left ventricle was improved and an augmentation in myocardial glucose uptake and glucose oxidation, and inhibition of fatty acid oxidation during ischemia and reperfusion. The protective effect of carvedilol was further supported in an in vivo regional ischemia model by ligation of left anterior descending coronary artery (LAD), mice treated with carvedilol followed by LAD occlusion and reperfusion showed significant size reduction in infarcted myocardium and improved cardiac functions. Therefore, Carvedilol as a clinical drug can modulate cardiac AMPK signaling pathway to reduce ischemic insults by ischemia and reperfusion.
1. Introduction
of myocardial damage and functional recovery after reperfusion. Therefore, metabolic interventions, aimed at enhancing glucose utilization and reducing fatty acid oxidation, have received attentions in recent years for the treatment of ischemia and reperfusion injury [3,4]. It has been demonstrated that the phosphorylation of AMP-activated protein kinase (AMPK) is not only functioning as an important regulator of intracellular energy metabolism, but also in reducing myocardial ischemia/reperfusion injury through the attenuation of oxidative stress and endoplasmic reticulum stress, inhibition of apoptosis and autophagy, and anti-inflammatory mechanisms to improve ischemic preconditioning or ischemic post-conditioning [5]. Activated AMPK can shift substrates utilization away from anabolic pathways and toward those specificities to upregulate the ATP-concentration in order to fulfill
Myocardial ischemia-reperfusion injury (MIRI) is one of the most important mechanisms of myocardial injury after reperfusion treatment. Upon reperfusion, the intracellular calcium overload, the altered myocardial energy metabolism, the release of inflammatory mediators and the formation of oxygen free radicals are all important factors contributing to aggravate the myocardial injury [1]. Therefore, novel pharmacological interventions are essential to further reduce infarct size, to preserve LV function, to mitigate adverse remodeling, and ultimately to improve survival in patients with acute myocardial infarction (MI) [2]. Complex alterations in myocardial energy substrate metabolism during myocardial ischemia can significantly affect the degree ⁎
Corresponding author at: Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510080, China. E-mail address: [email protected] (P. Zhu).
https://doi.org/10.1016/j.biopha.2019.109106 Received 16 May 2019; Received in revised form 6 June 2019; Accepted 6 June 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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echocardiography (VisualSonics, Toronto, ON, Canada) was performed to measure cardiac functions, wall thicknesses, and chamber volumes. LV wall thicknesses were measured by using a modified version of the leading-edge method from the American Society for Echocardiography using 3 consecutive cycles of M-mode tracing [19].
the needs of cardiac myocytes. Therefore, activation of AMPK has potential clinical significance in the prevention and treatment of myocardial ischemic heart disease [6]. Carvedilol (CAR) is a multifunctional cardiovascular drug licensed for the treatment of chronic heart failure, high blood pressure and myocardial infarction. During reperfusion, increased expression of β1and β2-adrenergic receptors on cardiomyocytes leads to the generation of reactive oxygen species (ROS) and cardiac myocyte apoptosis [7,8]. Carvedilol has been widely used in the treatment of cardiovascular diseases due to its function in blocking β1, β2 and α1 adrenergic receptors. In recent years, clinical and animal studies have confirmed that Carvedilol ameliorates ischemia and reperfusion injury by antioxidation, calcium antagonism, anti-arrhythmia, anti-apoptosis, and inhibiting neutrophil infiltration. Moreover, Carvedilol can shift myocardial substrates use from free fatty acid to glucose oxidation [9–12]. However, the relevant pharmacological effects of Carvedilol on cardiomyocytes have not yet been completely figured out. We hypothesized that Carvedilol could protect the heart from ischemia and reperfusion injury through activating AMPK signaling pathway, leading to increased myocardial salvage, decreased infarct size, and improved left ventricular contractility and remodeling.
2.6. Infarct size measurement Mice were anesthetized with isoflurane (1–3%) and placed on a ventilator (Harvard Apparatus, Holliston, MA, USA). Core temperature was maintained at 37 °C with a heating pad. After left lateral thoracotomy, the left anterior descending coronary artery was occluded for 45 min with an 8-0 nylon suture and polyethylene tubing to prevent arterial injury, and then reperfused for 24 h. An electrocardiogram and blanching of the LV confirmed ischemic repolarization changes (STsegment elevation) during coronary occlusion (ADInstruments). After reperfusion for 24 h, hearts were quickly excised, and the left anterior descending coronary artery was ligated, then hearts were stained with 2,3,5-triphenyltetrazolium (TTC) for 15 min to delineate the extent of myocardial necrosis as a percentage of the ischemic area at risk (AAR). Viable tissue in the ischemic region was stained red by TTC, and necrotic tissue was stained white (INF).
2. Material and methods 2.7. Fatty acid/glucose oxidation analysis 2.1. Isolation of cardiomyocytes The working heart preload was set up at 15 cm H2O, and the afterload was set at 80 cm H2O [20–22]. Heart function was monitored by a pressure transducer connected to the aortic outflow [9,10].-3Holeate (50 mci/L) and 14C-glucose (20 mci/L)–labeled BSA buffer was perfused into the heart via the pulmonary vein and pumped out through the aorta. Perfusate that was pumped out from the aorta and that outflowed from the coronary venous arteries was recycled and collected every 5 min to test the radioactivity. The fatty acid level was determined by the production of 3H2O from [9,10]-3H-oleate. Metabolized 3 H2O was separated from [9,10]-3H-oleate by filtering through anionexchange resin (Bio-Rad, Hercules, CA, USA). Glucose oxidation was measured and sampled every 5 min by both the metabolized 14CO2 that was dissolved in the perfusate buffer and by the gaseous 14CO2, which was further dissolved in sodium hydroxide. To separate 14CO2 from 14Cglocose, sulfuric acid was added to perfusate samples to release 14CO2. 3 H and 14C signals were detected to discriminate metabolic products from fatty acid and glucose respectively.
All animal protocols in this study were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. All C57BL/6 J mice (12 weeks) were obtained from Jackson Laboratory. Cardiomyocytes were isolated enzymatically as previously described [13,14]. 2.2. Measurement of contractile function and intracellular Ca2+ transient The mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix Corporation, Milton, Massachusetts) [13,14]. Cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus, IX-70, Center Valley, PA, USA) and stimulated with suprathreshold voltage at a frequency of 1 Hz, with a paI/R of platinum wires placed on the opposite sides of the chamber and connected to a stimulator (FHC Inc., Brunswick, NE, USA). For intracellular Ca2+ transient measurement, a dual-excitation and single-emission photomultiplier system (IonOptix) was applied for the purpose.
2.8. Statistical analysis
Immunoblots was performed as previously described [14,15]. Rabbit antibodies for p-AMPKα (Thr172), AMPKα, p-acetyl-CoA carboxylase (ACC; Ser79), ACC, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).
Results are presented as means ± SEM. Differences between treated groups and vehicle control groups were assessed by Wilcoxon test. One-way ANOVA with Turkey’s test was used to compare values between more than 2 groups. All statistical calculations were performed with Prism 7.0 (La Jolla, CA, USA). Differences were considered statistically significant at values of p < 0.05.
2.4. Isolated heart perfusions
3. Results
As previously described [16–18] mice were anesthetized with isoflurane (1–3%), and the isolated mouse hearts were perfused in the Langendorff mode with modified Krebs-Henseleit buffer containing 7 mM glucose, 0.4 mM oleate, 1% bovine serum albumin (BSA), and 10 mU/ml insulin at 37 °C. Hearts were perfused for 20 min at a flow of 4 ml/min, followed by 20 min of global, no-flow ischemia and 30 min of reperfusion.
3.1. Carvedilol triggers phosphorylation of AMPK in cardiomyocytes
2.3. Immunoblotting analysis
To determine whether CAR protects cardiomyocytes from hypoxia injury through AMPK signaling pathway, we first examined the effects of CAR on AMPK signaling pathway during normoxia and hypoxia. Isolated cardiomyocytes were subjected to either vehicle (DMSO) or 1 μM CAR treatment for 5 min at normal or hypoxic condition. In order to Inhibit the AMPK pathway, 10 mM compound C was administrated 5 min before CAR treatment, in both normoxia and hypoxia conditions. The Western blots results showed that, compared to normoxia, hypoxic conditions increased p-AMPK and p-ACC, but there are no obvious significant differences (Fig. 1A). However, the administration of CAR
2.5. In Vivo evaluation of heart function by echocardiography The representative, randomly selected animals from each group were anesthetized with isoflurane (1–3%), and transthoracic M-mode 2
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Fig. 1. CAR activated the phosphorylation of AMPK during hypoxia. Isolated cardiomyocytes were subjected to either vehicle (DMSO) or 1 μM CAR treatment with or without compound C (10 μM) for 5 min at normoxia or hypoxia conditions. (A) and (D) The level of p-AMPK and p-ACC; (B) and (E) The relative ratio of p-AMPK; (C) and (F) The relative ratio of p-ACC. Values are means ± SEM, n = 3–5 mice per group, *p < 0.01 vs. normoxia, respectively; #p < 0.05 vs. Vehicle hypoxia.
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Fig. 2. The contractile function and intercellular calcium signal of isolated cardiomyocytes with or without CAR treatment. Cardiomyocytes were subjected to either vehicle or CAR (0.1 μM, 1 μM, 5 μM) treatment for 5 min at normal or hypoxic condition. (A) The representative cell shortenings traces obtained after different concentrations of CAR treatment under normoxia; (B) The representative cell shortenings traces obtained after different concentration of CAR treatment under hypoxia; (C) Resting sarcomere length; (D) Peak height; (E) Maximal velocity of re-lengthening (+dL/dt); (F) Maximal velocity of shortening (−dL/dt); (G) Peak shortening (normalized to the resting sarcomere length); (H) The representative calcium signal traces obtained after different concentration of CAR treatment under normoxia; (I) The representative calcium signal traces obtained after different concentration of CAR treatment under hypoxia; (J) The baseline of Ca2+ signal (△F340/380); (K) Peak Ca2+ changes during contraction; (L) Maximum rate of Ca2+ change during contraction (+dF/dt); (M) Maximum rate of Ca2+ change during relaxation (−dF/dt). Values are means ± SEM, n = 30–70 cells per group derived from 5 to 8 mice. *p < 0.01 vs. normoxia, respectively; #p < 0.01 vs. Vehicle hypoxia; †p < 0.05 vs. Vehicle normoxia.
values in Fig. 2 indicated that CAR treatment did not affect cardiomyocyte contractile function under the normal condition. However, during hypoxic condition, 1 μM and 5 μM CAR significantly increased the peak height (Fig. 2D), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt) (Fig.2E and F), and the cardiomyocytes peak shortening (PS) (Fig. 2G) of cardiomyocytes without changing the resting sarcomere length (Fig. 2C) in cardiomyocytes. However, there were no differences between 1 μM and 5 μM, but there were differences in peak height and PS compared to 0.1 μM. From these results, we found that CAR had no effect on the contractile function on normoxia cells, but it protected cardiomyocytes from hypoxia-induced contractile dysfunction, and 1 μM had the most obvious effect. To explore the underlying mechanism involved in the role of CAR in inducing the contractility of cardiomyocytes, we evaluated the intracellular Ca2+ transients using the fura-2 fluorescence technique. Fig. 2A and B showed representative Ca2+ transients during normoxia and hypoxia with or without treatment of CAR. Average values shown
during the hypoxic condition significantly activated AMPK (Fig. 1B), and its downstream effector, ACC (Fig. 1C). Compound C, also called dorsomorphin, has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor [20]. The isolated mouse cardiomyocytes were exposed to 10 μM of compound C 5 min before CAR treatment, the results demonstrated that Compound C pretreatment significantly attenuated CAR-augmented phosphorylation of AMPK and downstream ACC under hypoxia conditions (Fig. 1D–F). 3.2. Carvedilol improves contractile functions of cardiomyocytes during hypoxia To investigate the potential role of CAR in regulating myocardial function during hypoxia, we tested the effects of three different concentrations of CAR on contractile function in myocytes isolated from mouse hearts. Fig. 2A and B showed the representative cell shortenings obtained after CAR treatment under normoxia or hypoxia. Average 4
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Fig. 3. CAR improves cardiac function and reduces myocardial infarction through AMPK signaling pathway during ischemia and reperfusion. (A) The representative echocardiography of the sham operations and I/R group with or without CAR and/or Compound C treatment; (B–C) Ischemia and reperfusion decreased the left ventricular ejection fraction (EF) and fractional shortening (FS) in vehicle group, CAR administration improved the EF and FS, but it was blocked in the Compound C pre-treatment. EF% and FS% have no differences between sham and Compound C alone treatment under both sham and I/R conditions. Values are means ± SEM, n = 3–4 mice per group. *p < 0.01 vs. Sham group; #p < 0.05 vs. Vehicle I/R; †p < 0.05 vs. CAR I/R group. (D) Representative sections of myocardial infarction. (E) The ratio of area at risk (AAR) to the myocardial area; (F) The ratio of infarction area (INF) to AAR. Values are means ± SEM, n = 3–4 mice per group. *p < 0.01 vs. Vehicle group; #p < 0.05 vs. CAR group.
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in Fig. 2H–M indicated that CAR elevated intracellular Ca2+ levels (Fig. 2K), +dF/dt (Fig. 2L) and -dF/dt (Fig. 2M) without changing the baseline of Ca2+ transient (Fig. 2J) as compared with vehicle group under both normal and hypoxic condition, but there were no significant differences between these three CAR concentrations.
oxidation to fatty acid oxidation was significantly reversed with CAR (1 μM) treatment during ischemia and reperfusion (Fig. 4B and C). The glucose oxidation of I/R with carvedilol treatment was increased to a comparable basal level (Fig. 4B), and the fatty acid oxidation of I/R with CAR treatment was markedly down-regulated (Fig. 4C). Of interest, there were no significant alterations in both glucose oxidation and fatty acid oxidation between vehicle and CAR groups under basal conditions (Fig. 4B and C).
3.3. Carvedilol improves cardiac function during ischemia and reperfusion In order to determine the effects of CAR on the heart tolerance to ischemic stress, mice were subjected to ligation of left anterior descending coronary artery (LAD) by suture to induce an in vivo regional ischemia of 45 min and then release of the suture to reperfusion for 24 h. Cardiac functions were measured by using echocardiography (Fig. 3A). The results showed that there was no significant difference on ejection fraction (EF) and fractional shortening (FS) among the sham and CAR groups. However, I/R challenge significantly reduced the LV FS and EF in vehicle group comparing to the CAR group. In the CAR and compound C groups, the EF and FS were found significantly decreased (Fig. 3B and C), but in the compound C basal group, the EF and FS showed no differences compared to the sham group. Furthermore, isolated hearts from C57BL/6 J mice were subjected to Langendorff heart perfusion to investigate the effect of CAR on the heart’s tolerance to ischemic injury and the recovery of post-ischemic cardiac functions. The isolated hearts were perfused for 20 min at basal condition, followed by 20 min of global, no-flow ischemia, and then reperfused for 30 min. Compared with the vehicle group, the post-ischemic cardiac function in CAR treatment group was significantly increased during reperfusion, and recovered as demonstrated by decreased heart rate–LV pressure products (RPP) (Fig. 4D). There were no significant changes in heart rate among the two group, which suggested that CAR improved cardiac function after ischemic insults.
4. Discussion This study examined the cardioprotective effects of CAR on myocardial ischemia and reperfusion injury through regulating AMPK signaling pathway. The main finding of our study is that CAR treatment significantly increased myocardial salvage, reduced infarct size, shifted metabolic consumption, and improved contractile function. The underlying mechanism for these effects is an activation of AMPK in cardiomyocytes. Previous reports and our earlier studies demonstrated that activation of AMPK exerts a protective effect toward ischemia and reperfusion (I/R) injury by decreasing the extent of myocardial necrosis, helping recovery of cardiac contractile function and altering metabolism [23,24]. Both animal and human studies have exhibited that, during hypoxia, CAR enhanced cardiomyocyte survival by antioxidative property, calcium antagonism, anti-arrhythmia, anti-apoptosis, and inhibition of neutrophil infiltration. Being consistent with this, our results in a mouse model of left coronary artery ligation, administration of CAR before ischemia and reperfusion significantly improved cardiac function after myocardial infarction, as shown by a significantly improved EF and FS of the CAR groups, compared to the vehicle group. However, after using an AMPK inhibitor-Compound C, the cardiac function improvement was blocked. Combined with the western blot results, we concluded that the cardioprotective effect of CAR may also have some relations with the activation of the AMPK signaling pathway. Alterations in myocardial energy metabolism may occur during ischemia and reperfusion stress conditions versus normal physiological conditions. In the early stage of myocardial ischemia, due to the depletion of oxyhemoglobin in the ischemic tissue, energy metabolism changes from aerobic oxidation to glycolysis, and the ATP generated through this anaerobic process becomes the only energy source for the survival of cardiomyocytes [25,26]. With the extension of ischemic time, the accumulation of metabolic end products such as lactic acid increases in the cells leading to a further decrease in intracellular pH resulting in acidosis and inhibition of glycolysis [27]. After reperfusion, as lactic acid and inorganic phosphoric acid are washed away, the inhibitory effect on the glycolysis process is weakened. In addition, mitochondria are damaged in ischemia, and are not able to utilize oxygen for aerobic oxidation during reperfusion. Therefore, myocardial cells still rely on glycolysis to provide energy for a considerable period of time after reperfusion [28]. AMPK is an energy sensor that controls ATP supply from substrate metabolism and protects the heart from energy stress. During myocardial ischemia, ATP is degraded into AMP, and then activates AMPK. Activated AMPK phosphorylates acetyl-CoA carboxylase (ACC) [29,30]. The effect of adrenergic signaling on AMPK activity appears to be tissue specific and may depend on the metabolic milieu. Our data supports the pharmacological utility of CAR in enhancing AMPK activation and reducing cardiac ischemic injury. Clinical experiments have confirmed that hemodynamic agents combined with metabolic agents used in ischemic heart disease patients performed with better efficacy and tolerability [31]. Thus, CAR may be a unique agent for the treatment of ischemia and reperfusion injury by both improving energy use and hemodynamics. In summary, the results support the concept that pharmacological activation of AMPK with carvedilol can reduce detrimental effects of ischemia and reperfusion injury. Carvedilol treatment improved myocardial salvage and reduced infarct size, leading to a robust
3.4. Carvedilol reduces myocardial infarction by ischemia and reperfusion To determine whether CAR can protect against myocardial injury, we examined the myocardial infarct size after regional ischemia and reperfusion (I/R) in vivo between the vehicle and CAR group. Anesthetized mice were subjected to 45 min of ligation of the LAD followed by 24 h reperfusion. For each group, CAR (2 mg/kg) or vehicle (saline) was injected intraperitoneally 30 min before surgery. In the compound C (0.1 μg/g) groups, mice were given Compound C through IP injection an hour before ischemia. Representative cardiac sections dually stained with TTC and Evans blue dye are shown in Fig. 3D. Ratios of the area at risk to the total myocardial area were similar among the 4 groups, indicating that an equal extent of ischemic stress was induced in all groups (Fig. 3E). Administration of CAR significantly reduced myocardial infarction (Fig., 3F), and this effect was attenuated with compound C pre-treatment, indicating that CAR reduces myocardial infarction injury partially through AMPK signaling pathway. 3.5. Carvedilol treatment shifts cardiac metabolism in response to I/R stress One of the most important functions of cardiac AMPK is to increase energy production during stress conditions. Activated AMPK can achieve this important physiologic process by modulating substrate metabolism via stimulating fatty acid uptake and oxidation and increasing glucose uptake. The glucose uptake measurement showed that ischemic stress triggered cardiac glucose uptake, and that glucose uptake by I/R was significantly increased in CAR group (Fig. 4A). To determine the effect of CAR treatment on glucose and fatty acid metabolism in ischemic hearts, glucose oxidation was measured by the amount of [14C]-glucose metabolism into 14CO2 in ex vivo working hearts. Fatty acid oxidation was measured by the incorporation of [9, 10-3H2O] oleate into 3H2O. The results showed that I/R caused a significant metabolic shift between glucose oxidation and fatty acid oxidation in the heart (Fig. 4B and C). The metabolic shift from glucose 6
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Fig. 4. Carvedilol shifts metabolism in response to I/R stress. (A) D-[2-3H] glucose were used for determining the glucose uptake; (B) Glucose oxidation and (C) Oleate oxidation were measured in the isolated working heart. Carvedilol (1 μM) or DMSO (Vehicle) were added in the perfusate. After balancing 20 min, isolated hearts were subjected to 10 min of ischemia and 20 min of reperfusion. Glucose oxidation was analyzed by measuring [14C] glucose metabolism into 14CO2. Oleate oxidation was measured by the incorporation of [9, 10-3H2O] oleate into 3H2O. Values are means ± SEM, n = 3–5 mice per group. *p < 0.05 vs. basal group, respectively; #p < 0.05 vs. Vehicle I/R group; (D) The Langendorff heart perfusion system showed a significant impaired post-ischemic recovery in vehicle hearts, and the post-ischemic recovery of LV contractility was significantly improved with CAR treatment. Values are means ± SEM, n = 3–4 mice per group. *p < 0.05 vs. Vehicle group.
Science Foundation of Guangdong Province of China (2017A030312007); The key program of Guangzhou Science Research Plan (805212639211).
improvement in systolic LV function, enhanced intrinsic LV mechanics, and metabolism. These cardioprotective effects of carvedilol in the reperfusion hearts are partially mediated by the activation of the AMPK signaling pathways, resulting in a reduction of fatty acid oxidation, increase glucose oxidation and glucose uptake. Our findings highlight the therapeutic potential of carvedilol in the peri-infarct period and warrant further investigations.
Conflicts of interest The authors declare no conflict of interest. References
Author contributions
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
The cardioprotective effects of carvedilol on ischemia and reperfusion injury by AMPK signaling pathway
T
Haiyan Hua,b,c, Xuan Lib, Di Renb, Yi Tand,e, Jimei Chenc, Lei Yangc, Ruiping Chenc, Ji Lib, ⁎ Ping Zhua,c, a Department of Cardiac surgery, Affiliated of South China Hospital, Southern Medical University (Guangdong Provincial People's Hospital), Southern Medical University/ The Second School of Clinical Medicine, Guangzhou 510515, China b Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, MS, United States c Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China d Pediatric Research Institute, Department of Pediatrics, University of Louisville, Louisville, KY, United states e Wendy L. Novak Diabetes Care Center, Louisville, KY, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: AMPK Carvedilol Cardioprotection Ischemia/reperfusion
Carvedilol, a third generation beta blocker, is in clinical use for heart failure patients. However, besides adrenergic receptor blockade, the pharmacological effects of carvedilol on cardiomyocytes remain unknown. AMPactivated protein kinase (AMPK) is an emerging target recognized for heart failure treatment. The mechanical properties and intracellular Ca2+ properties were measured in isolated cardiomyocyte contractile functions in response to ischemic stress. Treatment of cardiomyocytes with carvedilol augmented phosphorylation of AMPK and downstream acetyl CoA carboxylase (ACC), and ameliorated hypoxia-induced impairment in maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt), and the impaired peak height and peak shortening (PS) amplitude caused by hypoxia. Carvedilol treatment improved calcium homeostasis with rescuing the peak Ca2+ signal, the maximum rate of Ca2+ change during contraction (+dF/dt) and the maximum rate of Ca2+ change during relaxation (-dF/dt) under hypoxia conditions. In mouse hearts perfused ex vivo with carvedilol, the function of post-ischemia left ventricle was improved and an augmentation in myocardial glucose uptake and glucose oxidation, and inhibition of fatty acid oxidation during ischemia and reperfusion. The protective effect of carvedilol was further supported in an in vivo regional ischemia model by ligation of left anterior descending coronary artery (LAD), mice treated with carvedilol followed by LAD occlusion and reperfusion showed significant size reduction in infarcted myocardium and improved cardiac functions. Therefore, Carvedilol as a clinical drug can modulate cardiac AMPK signaling pathway to reduce ischemic insults by ischemia and reperfusion.
1. Introduction
of myocardial damage and functional recovery after reperfusion. Therefore, metabolic interventions, aimed at enhancing glucose utilization and reducing fatty acid oxidation, have received attentions in recent years for the treatment of ischemia and reperfusion injury [3,4]. It has been demonstrated that the phosphorylation of AMP-activated protein kinase (AMPK) is not only functioning as an important regulator of intracellular energy metabolism, but also in reducing myocardial ischemia/reperfusion injury through the attenuation of oxidative stress and endoplasmic reticulum stress, inhibition of apoptosis and autophagy, and anti-inflammatory mechanisms to improve ischemic preconditioning or ischemic post-conditioning [5]. Activated AMPK can shift substrates utilization away from anabolic pathways and toward those specificities to upregulate the ATP-concentration in order to fulfill
Myocardial ischemia-reperfusion injury (MIRI) is one of the most important mechanisms of myocardial injury after reperfusion treatment. Upon reperfusion, the intracellular calcium overload, the altered myocardial energy metabolism, the release of inflammatory mediators and the formation of oxygen free radicals are all important factors contributing to aggravate the myocardial injury [1]. Therefore, novel pharmacological interventions are essential to further reduce infarct size, to preserve LV function, to mitigate adverse remodeling, and ultimately to improve survival in patients with acute myocardial infarction (MI) [2]. Complex alterations in myocardial energy substrate metabolism during myocardial ischemia can significantly affect the degree ⁎
Corresponding author at: Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510080, China. E-mail address: [email protected] (P. Zhu).
https://doi.org/10.1016/j.biopha.2019.109106 Received 16 May 2019; Received in revised form 6 June 2019; Accepted 6 June 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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echocardiography (VisualSonics, Toronto, ON, Canada) was performed to measure cardiac functions, wall thicknesses, and chamber volumes. LV wall thicknesses were measured by using a modified version of the leading-edge method from the American Society for Echocardiography using 3 consecutive cycles of M-mode tracing [19].
the needs of cardiac myocytes. Therefore, activation of AMPK has potential clinical significance in the prevention and treatment of myocardial ischemic heart disease [6]. Carvedilol (CAR) is a multifunctional cardiovascular drug licensed for the treatment of chronic heart failure, high blood pressure and myocardial infarction. During reperfusion, increased expression of β1and β2-adrenergic receptors on cardiomyocytes leads to the generation of reactive oxygen species (ROS) and cardiac myocyte apoptosis [7,8]. Carvedilol has been widely used in the treatment of cardiovascular diseases due to its function in blocking β1, β2 and α1 adrenergic receptors. In recent years, clinical and animal studies have confirmed that Carvedilol ameliorates ischemia and reperfusion injury by antioxidation, calcium antagonism, anti-arrhythmia, anti-apoptosis, and inhibiting neutrophil infiltration. Moreover, Carvedilol can shift myocardial substrates use from free fatty acid to glucose oxidation [9–12]. However, the relevant pharmacological effects of Carvedilol on cardiomyocytes have not yet been completely figured out. We hypothesized that Carvedilol could protect the heart from ischemia and reperfusion injury through activating AMPK signaling pathway, leading to increased myocardial salvage, decreased infarct size, and improved left ventricular contractility and remodeling.
2.6. Infarct size measurement Mice were anesthetized with isoflurane (1–3%) and placed on a ventilator (Harvard Apparatus, Holliston, MA, USA). Core temperature was maintained at 37 °C with a heating pad. After left lateral thoracotomy, the left anterior descending coronary artery was occluded for 45 min with an 8-0 nylon suture and polyethylene tubing to prevent arterial injury, and then reperfused for 24 h. An electrocardiogram and blanching of the LV confirmed ischemic repolarization changes (STsegment elevation) during coronary occlusion (ADInstruments). After reperfusion for 24 h, hearts were quickly excised, and the left anterior descending coronary artery was ligated, then hearts were stained with 2,3,5-triphenyltetrazolium (TTC) for 15 min to delineate the extent of myocardial necrosis as a percentage of the ischemic area at risk (AAR). Viable tissue in the ischemic region was stained red by TTC, and necrotic tissue was stained white (INF).
2. Material and methods 2.7. Fatty acid/glucose oxidation analysis 2.1. Isolation of cardiomyocytes The working heart preload was set up at 15 cm H2O, and the afterload was set at 80 cm H2O [20–22]. Heart function was monitored by a pressure transducer connected to the aortic outflow [9,10].-3Holeate (50 mci/L) and 14C-glucose (20 mci/L)–labeled BSA buffer was perfused into the heart via the pulmonary vein and pumped out through the aorta. Perfusate that was pumped out from the aorta and that outflowed from the coronary venous arteries was recycled and collected every 5 min to test the radioactivity. The fatty acid level was determined by the production of 3H2O from [9,10]-3H-oleate. Metabolized 3 H2O was separated from [9,10]-3H-oleate by filtering through anionexchange resin (Bio-Rad, Hercules, CA, USA). Glucose oxidation was measured and sampled every 5 min by both the metabolized 14CO2 that was dissolved in the perfusate buffer and by the gaseous 14CO2, which was further dissolved in sodium hydroxide. To separate 14CO2 from 14Cglocose, sulfuric acid was added to perfusate samples to release 14CO2. 3 H and 14C signals were detected to discriminate metabolic products from fatty acid and glucose respectively.
All animal protocols in this study were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. All C57BL/6 J mice (12 weeks) were obtained from Jackson Laboratory. Cardiomyocytes were isolated enzymatically as previously described [13,14]. 2.2. Measurement of contractile function and intracellular Ca2+ transient The mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix Corporation, Milton, Massachusetts) [13,14]. Cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus, IX-70, Center Valley, PA, USA) and stimulated with suprathreshold voltage at a frequency of 1 Hz, with a paI/R of platinum wires placed on the opposite sides of the chamber and connected to a stimulator (FHC Inc., Brunswick, NE, USA). For intracellular Ca2+ transient measurement, a dual-excitation and single-emission photomultiplier system (IonOptix) was applied for the purpose.
2.8. Statistical analysis
Immunoblots was performed as previously described [14,15]. Rabbit antibodies for p-AMPKα (Thr172), AMPKα, p-acetyl-CoA carboxylase (ACC; Ser79), ACC, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).
Results are presented as means ± SEM. Differences between treated groups and vehicle control groups were assessed by Wilcoxon test. One-way ANOVA with Turkey’s test was used to compare values between more than 2 groups. All statistical calculations were performed with Prism 7.0 (La Jolla, CA, USA). Differences were considered statistically significant at values of p < 0.05.
2.4. Isolated heart perfusions
3. Results
As previously described [16–18] mice were anesthetized with isoflurane (1–3%), and the isolated mouse hearts were perfused in the Langendorff mode with modified Krebs-Henseleit buffer containing 7 mM glucose, 0.4 mM oleate, 1% bovine serum albumin (BSA), and 10 mU/ml insulin at 37 °C. Hearts were perfused for 20 min at a flow of 4 ml/min, followed by 20 min of global, no-flow ischemia and 30 min of reperfusion.
3.1. Carvedilol triggers phosphorylation of AMPK in cardiomyocytes
2.3. Immunoblotting analysis
To determine whether CAR protects cardiomyocytes from hypoxia injury through AMPK signaling pathway, we first examined the effects of CAR on AMPK signaling pathway during normoxia and hypoxia. Isolated cardiomyocytes were subjected to either vehicle (DMSO) or 1 μM CAR treatment for 5 min at normal or hypoxic condition. In order to Inhibit the AMPK pathway, 10 mM compound C was administrated 5 min before CAR treatment, in both normoxia and hypoxia conditions. The Western blots results showed that, compared to normoxia, hypoxic conditions increased p-AMPK and p-ACC, but there are no obvious significant differences (Fig. 1A). However, the administration of CAR
2.5. In Vivo evaluation of heart function by echocardiography The representative, randomly selected animals from each group were anesthetized with isoflurane (1–3%), and transthoracic M-mode 2
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Fig. 1. CAR activated the phosphorylation of AMPK during hypoxia. Isolated cardiomyocytes were subjected to either vehicle (DMSO) or 1 μM CAR treatment with or without compound C (10 μM) for 5 min at normoxia or hypoxia conditions. (A) and (D) The level of p-AMPK and p-ACC; (B) and (E) The relative ratio of p-AMPK; (C) and (F) The relative ratio of p-ACC. Values are means ± SEM, n = 3–5 mice per group, *p < 0.01 vs. normoxia, respectively; #p < 0.05 vs. Vehicle hypoxia.
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Fig. 2. The contractile function and intercellular calcium signal of isolated cardiomyocytes with or without CAR treatment. Cardiomyocytes were subjected to either vehicle or CAR (0.1 μM, 1 μM, 5 μM) treatment for 5 min at normal or hypoxic condition. (A) The representative cell shortenings traces obtained after different concentrations of CAR treatment under normoxia; (B) The representative cell shortenings traces obtained after different concentration of CAR treatment under hypoxia; (C) Resting sarcomere length; (D) Peak height; (E) Maximal velocity of re-lengthening (+dL/dt); (F) Maximal velocity of shortening (−dL/dt); (G) Peak shortening (normalized to the resting sarcomere length); (H) The representative calcium signal traces obtained after different concentration of CAR treatment under normoxia; (I) The representative calcium signal traces obtained after different concentration of CAR treatment under hypoxia; (J) The baseline of Ca2+ signal (△F340/380); (K) Peak Ca2+ changes during contraction; (L) Maximum rate of Ca2+ change during contraction (+dF/dt); (M) Maximum rate of Ca2+ change during relaxation (−dF/dt). Values are means ± SEM, n = 30–70 cells per group derived from 5 to 8 mice. *p < 0.01 vs. normoxia, respectively; #p < 0.01 vs. Vehicle hypoxia; †p < 0.05 vs. Vehicle normoxia.
values in Fig. 2 indicated that CAR treatment did not affect cardiomyocyte contractile function under the normal condition. However, during hypoxic condition, 1 μM and 5 μM CAR significantly increased the peak height (Fig. 2D), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt) (Fig.2E and F), and the cardiomyocytes peak shortening (PS) (Fig. 2G) of cardiomyocytes without changing the resting sarcomere length (Fig. 2C) in cardiomyocytes. However, there were no differences between 1 μM and 5 μM, but there were differences in peak height and PS compared to 0.1 μM. From these results, we found that CAR had no effect on the contractile function on normoxia cells, but it protected cardiomyocytes from hypoxia-induced contractile dysfunction, and 1 μM had the most obvious effect. To explore the underlying mechanism involved in the role of CAR in inducing the contractility of cardiomyocytes, we evaluated the intracellular Ca2+ transients using the fura-2 fluorescence technique. Fig. 2A and B showed representative Ca2+ transients during normoxia and hypoxia with or without treatment of CAR. Average values shown
during the hypoxic condition significantly activated AMPK (Fig. 1B), and its downstream effector, ACC (Fig. 1C). Compound C, also called dorsomorphin, has been widely used in cell-based, biochemical, and in vivo assays as a selective AMPK inhibitor [20]. The isolated mouse cardiomyocytes were exposed to 10 μM of compound C 5 min before CAR treatment, the results demonstrated that Compound C pretreatment significantly attenuated CAR-augmented phosphorylation of AMPK and downstream ACC under hypoxia conditions (Fig. 1D–F). 3.2. Carvedilol improves contractile functions of cardiomyocytes during hypoxia To investigate the potential role of CAR in regulating myocardial function during hypoxia, we tested the effects of three different concentrations of CAR on contractile function in myocytes isolated from mouse hearts. Fig. 2A and B showed the representative cell shortenings obtained after CAR treatment under normoxia or hypoxia. Average 4
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Fig. 3. CAR improves cardiac function and reduces myocardial infarction through AMPK signaling pathway during ischemia and reperfusion. (A) The representative echocardiography of the sham operations and I/R group with or without CAR and/or Compound C treatment; (B–C) Ischemia and reperfusion decreased the left ventricular ejection fraction (EF) and fractional shortening (FS) in vehicle group, CAR administration improved the EF and FS, but it was blocked in the Compound C pre-treatment. EF% and FS% have no differences between sham and Compound C alone treatment under both sham and I/R conditions. Values are means ± SEM, n = 3–4 mice per group. *p < 0.01 vs. Sham group; #p < 0.05 vs. Vehicle I/R; †p < 0.05 vs. CAR I/R group. (D) Representative sections of myocardial infarction. (E) The ratio of area at risk (AAR) to the myocardial area; (F) The ratio of infarction area (INF) to AAR. Values are means ± SEM, n = 3–4 mice per group. *p < 0.01 vs. Vehicle group; #p < 0.05 vs. CAR group.
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in Fig. 2H–M indicated that CAR elevated intracellular Ca2+ levels (Fig. 2K), +dF/dt (Fig. 2L) and -dF/dt (Fig. 2M) without changing the baseline of Ca2+ transient (Fig. 2J) as compared with vehicle group under both normal and hypoxic condition, but there were no significant differences between these three CAR concentrations.
oxidation to fatty acid oxidation was significantly reversed with CAR (1 μM) treatment during ischemia and reperfusion (Fig. 4B and C). The glucose oxidation of I/R with carvedilol treatment was increased to a comparable basal level (Fig. 4B), and the fatty acid oxidation of I/R with CAR treatment was markedly down-regulated (Fig. 4C). Of interest, there were no significant alterations in both glucose oxidation and fatty acid oxidation between vehicle and CAR groups under basal conditions (Fig. 4B and C).
3.3. Carvedilol improves cardiac function during ischemia and reperfusion In order to determine the effects of CAR on the heart tolerance to ischemic stress, mice were subjected to ligation of left anterior descending coronary artery (LAD) by suture to induce an in vivo regional ischemia of 45 min and then release of the suture to reperfusion for 24 h. Cardiac functions were measured by using echocardiography (Fig. 3A). The results showed that there was no significant difference on ejection fraction (EF) and fractional shortening (FS) among the sham and CAR groups. However, I/R challenge significantly reduced the LV FS and EF in vehicle group comparing to the CAR group. In the CAR and compound C groups, the EF and FS were found significantly decreased (Fig. 3B and C), but in the compound C basal group, the EF and FS showed no differences compared to the sham group. Furthermore, isolated hearts from C57BL/6 J mice were subjected to Langendorff heart perfusion to investigate the effect of CAR on the heart’s tolerance to ischemic injury and the recovery of post-ischemic cardiac functions. The isolated hearts were perfused for 20 min at basal condition, followed by 20 min of global, no-flow ischemia, and then reperfused for 30 min. Compared with the vehicle group, the post-ischemic cardiac function in CAR treatment group was significantly increased during reperfusion, and recovered as demonstrated by decreased heart rate–LV pressure products (RPP) (Fig. 4D). There were no significant changes in heart rate among the two group, which suggested that CAR improved cardiac function after ischemic insults.
4. Discussion This study examined the cardioprotective effects of CAR on myocardial ischemia and reperfusion injury through regulating AMPK signaling pathway. The main finding of our study is that CAR treatment significantly increased myocardial salvage, reduced infarct size, shifted metabolic consumption, and improved contractile function. The underlying mechanism for these effects is an activation of AMPK in cardiomyocytes. Previous reports and our earlier studies demonstrated that activation of AMPK exerts a protective effect toward ischemia and reperfusion (I/R) injury by decreasing the extent of myocardial necrosis, helping recovery of cardiac contractile function and altering metabolism [23,24]. Both animal and human studies have exhibited that, during hypoxia, CAR enhanced cardiomyocyte survival by antioxidative property, calcium antagonism, anti-arrhythmia, anti-apoptosis, and inhibition of neutrophil infiltration. Being consistent with this, our results in a mouse model of left coronary artery ligation, administration of CAR before ischemia and reperfusion significantly improved cardiac function after myocardial infarction, as shown by a significantly improved EF and FS of the CAR groups, compared to the vehicle group. However, after using an AMPK inhibitor-Compound C, the cardiac function improvement was blocked. Combined with the western blot results, we concluded that the cardioprotective effect of CAR may also have some relations with the activation of the AMPK signaling pathway. Alterations in myocardial energy metabolism may occur during ischemia and reperfusion stress conditions versus normal physiological conditions. In the early stage of myocardial ischemia, due to the depletion of oxyhemoglobin in the ischemic tissue, energy metabolism changes from aerobic oxidation to glycolysis, and the ATP generated through this anaerobic process becomes the only energy source for the survival of cardiomyocytes [25,26]. With the extension of ischemic time, the accumulation of metabolic end products such as lactic acid increases in the cells leading to a further decrease in intracellular pH resulting in acidosis and inhibition of glycolysis [27]. After reperfusion, as lactic acid and inorganic phosphoric acid are washed away, the inhibitory effect on the glycolysis process is weakened. In addition, mitochondria are damaged in ischemia, and are not able to utilize oxygen for aerobic oxidation during reperfusion. Therefore, myocardial cells still rely on glycolysis to provide energy for a considerable period of time after reperfusion [28]. AMPK is an energy sensor that controls ATP supply from substrate metabolism and protects the heart from energy stress. During myocardial ischemia, ATP is degraded into AMP, and then activates AMPK. Activated AMPK phosphorylates acetyl-CoA carboxylase (ACC) [29,30]. The effect of adrenergic signaling on AMPK activity appears to be tissue specific and may depend on the metabolic milieu. Our data supports the pharmacological utility of CAR in enhancing AMPK activation and reducing cardiac ischemic injury. Clinical experiments have confirmed that hemodynamic agents combined with metabolic agents used in ischemic heart disease patients performed with better efficacy and tolerability [31]. Thus, CAR may be a unique agent for the treatment of ischemia and reperfusion injury by both improving energy use and hemodynamics. In summary, the results support the concept that pharmacological activation of AMPK with carvedilol can reduce detrimental effects of ischemia and reperfusion injury. Carvedilol treatment improved myocardial salvage and reduced infarct size, leading to a robust
3.4. Carvedilol reduces myocardial infarction by ischemia and reperfusion To determine whether CAR can protect against myocardial injury, we examined the myocardial infarct size after regional ischemia and reperfusion (I/R) in vivo between the vehicle and CAR group. Anesthetized mice were subjected to 45 min of ligation of the LAD followed by 24 h reperfusion. For each group, CAR (2 mg/kg) or vehicle (saline) was injected intraperitoneally 30 min before surgery. In the compound C (0.1 μg/g) groups, mice were given Compound C through IP injection an hour before ischemia. Representative cardiac sections dually stained with TTC and Evans blue dye are shown in Fig. 3D. Ratios of the area at risk to the total myocardial area were similar among the 4 groups, indicating that an equal extent of ischemic stress was induced in all groups (Fig. 3E). Administration of CAR significantly reduced myocardial infarction (Fig., 3F), and this effect was attenuated with compound C pre-treatment, indicating that CAR reduces myocardial infarction injury partially through AMPK signaling pathway. 3.5. Carvedilol treatment shifts cardiac metabolism in response to I/R stress One of the most important functions of cardiac AMPK is to increase energy production during stress conditions. Activated AMPK can achieve this important physiologic process by modulating substrate metabolism via stimulating fatty acid uptake and oxidation and increasing glucose uptake. The glucose uptake measurement showed that ischemic stress triggered cardiac glucose uptake, and that glucose uptake by I/R was significantly increased in CAR group (Fig. 4A). To determine the effect of CAR treatment on glucose and fatty acid metabolism in ischemic hearts, glucose oxidation was measured by the amount of [14C]-glucose metabolism into 14CO2 in ex vivo working hearts. Fatty acid oxidation was measured by the incorporation of [9, 10-3H2O] oleate into 3H2O. The results showed that I/R caused a significant metabolic shift between glucose oxidation and fatty acid oxidation in the heart (Fig. 4B and C). The metabolic shift from glucose 6
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Fig. 4. Carvedilol shifts metabolism in response to I/R stress. (A) D-[2-3H] glucose were used for determining the glucose uptake; (B) Glucose oxidation and (C) Oleate oxidation were measured in the isolated working heart. Carvedilol (1 μM) or DMSO (Vehicle) were added in the perfusate. After balancing 20 min, isolated hearts were subjected to 10 min of ischemia and 20 min of reperfusion. Glucose oxidation was analyzed by measuring [14C] glucose metabolism into 14CO2. Oleate oxidation was measured by the incorporation of [9, 10-3H2O] oleate into 3H2O. Values are means ± SEM, n = 3–5 mice per group. *p < 0.05 vs. basal group, respectively; #p < 0.05 vs. Vehicle I/R group; (D) The Langendorff heart perfusion system showed a significant impaired post-ischemic recovery in vehicle hearts, and the post-ischemic recovery of LV contractility was significantly improved with CAR treatment. Values are means ± SEM, n = 3–4 mice per group. *p < 0.05 vs. Vehicle group.
Science Foundation of Guangdong Province of China (2017A030312007); The key program of Guangzhou Science Research Plan (805212639211).
improvement in systolic LV function, enhanced intrinsic LV mechanics, and metabolism. These cardioprotective effects of carvedilol in the reperfusion hearts are partially mediated by the activation of the AMPK signaling pathways, resulting in a reduction of fatty acid oxidation, increase glucose oxidation and glucose uptake. Our findings highlight the therapeutic potential of carvedilol in the peri-infarct period and warrant further investigations.
Conflicts of interest The authors declare no conflict of interest. References
Author contributions
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H.H. and P.Z. wrote the first draft of the manuscript; X.L, D.R., Y.T., J.C., L.Y., R.C. contributed to collect experiments and discussion; and J.L. and P.Z. contributed to research design and finalized the draft of the manuscript. Funding Financial support for the study was provided grants awarded by National Natural Science Foundation of China81570279; National Key Research and Development Program of China (2018YFA0108700, 2017YFA0105602); NSFC Projects of International Cooperation and Exchanges (81720102004); The Research Team Project of Natural 7
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