The chemical chaperon 4-phenylbutric acid improves cardiac function following isoproterenol-induced myocardial infarction in the rat

Purpose 4-phenyl butyric acid (4-PBA) is a chaperone-mediated autophagy (CMA) inducer, which eliminates unnecessary and damaged cellular components through lysosomal enzymes. It could reduce misfolded and unfolded proteins produced after myocardial infarction (MI) and can improve cardiac function. We aimed to investigate the effect of 4-PBA on isoproterenol-induced MI in the rat. Methods Isoproterenol (100 mg/kg) was injected subcutaneously for two consecutive days simultaneous with an intraperitoneal (IP) injection of 4-PBA at 20, 40, or 80 mg/kg with 24-h intervals for ve days. On day 6, hemodynamic parameters, histopathological changes, peripheral neutrophil count, and total antioxidant capacity (TAC) evaluated. The expression of autophagy proteins measured by using western blotting. 4-PBA signicantly improved post-MI changes in hemodynamic parameters. Results Histological improvement found in 4-PBA 40 mg/kg (P<0.05). The neutrophils count in the peripheral blood signicantly decreased in the treatment groups compared with the isoproterenol. Furthermore, 4-PBA at 80 mg/kg signicantly increased the serum TAC compared to isoproterenol (P<0.001). Western blotting results showed a signicant decrease in the P62 level (P<0.05) of 40 and 80 mg/kg 4-PBA treated groups. Conclusion This study demonstrated that 4-PBA could have a cardio-protective effect against isoproterenol-induced MI, which can be due to the modulation of autophagy and inhibition of oxidative stress. Obtaining effective results in different doses shows the need of optimum degree of authophagic activities in cells.


Introduction
Myocardial ischemia is among the most important causes of morbidity and mortality worldwide, with a 42% increased prevalence in recent years [1]. Myocardial ischemia occurs due to an obstruction of coronary arteries and consequently decreased blood ow to the coronary arteries, leading to myocardial infarction (MI). Tissue remodeling, necrosis, brosis, hypertrophy, in ammation, and accumulation of neutrophils at the site of infarction and subsequently increased destruction due to the activity of proteolytic enzymes are among the consequences of MI [2].
Physiological stresses such as oxidative stress occur in MI, causing several changes in cells, and affecting the structure and function of proteins. In this process, unfolded and misfolded proteins also synthesized, which can cause further cardiac damage and apoptosis of cardiac cells. These proteins activate the unfolded protein response pathway, which can cause further tissue damages [3,4]. Autophagy is an intracellular pathway that helps to eliminate the unfolded and misfolded proteins and plays a critical catabolic role for cell survival against different stress types such as the endoplasmic reticulum stress [3,5]. The process of autophagy can activate programmed cell death under speci c circumstances, and serves as an alternative to the apoptotic pathway [6]. Autophagy is divided into three main types of macro-autophagy, micro-autophagy, and chaperone-mediated autophagy (CMA) [7]. Microautophagy is mediated by lysosomal action through a direct engulfment of the cytoplasmic cargo. In macro-autophagy, an autophagosome with a bilayer membrane is formed and engulfs the damaged organelles and the unusable proteins.
It then delivers them to a vacuole to destroy and discard them [8][9][10]. CMA is a selective form of autophagy in mammals. In this process, some speci c proteins containing the KFERQ amino-acid sequence are detected by the chaperones and unfolded. Subsequently, they are delivered into the lysosomes by lysosome-associated membrane protein type 2A and degraded [11]. Macroautophagy and CMA, both maximally switched on in response to stress. As the sequential activation of these two pathways, cross-talk between these stress-related autophagic pathways are investigated in different studies [12].
The effective proteins in the macro-autophagy pathway include the Beclin1, P62, and LC3. By measuring the level of these proteins, the autophagic activity of the cells can be quanti ed [13]. Evidence shows that Beclin1 regulates autophagy and membrane tra cking involved in physiological and pathological processes [14]. P62 identi es the intracellular residues, and takes the role in their elimination. Impairment of autophagy would lead to accumulation of P62 in cells, leading to cellular stress and eventual disease [15,16]. Thus, an increased level of this protein indicates decreased autophagy [17,18]. It was reported that blockage of CMA activates macroautophagy in cultured cells [19].
Furthermore, a bidirectional cross-talk between macroautophagy and CMA was established which means changes in the activity of one of these pathways will affect the protein breakdown contribution of the other pathway [20].
In the process of autophagy, the cytoplasmic form of LC3 (LC3-I) is converted to conjugated LC3 (LC3-II), which is absorbed by the autophagosomal membrane. Since LC3-II within the autophagosomal membrane is degraded by the lysosomal hydrolase, the transient amount of LC3-II cannot indicate autophagic activity. Thus, LC3-II lysosomal turnover is estimated to determine the autophagic activity [21,22]. The GAPDH protein is used as a standard along with the aforementioned three proteins to assess their relative amount [23].
When autophagy moderately augmented, it exerts protective effects on cell function, such as ATP production and clearance of oxidized proteins and injured organelles. On the other hand, excessive activation of autophagy is associated with the risk of eliminating of essential proteins and intracellular organelles, which can cause cell death [24].
Chemical chaperones induce the CMA pathway, as the ability of mimicking the function of chaperones in the autophagy pathway. They enhance the transfer of mutated and misfolded proteins and improve the folding capacity of the endoplasmic reticulum [25]. The 4-phenyl butyric acid (4-PBA) is a non-toxic chemical chaperone and a type of fatty acid with low molecular weight. The American Food and Drug Administration has approved the application of 4-PBA [26], and is clinically used as an ammonia scavenger in children with urea cycle disorder and the treatment of sickle cell anemia and thalassemia [27]. It has maximum uptake by the heart and kidneys and was recently manufactured in controlled release form due to its short half-life [28,29]. Previous in vivo studies have documented the potential e cacy of 4-PBA in reducing the complications due to ischemia-reperfusion, brosis, and cardiac necrosis in rats [30,31]. There is a gap in existing knowledge in the area of effects of chemical chaperones such as 4-PBA on MI. Thus, this in vivo study aimed to assess the role of 4-PBA on hemodynamic and histopathological factors following induction of MI by isoproterenol in the rats. In addition, proteins involved in autophagy such as Beclin1, LCII/I, and P62 are evaluated to further understanding the compensatory mechanisms between the autophagic pathways in MI.

Experimental animals
In this study, a total of 30 male Wistar rats weighing 270-300 g were selected and randomly assigned to six standard propylene cages (n=5 in each cage). The rats were housed at 25-27°C and 50±10% humidity under 12 h light/12 h dark cycles with ad libitum access to food and water. The technician taking care of the rats was blinded to the study objectives. In case of the appearance of wound, infection, or death of a rat during the study period, it was excluded from the study and replaced with another rat.
This study was conducted under the institutional guidelines for the care and use of animal models. The approvement by the ethics committee of Tabriz University of Medical Sciences obtained (IR.TBZMED.VCR.REC.1397.139 and IR.TBZMED.VCR.REC.1397.048). In addition, the study designed and implemented under the recommendations of the ARRIVE guidelines for reporting animal research [32].
The healthy control rats received twice-subcutaneous injection of 0.3 ml saline at the back of their neck with a 24-h interval. In addition, they received ve injections of saline (0.3 ml, IP) with 24-h intervals. In the isoproterenol group to induce MI, isoproterenol dissolved in saline at a dosage of 100 mg/kg and subcutaneously injected into the back of rat's neck (in two consecutive days with a 24-h interval). In addition, similar to the control group, saline (0.3 ml, IP) was injected daily for ve days starting from the rst day.
The rats in groups P20, P40, and P80 received ve intraperitoneal injections of 4-PBA at a dosage of 20, 40, and 80 mg/kg, respectively. Simultaneously, all 4-PBA groups received two injections of isoproterenol as the same of isoproterenol group. The surgical procedure was performed 96 h after the second isoproterenol injection. NaOH was used for better dissolution of 4-PBA and the pH was adjusted at 7.4 [33]. The addition of NaOH converted the weak acid of 4-PBA, to sodium salt, similar to the saline group.
The rats in all ve groups were anesthetized by intraperitoneal injection of ketamine, xylazine and acepromazine.
Afterward rats underwent a surgical procedure on day 6 (96 h after the second isoproterenol injection).

Measurement of hemodynamic parameters
The open chest technique was used to assess the left ventricle function and direct measurement of left ventricle pressure alterations. For this purpose, the chest was opened and a catheter was directly inserted into the left ventricle. The pressure change at a xed ventricular pressure (LV dP/dt/P), left ventricular maximal and minimal rates of pressure increase (LV dP/dt min and LV dP/dt max ), left ventricular systolic pressure (LVSP) and left ventricular enddiastolic pressure (LVEDP), mean arterial pressure (MAP) and heart rate (HR) were recorded [34,35].

Blood collection and preparing the slides
Once the abdomen of the rats was opened, the blood was collected from the hepatic portal vein using a 10-ml syringe. One drop of blood was placed on a slide and spread. After drying, it was xed by dripping methanol over it, and stained by Giemsa stain (Merck). The peripheral blood neutrophils were evaluated under a microscope (Olympus, Japan) at x100 magni cation. The remaining blood was stored in the blood collecting tubes and plasma was separated from the blood cells by cold centrifugation (Eppendorf AG, Germany) at 3200 rpm for 20 min.

Cardiac examination
After opening the chest, the heart was removed and rinsed with cold saline at 4°C and weighed. To assess cardiac hypertrophy and edema, the ratio of the heart weight (in grams) to the rat body weight (in kilograms) was calculated. The cardiac tissue of 15 rats were frozen at -70°C for western blotting and the others were xed in 10% formalin for histopathological studies. Fixed tissues, embedded in para n, and sectioned into pieces with 5-µm thickness. The specimens were stained with hematoxylin and eosin (Sigma Aldrich, USA) and inspected under a microscope at x40 magni cation. Cardiac brosis and necrosis were assessed in each section by the morphometric point-counting procedure [36]. A blinded technician scored the histopathological changes as mild (score 1), moderate (score 2), severe (score 3), or very severe (score 4).

Measuring the serum TAC
The serum TAC was measured in plasma samples and assessed using the Ferric reducing ability of plasma (FRAP) method [37]. The method is based on the principle of the reduction of the ferric-tripyridyltriazine complex to the ferrous form by the antioxidants of a sample, which causes the development of an intense blue color, which is spectrophotometrically measurable at 593 nm and the change in absorbance is related to the antioxidant capacity of sample.
Western blotting A total of 40 mg/kg of the cardiac tissue apex was dissected and lysed in 2 ml of the lysis buffer using a homogenizer. Then centrifuged at 1000 rpm at 4°C for 10 minutes and the supernatant was stored at -70°C for further analysis. For western blotting, rst, total protein was quanti ed by NanoDrop (Thermo, USA). Next, 50-100 µg protein was removed from the solution and electrophoretically transferred onto a polyvinylidene uoride membrane using SDS-PAGE (12%). The membranes were blocked with 6% nonfat dried milk in phosphate-buffered saline (pH of 7.2) for one hour at room temperature. After blocking the non-speci c binding sites, the membranes were rinsed with the wash buffer and placed in a solution containing primary antibodies, and incubated overnight at 4°C. The primary antibodies included the rabbit polyclonal anti-Beclin1 (1:1000; Cell Signaling, USA), rabbit polyclonal anti-P62 (1:1000; Cell Signaling, USA), rabbit polyclonal anti-LC3 (1:1000; Cell Signaling, USA), and mouse polyclonal anti-GAPDH (1:2500; Abcam, England). Next, all membranes were rinsed with the wash buffer and incubated at room temperature for one hour in a solution containing secondary antibodies such as goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2000; Cell Signaling, USA) for Beclin1, P62, and LC3, and goat antimouse IgG horseradish peroxidase-conjugated secondary antibody (1:5000; Abcam, England) for GAPDH. The proteins were detected by the chemiluminescence method, and the appeared bands were observed by an imaging device. The optical density of the bands was quanti ed by an image scanning analysis software (Image j; Wayne Rasband, National Institute of Health, USA) and reported as optical density per square millimeter.

Statistical analysis
Data were analyzed using SPSS version 25 (SPSS Inc., IL, USA). The data were presented as mean ± standard error of the mean (SEM). ANOVA was used to compare the data among the study groups. In case the presence of a signi cant difference, the Fisher's least signi cant difference (LSD) test was applied. Differences between groups were considered signi cant if p < 0.05.
Heart rate decreased in the isoproterenol group (P<0.01). No signi cant change was documented in HR in groups P20, P40 and P80 compared to isoproterenol.
Isoproterenol group had lower LV dP/dt max and LV dP/dt min than the control group (P<0.01, P<0.05 respectively). P20 increased both the max and min LV dP/dt (P<0.01, P<0.05 respectively). P40, only made signi cant increase in LV dP/dtmax (P<0.05). In contrast, max and min LV dP/dt were not effectively changed by P80, compared to isoproterenol. The results showed that the changes trend in LV dP/dt/P were similar to the changes in min LV dP/dt. Effect of 4-PBA on the heart weight/body weight ratio To assess cardiac hypertrophy and edema, the ratio of the heart weight (in grams) to the rat body weight (in kilograms) was calculated and demonstrated in gure 1. The heart weight/body weight ratio increased in the isoproterenol group (P<0.01). 4-PBA in none of the treated groups could remarkably in uence this ratio compared to isoproterenol (P>0.05).
Effect of 4-PBA on cardiac tissue histopathology As shown in gure 2, in the control group, myocardial bers were arranged regularly with distinctive striation. No clear degeneration or necrosis was noted. Histological sections of cardiac tissue in the isoproterenol group showed extensive subendocardial necrosis, hypertrophy, abundant broblastic hyperplasia, and capillary dilation and leukocyte in ltration. P40 with less amount of these isoproterenol-induced injuries, signi cantly decreased the in ammatory response and destruction (P<0.001). In contrast, changes in groups P20 and P80 were not signi cant (P>0.05). To report the quantitative analysis of histological results, grading of histopathological changes of heart tissue after isoproterenol injection and in the presence of different doses of 4-PBA are also shown in gure 2.
Effect of 4-PBA on the peripheral blood neutrophils and serum TAC The peripheral blood neutrophils count and serum TAC are demonstrated in gure 3. Following the injection of isoproterenol and induction of in ammation, the percentage of peripheral blood neutrophils in the isoproterenol group was signi cantly augmented compared to the control group (P<0.001). This ratio decreased in all 4-PBA receiving groups with a signi cant difference in P40 (P<0.01) and P80 (P<0.001).
The serum TAC signi cantly decreased in the isoproterenol group compared with the control group (P<0.001). Using the 80 mg/kg of 4-PBA, made a notable serum TAC augmentation (P<0.001 versus isoproterenol group). P20 and P40 had not considerable TAC variation (P>0.05).

Results of western blotting
The Beclin1/GAPDH protein ratio increased in the isoproterenol group compared to the control. An insigni cant change in P20, P40 and P80 groups was documented for Beclin1/GAPDH compared to the isoproterenol group (P>0.05, Figure 4).
The difference in P62/GAPDH protein level between the isoproterenol and the healthy control groups was not remarkable (P>0.05). However, the P40 and P80 signi cantly decreased the P62 expression compared to the isoproterenol group (P<0.05, Figure 5).
The ratio of LC3-II/LC3-I/GAPDH protein was insigni cantly higher in the isoproterenol group compared to the control (P>0.05). In groups receiving 4-PBA, this amount decreased in the P20 group and again increased in the P40 and P80 groups compared with the isoproterenol group, however none of these changes were statistically signi cant (P>0.05, Figure 6).

Discussion
This study assessed the cardiac pharmacological effects of 4-PBA in rats with isoproterenol-induced MI. The hypothesis was that injection of 4-PBA by interfering in autophagy pathways would improve the pathological status of the heart. The results showed that the cardiac injury caused by isoproterenol signi cantly decreased following the administration of 4-PBA.
Isoproterenol is a beta-agonist sympathetic stimulant that induces MI, changes the hemodynamic parameters, and causes necrosis, brosis, in ammation, and neutrophil accumulation [38]. An increase in incompetent proteins level occurs in oxidative stress and MI, which result in further damage [39]. The cardiac status after MI may be improved by potential of 4-PBA in the regulation of oxidative stress and elimination of unfolded and misfolded proteins [40].
In this study, administration of 4-PBA in dosage of 20 and 40 mg/kg signi cantly improved the hemodynamic parameters. They reversed all the heart-weakening effects of isoproterenol by increasing the MAP, LV dP/dt/P, LV dP/dt max , LV dP/dt min , and LVSP and decreasing the LVEDP. Considering the LV dp/dt/P results, the mechanism of 4-PBA to demonstrate these hemodynamic in uences is highly by enhancing the cardiac muscle power and less likely through changing the volume or preload. In contrast, the variation in hemodynamic parameters was not signi cant following the injection of 80 mg/kg of 4-PBA. This controversy can be due to excessive stimulation of autophagy by the higher dose of 4-PBA and consequent elimination of essential proteins of cells, which could induce apoptosis in cardiac cells [41,42].
Administration of 4-PBA in concentration of 40 mg/kg decreased necrosis, brosis, and in ammation caused by isoproterenol in cardiac tissue. A previous research on the effect of 4-PBA on cardiac necrosis and brosis indicated that injection of 80 mg/kg 4-PBA in 1 h before induction of MI, decreased brosis and necrosis in rats by minimizing the oxidative stress [31]. Another study assessed the e cacy of 4-PBA to prevent the effects of thapsigargin on cardiac broblasts isolated from rats. It showed that 4-PBA decreased the endoplasmic reticulum stress and accumulation of procollagen in cardiac broblasts and consequently decreased the cell death [43]. Our results were in line with their ndings with the difference that 40 mg/kg of 4-PBA in our study corresponded to approximately 80 mg/kg dosage of 4-PBA sodium salt in previous studies.
Administration of 40 and 80 mg/kg of 4-PBA had striking effect in reducing the percentage of peripheral blood neutrophils, while this uctuation was not signi cant following the injection of 20 mg/kg of 4-PBA. This nding can be due to the demonstrating effective anti-in ammatory role in higher doses of 4-PBA, which have been previously reported. A study performed to assessment of the e cacy of 4-PBA in palliation of in ammation caused by dextran sulfate sodium (DSS) in mice colon. It exerted anti-in ammatory effects in higher dose of 150 mg/kg by suppressing the nuclear factor-κB activation and inhibiting the pro-in ammatory cytokines, interleukin-6, interleukin-1B, and tumor necrosis factor-alpha [44].
Compatible with neutrophils count abatement, the increase in serum TAC was only signi cant in high dose of 80 mg/kg of 4-PBA. This result can be related to the declining of oxidative stress, which result in increasing the antioxidant capacity and decreasing cell damages [45].
The ratio of heart weight/body weight signi cantly increased following the injection of isoproterenol compared with the control group. This increase, seems to be due to the edema and cardiac tissue in ammation, and less likely related to cardiac tissue hypertrophy. As we assessed acute MI and there was not su cient time for the cardiac hypertrophy occurrence. Injection of 4-PBA did not cause a signi cant change in this ratio, which indicates ine cacy of 4-PBA for tissue edema.
The results of western-blotting tests showed that the level of P62 signi cantly decreased following the 40 and 80 mg/kg of 4-PBA, indicating the induction of the macro-autophagy pathway. In addition, the variation trend of other proteins in this cascade were also towards the induction of macro-autophagy, though not signi cant. There are different reports introducing the 4-PBA as a chemical chaperon with potential of inducing the CMA pathway. Considering the correlation of CMA with the macro-autophagy pathways, by induction of CMA, the macro-autophagy pathway is suppressed [46][47][48]. In this study, in contrast to our expectations, injection of 4-PBA induced the macroautophagy pathway. This may be due to a sudden increase in unfolded or misfolded proteins in MI, which can activate both macro-autophagy and CMA pathways at the same time [49]. Moreover, P62 can serve as a mediator between the CMA and macro-autophagy pathways, suggesting the cross-talking of these two pathways

Conclusion
The current results showed that injection of 4-PBA caused signi cant improvements in hemodynamic factors, histopathological changes, in ammation, serum TAC, and neutrophil accumulation, which demonstrated the cardioprotective effects in isoproterenol-induced MI. As the autophagy modulation potential of 4-PBA, understanding its compensatory mechanisms between the different autophagic pathways is important in light of the development of interventions aimed for therapeutic purposes with altering autophagy especially in cardiac disorders.

Declarations
FundingThis work was supported by the vice chancellor for research of Tabriz University of medical sciences. It belonged to study design number 60013, which was con rmed by the ethical committee of Tabriz medical university (IR.TBZMED.VCR.REC.1397.048).
Competing Interests The authors have no relevant nancial or non-nancial interests to disclose.
Author contributions All authors contributed to the study conception and design. Fatemeh Vatankhah performed most of the experiments and drafted the manuscript; Haleh Vaez designed, conceived the experiments and analyzed the study and corrected the manuscript; Alireza Garjani supervised the study and corrected the manuscript.