The Effect of Chemerin on Cardiac Parameters and Gene Expressions in Isolated Perfused Rat Heart

Background: Chemerin is a novel chemoattractant adipokine expressed in cardiovascular system, and its receptor has been detected in the epicardial adipose tissue. Aims: To determine the effects of chemerin on the cardiac parameters and gene expressions in the isolated perfused rat heart. Study Design: Animal experiment. Methods: The hearts were retrogradely perfused with Langendorff technique to measure the cardiac parameters. The experimental groups were acutely treated with 10, 100, and 1000 nM doses of chemerin. Another group was given 10 μM L-nitric oxide synthase inhibitor for 5 min before 1000 nM chemerin administration. The real-time polymerase chain reaction was performed for detecting the expression of target genes. Results: All doses of chemerin significantly decreased the left ventricular developed pressure (max 35.33 Δ%, p<0.001), and +dP/dtmax (max 31.3 Δ%, p<0.001), which are the indexes of cardiac contractile force. In addition, 1000 nM chemerin reduced the coronary flow (max 31 Δ%, p<0.001). N(W)-nitro-L-arginine methyl ester antagonized the negative inotropic effect of chemerin on contractility. Chemerin induced a 2.16-fold increase in endothelial nitric oxide synthase mRNA and increased the cyclic guanosine monophosphate levels (p<0.001) but decreased the PI3Kγ gene expression (1.8-fold, p<0.001). Furthermore, all doses of chemerin decreased the CaV1.2 gene expression (1.69-fold, p<0.001). Conclusion: Acute chemerin treatment induces a negative inotropic action with the involvement of nitric oxide pathway, CaV1.2, and PI3Kγ on isolated rat heart.

The adipose tissue not only serves as an energy storage but also acts as an endocrine tissue that releases various proteins called adipocytokines (1). These proteins exhibit endocrine, autocrine, and paracrine effects and are involved in various physiological and pathophysiological events, such as food intake, insulin sensitivity, immunity, and inflammation (2). Chemerin is a 16 kDa adipocytokine consisting of 137 amino acid sequences (3); its receptors are synthesized mainly in the liver, adipose tissue, and cardiovascular system (4). Chemerin acts as a crucial protein in terms of physiological responses, such as inflammation and insulin resistance, adipose differentiation, and regulation of immune response, maturation, and metabolism (5). Chemerin may regulate the cardiovascular functions and gene expressions. Studies have reported that chemerin is associated with obesity, metabolic syndrome, and diabetes (6,7). In addition, circulating chemerin concentrations has been positively correlated to many obesity-related parameters, including body mass index, blood pressure, serum lipid, insulin, and cholesterol concentration (8).

MATERIALS AND METHODS
Female Sprague-Dawley rats (250-350 g) were fed with a standard rat diet and housed in cages under 12 h light/12 h dark at 20 °C-25 °C. The procedures in the present study were conducted in accordance to the "Guide to the Care and Use of Experimental Animals" by the Canadian Council of Animal Care and after receiving the approval of the Institutional Animal Care and Use Committee (IACUC) in Eskişehir Osmangazi University (IACUC approval no. 385/2014).

Experimental groups
Four experimental groups were studied. Group 1: The hearts were perfused with only Krebs Henseleit solution (mK-Hs) for 30 min (control group). Groups 2, 3, and 4: The hearts were perfused with mK-Hs containing 10, 100, and 1000 nM chemerin, respectively. Group 5: The hearts were perfused with mK-Hs containing 10 μM NO synthase inhibitor N(W)-nitro-L-arginine methyl ester (L-NAME) for 5 min before 1000 nM chemerin administration.

Experimental protocol
The animals were anesthetized with intraperitoneal injection of sodium thiopental (50 mg/kg) and given heparin (1000 IU). After sufficient anesthesia administration, the chest of the rats was opened. The heart was rapidly removed and arrested in ice-cold mK-Hs and then transferred to the Langendorff apparatus while perfusing with mK-Hs. The retrograde perfusion was performed in the noncirculating Langendorff mode under a constant pressure (60 mmHg), whereas the pulmonary artery was incised for the complete coronary drainage of the ventricles. To measure the cardiac contractile force, this study used the method previously described by He and Downey (12). A water-filled latex balloon was set to achieve a stable left ventricular end-diastolic pressure of 8 mmHg and then inserted into the left ventricle via the mitral valve and connected to a pressure transducer. Data acquisition was performed with a data acquisition and analysis system (Isoheart Software, Germany) to determine the pressure measurements. LVDP and +dP/dt max were monitored and used as the contractility indexes. The contact electrode technique was applied by using MAP electrodes (Ag/AgCl 2 ) to measure the MAPamp and MAP 90 recordings. The hearts were included from the study if normal sinus rhythm or LVDP, +dP/dt max , and heart rate after the first 30 min of perfusion were more than 60 mm Hg, 2800 mmHg s -1 , and 200 beat/min, respectively. mRNA preparation and real-time quantitative polymerase chain reaction

Enzyme-linked immunosorbent assay
The frozen tissues (50-100 mg) were treated with phosphatebuffered saline at 0 °C and centrifuged for 20 min at 446-1006×g. After the supernatant was extracted, the cAMP and cGMP concentrations of the aqueous phase were measured with a commercial enzyme immunoassay (YH Biosearch, Shanghai, China).

Statistical analysis
SPSS for Windows (version 13.0, SPSS Inc, Chicago, USA) was used to conduct the statistical analysis. The normality of data distribution was evaluated with Shapiro-Wilk test and Kolmogorov-Smirnov test with Lilliefor's correction. Thereafter, one-way analysis of variance and Tukey's honest significant difference multiple comparisons post hoc test were used for the data analysis. The findings were reported as the mean ± standard deviation, and p values were considered statistically significant at less than 0.05.

DISCUSSION
The present study showed that chemerin significantly reduced LVDP and +dP/dt max values in isolated perfused rat hearts. This study also demonstrated that the treatment of isolated hearts with chemerin protein increased the eNOS mRNA and cGMP levels and reduced the expression of CaV1.2 gene in cardiac tissues. This research reported for the first time that acute chemerin treatment of isolated perfused rat leads to decreases in cardiac contractility. In addition, in this work, the negative inotropic effects of chemerin were antagonized with L-NAME. The findings revealed that NO may mediate the negative inotropic effects of chemerin.
Considering that adipocytokines activate several signaling pathways, chemerin could induce the NO synthesis through activation of the PI3K/Akt/eNOS pathways. The activation of the PI3K/Akt/eNOS signaling pathway results in the phosphorylation of Akt; phosphorylated Akt enhances the NO production (13). NO activates the soluble guanyl cyclase, subsequently leading to the production of cGMP. Subsequently, the cGMP-dependent protein kinase G (PKG) is activated. PKG reduces the amount of CaV1.2 mRNA and consequently reduces the amount of Ca 2+ entering the heart muscle cells by decreasing the Ca 2+ channel flow. PKG also exerts negative inotropic effects by diminishing the L-type Ca 2+ channel currents and through the desensitization of cardiac myofilaments to Ca 2+ (13,14). In this manner, the reduction of  Ca 2+ entering the cells results in a negative inotropic effect on the heart. Therefore, whether changes in the expression of genes are related to the PI3K/Akt/eNOS pathways in chemerin treatment isolated rat hearts must be investigated. To this end, we studied the expression of the genes of Cav1.2, PI3Kα, PI3Kγ, and eNOS on isolated rat hearts. The application of chemerin to isolated rat hearts resulted in a statistically significant increase in the eNOS mRNA levels in comparison with the controls. In addition, with the increase in eNOS gene expression, the amount of cGMP in the cell also increased. NO contributes to the fine-tuning of the excitation-contraction coupling in the myocardium (15). The low concentrations of NO lead to positive inotropic effects, (16) whereas its high concentrations of NO result in the opposite trend. According to recent studies, the general idea regarding NO is that it causes biphasic effects on the myocardial function (15)(16)(17)(18). These findings also support that NO mediates the negative inotropic effects of chemerin. NO is an important biological messenger which plays an important role in regulating cardiac functions, such as cardiac contraction, heart rate, and vascular tone. The disequilibrium of NO production is associated with cardiovascular disorders, including ischemic heart disease, hypertension, heart failure, coronary artery disorder, and arrhythmia (16). Studies have reported that endogenous eNOS activation is associated with ventricular arrhythmias in dogs. Massion ve ark. (15) observed that the amount of eNOS mRNA in myocytes decreased in the late phase of hypertrophic cardiomyopathy and heart failure. In the early period of ischemia and reperfusion, the amount of eNOS in the heart increases; the increase in NO protects against cardiac ischemia and reperfusion injury with various mechanisms (18). The cardiovascular system is complex given its own system and functions similar to those of the endothelial cells. In addition, the cardiovascular system plays an important role by interfering with other systems thorough various mechanisms. Research suggests that chemerin acts as a central actor in human physiology and pathology. For this reason, understanding chemerin, as a global protein in the cardiovascular system, will allow us to better understand the pathologies associated with chemerin and to apply appropriate treatments (3). PI3Kα is a subunit of the PI3K signaling pathway, and the decrease in its signal reduces the number of L-type Ca +2 channels present in the heart cells; a decreased L-type Ca +2 channel current results in reduced contractility (19). In other words, PI3Kα participates in the positive regulation of heart contraction. In vitro contractility also increased due to the increased PI3Kα expression in the adult mice (20). In our study, we determined no statistically significant change in the PI3Kα gene expression levels in the chemerin groups in comparison with the control group. Considering the positive relationship between PI3Kα and the L-type Ca +2 channel current, we concluded that the significant decrease in the mRNA level of CaV1.2 gene in chemerin-treated groups is unrelated to PI3Kα. PI3Kγ is another subunit of the PI3K signaling pathway. This subunit plays a role in regulating cardiac contraction. The catalytic subunit of PI3Kγ, p110γ, regulates the amount of cAMP (21). p110γ activates phosphodiesterases (PDEs); activated PDEs cause the destruction of cAMP. This condition negatively affects the contractility by reducing the amount of cAMP in the cell (21,22).
In our study, statistically significant reductions in PI3Kγ gene expression levels were observed in the chemerin-treated groups. The literature shows that intracellular cAMP levels increase with decreasing PI3Kγ gene expression levels. The amounts of cAMP that is required to be evaluated together with the decreased PI3Kγ gene expression showed no statistically significant increase but demonstrated a 2-fold increase in comparison with the control values (23)(24)(25). The insignificant increase in the amount of cAMP and decrease in the PI3Kγ levels are consistent with the observed negative inotropic effects of chemerin. The cardiac ARs in the myocardium contribute to the control of inotropy, rate of relaxation with the changing levels of intracellular Ca 2+ , and heart rate. Approximately 75% of the cardiac ARs are β1-AR, and the rest comprise β2-AR (26). In this research, the β1-AR and β2-AR gene expressions were studied to investigate whether changes in the expression of the genes are related to the contraction of isolated rat heart treated with chemerin. However, upon comparison of the chemerin-treated and control groups, no statistically significant difference was found in both gene expressions. In addition, no statistically significant difference in heartbeats was determined between the chemerin-administered and control groups. These findings suggest that chemerin could not mediate the effects of β1-AR and β2-AR on heart contractility. This compound also plays no part in the regulation of heart rate. As a result of the stimulation of β-ARs, the literature suggests that the level of cAMP in the cell increases; consequently, the contraction force of the heart increases (26). However, in our study, no significant increase was observed in the cAMP level. The statistically insignificant increase in the amount of cAMP in the chemerin-treated groups may be due to a decrease in the PI3Kγ gene expression level, leading to an increase in cAMP (25). In our study, 2 min after the application of 1000 nM chemerin, the volume of coronary flow decreased by 30%-40%, and this decrease showed statistical significance. In addition, coronary flow volumes during the application of the chemerin remained below the control values. Lobato et al. (27) have demonstrated that chemerin enhanced the vasoconstriction responses of the α1adrenergic agonist phenylephrine and endothelin-1 in rat aorta. Watts et al. (28) showed that the application of 1000 nM dose of chemerin caused contraction on the isolated thoracic aorta from obese and hypertensive rats and the isolated mesenteric arteries from obese humans. They also observed that the NO synthase inhibitor N-ω-nitro-L-arginine increased the contractile responses caused by chemerin. In our study, the reduction in the coronary flow may be attributed to the reduced heart contractility and the vasoconstrictor effect of chemerin on the 1000 nM dose group. The expectation was that eNOS would lead to a rise in coronary flow; however, the 10 and 100 nM chemerin doses caused no alterations on this variable. The coronary perfusion pressure is a good index of coronary vascular tone. However, in our study, the coronary effluent amounts could be measured from the collection of the coronary effluent after 1 min in a graduated cylinder instead of coronary perfusion pressure. Although the eNOS mRNA quantities increased, no increase was observed in the coronary flow. Thus, the unexpected results may be also the outcomes of the differences in the methods employed. MAPamp depends on the intracellular diffusion of Na + ions from Na + channels in the first phase of MAP (phase 0) (29). In our study, chemerin caused no significant change in the values of MAPamp. This finding suggests that chemerin protein caused no alteration in the Na + channel current during phase 0. On the other hand, MAP90 provides information about the repolarization phase of AP; it is inversely proportional to the number of heartbeats (30). The MAP90 showed a statistically insignificant change in the chemerin-treated groups compared with the control groups. The non-significant changes in the MAP90 in this study may be due to the insignificant change in the gene expression levels of β1-2-ARs and consequent negligible changes in heartbeats. This study is the first attempt to demonstrate that chemerin exerts negative inotropic effects while decreasing the LVDP, +dP/ dt max , and coronary flow. On the other hand, L-NAME totally reversed the negative inotropic effects of chemerin. We suggest that chemerin possesses a negative inotropic action, and NO may mediate this action. For the first time, we observed that chemerin reduces the PI3Kγ and CaV1.2 gene expressions but enhances the eNOS mRNA and tissue cGMP levels. This study argues that the negative inotropic effects generated by chemerin is associated with the increased amount of eNOS mRNA and decreased CaV1.2 mRNA levels. However, the relationship of negative inotropic effect induced by chemerin to cardiac and vascular pathology must be demonstrated in vivo and replicated in human tissues. Further and more advanced research is required to explore the potential impact of our findings on the pathology of cardiovascular illnesses and the potential role of chemerin in the treatment processes.