INTRODUCTION

Myocardial infarction (MI) is one of the ischemic heart diseases. Insufficient oxygenated blood flow to the myocardium leads to necrosis and apoptosis inducing myocardial injury in MI¹. MI also causes edema and it has been reported that ischemic injury extends after longer than 15 min following myocardial edema². Slight increase of myocardial water content has devastating effects on systolic and diastolic functions whereas the 3.5% increase of myocardial edema may cause 30-50% decrease in cardiac output^3,4.

Aquaporins (AQPs), are a family of small integral membrane proteins, facilitating bidirectional flow of water. In mammals, 13 AQPs channels have been identified (AQP 0-12). AQP3, AQP7, AQP9 and AQP10 are called aquaglyceroporins, which permit transcellular passage of glycerol, urea, and other solutes⁵. AQP channels were previously described in the myocardium of rats and cardiac AQPs were associated with myocardial edema after MI and doxorubicin, or cisplatin induced myocardial injury^6-8. However, the role of AQP channels in myocardial edema have not been fully clarified.

Isoproterenol (ISO; 1-(3,4-dihydroxyphenyl)-2-isopropylamino-ethanol hydrochloride), is a β-adrenergic agonist, and the administration supramaximal dose of ISO provides a rapidly, easy, and non-invasive MI method with the low mortality and high effectiveness. The pathophysiological alterations such as increased oxidative stress, lipid peroxidation, increase of myocardial injury markers [troponin I (TnI), lactate dehydrogenase, creatine kinase (CK-MB)] and pro-inflammatory cytokines [C-reactive protein (CRP), interleukin-6, and tumor necrosis factor-α (TNF-α)], necrosis and apoptosis may occur in ISO induced MI model^7-9.

MEL has cardioprotective effects against environmental chemicals and drugs induced cardiotoxicity⁹, cardiac ischemia-reperfusion injury¹⁰, diabetic cardiomyopathy¹¹ and MI. With those effects, MEL was suggested as a potential therapeutic agent for cardiovascular diseases¹². However, the effects of MEL on cardiac AQP channels have not been adequately clarified.

Therefore, we aimed to investigate the changes of AQP1, AQP3, AQP4 and AQP7 channels in ISO induced MI model and to investigate the effects of MEL on these channels and preventive effects on myocardial edema and damage.

MATERIALS AND METHODS

Animals

In this study, 28 Wistar albino male rats (2-3 months old) were used. Animals were purchased from Çanakkale Onsekiz Mart University Experimental Research Centre. During the experiment, rats were fed ad libitum. Temperature was maintained at 22±2 °C with a 12 h light/dark cycle. All animal experimental procedures and protocols were approved by Çanakkale Onsekiz Mart University Animal Experiments Local Ethics Committee (decision number 2019/07-04, date: 25.09.2019).

Group Design

Rats were randomly divided into four groups. All experimental procedures were applicated at 08:00-10:00 a.m.

Control group (n=7): The vehicles for MEL and isoproterenol were administrated for 7 days intraperitoneally (ip) in this group.

MEL group (n=7): MEL was administrated with 10 mg/kg/day ip for 7 days.

ISO group (n=7): Isoproterenol was administrated (85 mg/kg) at the 6^th and 7^th days of the experiment.

Isoproterenol and MEL group (ISO+MEL; n=7): MEL was administrated 10 mg/kg/day ip for 7 days. One hour after MEL administration, ISO was administrated (85 mg/kg) at the 6^th and 7^th days of the experiment.

Electrocardiographic (ECG) recording was obtained on the 8^th day of the experiment.

Preparation and Administration of Drugs

Isoproterenol (Sigma-Aldrich, I6504) was dissolved with 1 mL 0.9% NaCl and freshly prepared before administration during the whole experiment¹³.

MEL (N-acetyl-5-methoxytryptamin; product code: M5250-1G) was dissolved in absolute ethanol (absolute GR for analysis, MERCK) and further dilutions were prepared in isotonic saline for final concentration of 2.4% (v/v) ethanol. MEL was daily prepared and administrated freshly¹⁴.

Induction of Myocardial Infarction

The MI was induced with ISO treatment (85 mg/kg, ip) on the 6^th and 7^th days of the experiment¹³.

ECG Recording and Evaluation

ECG recording was obtained at 09:00-09:30 on the 8^th day of the experiment after 24 h following the last ISO administration. The extremity derivations DI, DII and DIII, aVR, aVL and aVF were recorded noninvasively (Poly-Spectrum 12 channel ECG-System, Poly-Spectrum-8, Neurosoft, 5, Voronin str., Ivanovo, Russia). Before 10 min of ECG recording, animals were treated with 30 mg/kg ketamine and 5 mg/kg xylazine for sedation¹⁵. After evaluation of ECG, morphology of ST segment was examined.

Euthanasia and Tissue Harvesting

At the end of the experiment (8^th day), blood samples were obtained from the heart under the anesthesia with ketamine and xylazine . Then, animals were sacrificed. Heart specimen were harvested and left ventricles (LV) were separated. The LV samples were sliced into three pieces for histopathological, genetic analysis and water content examination.

Water Content

LV slices were weighed for wet weight evaluation immediately, then kept for 24 h in an oven at +80 °C. Then, LV slices were used for dry/weight evaluation. Water content of LV samples were calculated as [(wet weight-dry weight)/wet weight] x 100%¹⁶.

Histopathological Evaluation

LVs were fixed in 10% formaldehyde, embedded in paraffin, and processed for staining with hematoxylin&eosin (H&E) for histopathological evaluation.

Biochemical Analysis

cTnI (Catalog≠:MBS2104797) and CK-MB (Catalog≠ :MBS2515061) levels were measured from serum samples using ELISA kits provided according to the manufacturer procedure.

Total RNA Isolation

Total RNA isolation was performed from LV samples. Briefly, 25-30 mg tissue samples were put into 2 mL Eppendorf tubes and incubated for 10 sec in liquid nitrogen, then homogenized. Later, homogenates were manually used for total RNA isolation by PURE Link RNA MiniKit (CatNo.12183018A). Purification and concentration analysis of total RNAs were performed with NanoDrop ND-1000 spectrophotometer. Samples were kept at -80 °C until genetic evaluation. RNAs scored between 1.8 and 2.1 were purified at 260/280 nm and considered appropriate for analysis.

cDNA Isolation

After spectrophotometric evaluation, isolated RNAs were put in 0.2 mL PCR tubes according to RNA concentrations. cDNA isolation was performed manually with a kit. The mixture was put into PCR tubes and evaluation was performed at PCR device (the Applied Biosystems^®, 2720 Thermal Cycler 96-Well PCR).

Real-Time PCR (qRT-PCR)

Quantitative real-time PCR (StepOnePlus™ Real-Time PCR System) was performed with DNA samples. TaqMan (TaqManTM Fast Advanced Master Mix, Ampliqon, Lithuania) was used for gene expression levels.

AQP1, AQP3, AQP4, AQP7, TNF-α, Caspase-3 and Bax, gene expression levels were evaluated with qRT-PCR against normalization by β-actin the housekeeping gene.

Taqman primer-probs were used for specific genes in this study given below.

TaqMan^® Gene Expression Assays, 250 rxns AQP1..Rn00562834_m1

TaqMan^® Gene Expression Assays, 250 rxns AQP3..Rn00581754_m1

TaqMan^® Gene Expression Assays, 250 rxns AQP4..Rn01401327_s1

TaqMan^® Gene Expression Assays, 250 rxns AQP7..Rn00569727_m1

TaqMan^® Gene Expression Assays, 250 rxns TNF-α..01525859_g1

TaqMan^® Gene Expression Assays, 250 rxns Caspase-3..Rn00563902_m1

TaqMan^® Gene Expression Assays, 250 rxns Bax..Rn01480161_g1

TaqMan^® Gene Expression Assays, 250 rxns ACTB..Rn00667869_m1

Statistical Analysis

Genetic analysis was performed with qRT-PCR and relative fold changes were estimated with the 2^-ΔΔCt formula. The other results were presented as mean±standard error. Data were analyzed by using IBM Statistics Statistical Package for the Social Sciences 20.0. The Kruskal-Wallis test and Mann-Whitney U test were used for the comparison of groups. The significance level was considered as p<0.05.

RESULTS

ECG Determination

ST segment elevation was not observed on the 8^th day in the control and MEL groups. However, ST segment elevation was determined in all rats of ISO and 2 rats of 7 in the ISO-MEL groups on the 8^th day of the experiment. ECG recording obtained from control and ST elevated animals were represented in Figure 1.

LV Water Content Results

LV water content outcomes were presented in Figure 2. The water content levels of the MEL and Control groups were observed to be similar. However, higher water content values were obtained both in the ISO and ISO+MEL (p<0.01) groups compared to other groups.

Biochemical Analysis

Plasma CK-MB and cTnI levels were presented in Table 1. There was no significant difference between the groups in terms of CK-MB levels (p>0.05). Higher and statistically significant cTnI levels were observed in the ISO and ISO+MEL group compared to the control group (p<0.01).

Genetic Analysis

Inflammation and Apoptosis Related Molecules

The change of inflammation (TNF-α) and apoptosis (Caspase-3 and BAX) related molecules gene expression levels were submitted in Figure 3. The higher and significantly increasing values were observed in the ISO group for all mentioned molecules compared to the control (p<0.05 or p<0.01). MEL reduced TNF-α, Caspase-3 and BAX gene expression levels.

AQP Channels

Higher AQP1, AQP3, AQP4 and AQP7 levels were observed in the ISO group compared to the control (p<0.01), MEL (p<0.05) and ISO+MEL groups (p<0.01), as represented in Figure 4. MEL treatment significantly decreased all AQP channels gene expression levels.

Histopathological Results

H&E staining results were represented in Figure 5. Our results indicated that there was no edematous area in the control and melatonin groups (Figure 5A, 5B and 5E). However, several edematous areas were obtained in the ISO group (p<0.01, Figure 5C and 5E). MEL treatment significantly decreased edema score compared to ISO (p<0.01, Figure 5E). These results indicated that MEL had an ameliorative effect on ISO induced myocardial edema at histopathological level. Mononuclear cellular infiltration examination of LV was performed by H&E staining and results were also presented in Figure 5F. There was no mononuclear cell infiltration in the control group while there was a quite low mononuclear cell infiltration in the MEL group (Figure 5A, 5B). Our results indicated that there was a significant increase of mononuclear cell infiltration in the ISO group (p<0.01, Figure 5C and 5F). However, MEL administration had a slight and insignificant decreasing effect on mononuclear cellular infiltration score in the ISO+MEL group (Figure 5F). These results pointed out that MEL had an ameliorative effect on mononuclear cellular infiltration but this effect was not as strong as on myocardial edema.

DISCUSSION

This is the first study evaluated the changes of AQP1, AQP3, AQP4 and AQP7 gene expression levels in myocardium after ISO induced MI model in rats and the effects of MEL on AQP channels.

ISO is a widely used agent for inducing experimental MI model in rats. ISO induced MI model provides an advantage for the investigation of cardioprotective agents¹⁷. ST segment elevation is considered a prominent indicator of ISO induced MI^18,19. In our study, we observed ST segment elevation in all 7 rats in the ISO group (Figure 1). Increases of both cTnI and CK-MB^20,21 or only CK-MB levels²² were reported after ISO induced MI in rats. Also, it has been suggested that increased CK-MB can usually be detected in about 4-8 hours after the onset of MI; however, the cTnI is a more specific marker for the determination of MI²³. In our study, cTnI levels were found significantly higher in the ISO-treated group compared to the control group (p<0.01; Table 1). We could not obtain significant changes in blood CK-MB levels. Histopathological changes such as irregular manner of myocardial cells, inflammatory cell infiltration (mononuclear cellular infiltration), and deterioration in myocardial fibers were observed in the myocardium after ISO-induced MI^18,24. In accordance with previous studies, we determined edema and cellular infiltration in the myocardium after ISO administration. In our study, melatonin administration reduced inflammatory cell infiltration, but this result was statistically insignificant. This outcome indicated that the protective effect of melatonin on inflammatory cell infiltration might not strongly come out in the first 24 hours after MI.

The cardioprotective effects of MEL are well known with antioxidant, anti-inflammatory and endothelial protective properties²³. In our study, the cardioprotective effects of MEL were determined with ECG, biochemical, and histopathological evaluation. ST segment elevation was observed in all 7 rats in the ISO group; however, ST elevation was obtained only in two rats in the MEL group (Figure 1). Histopathological evaluation indicated that MEL administration reduced cardiac edema (Figure 5B, 5E). However, the results for the heart water content were not fully compatible with edema score. We observed high edema score in the ISO group, but we did not obtain higher water content in this group. Thus, we suggested that there could be other effective factors on water content without edema score. MEL treatment continued after two days of MI inducement, suggesting that MEL has also a protective effect on ISO induced MI.

Apoptosis has an important role in the pathogenesis of various heart diseases including MI²⁵. The role of BAX and Caspase-3 in cardiomyocyte apoptotic pathway is well known²⁶. It is reported that increased BAX and Caspase-3 gene expression levels were determined in MI²⁷. In our study, the highest levels of Caspase-3 and BAX levels were investigated in the ISO group. MEL administration reduced the increased levels of Caspase-3 and BAX levels. MEL is a well-known anti-apoptotic agent in other organs²⁸, also in the heart^8,29. It was investigated in our study that MEL ameliorated ISO induced apoptosis.

Ischemia/reperfusion may cause fluid accumulation through both interstitial and intracellular compartments and produce edema and cardiomyocyte damage³⁰. It has been reported that increase of sarcolemmal breakdown, stimulation of phospholipases activity and dysfunction of Na+-K+ ATPase might trigger myocardial edema and cardiomyocyte damage³¹. Eventually, an increase of cardiac edema significantly reduces the systolic and diastolic functions of the heart². Although several AQPs are determined in the myocardium and AQPs play a pivotal role in body water balance, the physiological role of cardiac AQPs in the heart remains unknown. In the present study, we investigated AQP1, AQP3, AQP4 and AQP7 gene expression levels in LVs after ISO induced MI and effects of MEL on these AQP channels.

Previous studies clarified the relationship between the AQP1 channel and myocardial edema. High AQP1 levels directly increased water permeability and caused myocardial edema³¹. Yan et al.³² observed a significant increase both in AQP1 gene expression level and myocardial edema in goats undergoing cardiopulmonary bypass. In a similar study, an increase of myocardial edema and cGMP related AQP1 were observed in LV after the cardiopulmonary bypass in sheep. On the other hand, the administration of ODQ (a specific inhibitor of soluble guanylate cyclase) caused a decrease both in myocardial edema and AQP1 level³³. It was previously determined that the effects of AQP1 on ischemia/reperfusion induced myocardial edema and high levels of AQP1 increased LV water content in rats³¹. In our study, we obtained higher AQP1 and LV water content in the ISO induced MI group compared to the control group (p<0.01, Figure 4A). MEL significantly decreased AQP1 in the ISO+MEL group (p<0.01). MEL administration did not significantly decrease LV water content compared to the ISO group; however, we observed significantly lower edema score in the MEL administrated group at histopathological level. Although MEL and AQP1 interaction was investigated in the kidney³⁴ and medulla spinalis³⁵, there is scarcity about the effects of MEL on myocardial AQP1. Furthermore, there are limited studies that reported both cardiac AQP1 existence and function of myocardial AQP1. Best of our knowledge, our study was the first research representing the interaction between MEL and AQP1 in MI.

AQP3 is known as a member of aquaglyceroporin family and was determined at the kidney, skin, digestive tract, and salivary gland in rats^36-39. Nevertheless, there are controversial and limited studies focusing on cardiac AQP3². Also, the role of AQP3 in cardiovascular diseases and MEL-AQP3 interaction in the myocardium remains unknown. Most recently, our group has previously observed AQP3 expression in LV and MEL has a decreasing effect on AQP3 expression in cisplatin induced cardiac injury of rats⁸. To the best of our knowledge, our study is the first research investigating MEL and AQP3 relationship in ISO induced MI. AQP3 transports H₂O₂ to intracellular compartment and H₂O₂ acts as a secondary messenger on several pathways including inflammation, cell development and migration⁴⁰. However, an increase of AQP3 expression leads to transferring of large amounts of H₂O₂ into the cell and higher intracellular H₂O₂ stimulates cellular damage^41,42. It has been determined that quercetin, a powerful antioxidant, has cellular protective effect against H₂O₂ toxicity through the downregulation of AQP3 channels in the intestinal tract. Another study reported that the increase of AQP3 expression in the kidney was related to oxidative effects of fructose in metabolic syndrome⁴³. In our study, we obtained significantly higher AQP3, TNF-α and Caspase-3 expression levels and widespread myocardial edema in the ISO administrated group compared to other (p<0.01). We also determined powerful inhibitory effect of MEL on AQP3 and TNF-α expression and reduced myocardial edema. Furthermore, MEL moderately decreased Caspase-3 and mononuclear cell infiltration. These outcomes indicate that MEL affects the AQP3 channel by reducing inflammation and apoptosis.

The AQP4 is the most investigated AQP channels in respect with myocardium and in relation to MI. AQP4 is an important AQP channel for the heart and has widespread distribution in the myocardium, such as cardiac muscle, intercalated discs, endothelial cells, sarcolemma, and serosa⁴⁴. Zhang et al.⁷ observed an increase in AQP4 within 1 week after MI. In another study, it was reported that there was an increase in AQP4 at the 15^th minute and reached the highest level at the 45^th minute during the acute period of MI. These results indicated that AQP4 was a crucial component of myocardial edema. There was a relationship between AQP4 and infarct size, which was also reported⁴⁵. Both studies mentioned above were conducted on mice. One of other studies⁴⁶ determined that AQP4 expression increased in a cardiopulmonary resuscitation model of rats. They revealed that there was a relationship between the PI3K/Akt pathway and AQP4, and this pathway had a reducing effect on myocardial damage. The increase of AQP4 was also reported in cardiopulmonary resuscitation and considered as an important criterion of myocardial damage⁴⁷. Novel studies reported the relationship between MEL and AQP4 in the medulla spinalis^48,49 and brain⁵⁰. However, AQP4 and its relationship with MEL in the heart remains unknown. We observed that MEL had a decreasing effect on myocardial edema and AQP4. These are the first findings indicating that MEL has ameliorative effects on ISO induced myocardial edema and increase of AQP4.

There are more researches on AQP7 channel in the myocardium compared to AQP1, AQP3 and AQP^42,30. AQP7 is permeated glycerol as well as water and subcategorized as aquaglyceroporins⁵¹. Hibuse et al.⁵² revealed that AQP7 deficiency in the heart significantly reduced glycerol consumption and ATP content. However, they suggested that there was no relationship between AQP7 and myocardial edema. Higher AQP7 levels were observed in rats consuming high protein diet⁵³. It is generally accepted that AQP7 channels are effective for metabolic adaptation of the heart. Glycerol is one of the main myocardium energy sources transported through AQP7⁵⁴. We observed a significant increase of AQP7 gene expression (p<0.01) and ameliorative effects of MEL on AQP7 (p<0.01, Figure 4D). We focused on the change of LV AQP channels and we did not investigate the metabolic condition of the myocardium. Therefore, we could not explain the inducible factor of changing in AQP7 channel in ISO induced MI.

Study Limitations

In our study, we applied a chemical method for inducing MI.

CONCLUSION

In this study, we demonstrated that MEL administration had protective effects against the increase in AQP1, AQP3, AQP4 and AQP7 channels. MEL also decreased myocardial edema in ISO induced MI model. This is the first study investigated the protective effects of MEL on the level of myocardial edema and the changes of AQP1, AQP3, AQP4 and AQP7 channels after MI. Besides the other cardioprotective effects, according to our results, MEL was also suggested as a potential adjuvant agent, which decreases cardiac edema and regulates the cardiac AQP channels.

Ethics

Ethics Committee Approval: All animal experimental procedures and protocols were approved by Çanakkale Onsekiz Mart University Animal Experiments Local Ethics Committee (decision number 2019/07-04, date: 25.09.2019).

Informed Consent: Animal experiment.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: N.K.T., M.U., Design: N.K.T., M.U., Data Collection or Processing: N.K.T., B,B., M.U., Analysis or Interpretation: N.K.T., B,B., M.U., Literature Search: N.K.T., B,B., M.U., Writing: N.K.T., B,B., M.U.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This work was funded by the Scientific Research Coordination Unit (BAPD) of Çanakkale Onsekiz Mart University (project number: TYL-2019-3081).