Cramming the causative mechanism of glycogen synthase kinase-3β mediated by ischemic preconditioning against ovariectomy challenged rat heart

Vishal Kumar Vishwakarma1, Tarique Mahmood Ansari*2, Prabhat Kumar Upadhyay3, Ritesh Kumar Srivastav4, Farogh Ahsan2, Arshiya Shamim2 1Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India 2Faculty of Pharmacy, Integral University, Lucknow, Uttar Pradesh-226021, India 3Institute of Pharmaceutical Research, GLA University, Mathura, Uttar Pradesh-281406, India 4Faculty of Pharmacy, Kamla Nehru Institute of Management and Technology, Sultanpur-228119, India


INTRODUCTION
The clinical effects of cardiovascular studies in postmenopausal women are disappointing and inconsistent yet. After menopause, the lower level of estrogens in females increases the risk of cardiovascular diseases. It creates new studies on the actions of 17β-estradiol on the heart. It has also been reported that estrogens have a protective role in animal models associated with cardiac complications like arrhythmia, atherosclerosis (Booth et al., 2008). Estrogen also reduces the episode of ischemiareperfusion (IR) injury, myocardial infarct size, and also neutrophil in iltration in the cardiac muscles (Posa et al., 2017). The normal functioning of the myocardium is notably restored by reperfusion of ischemic heart (Topol et al., 1992). While sudden reperfusion of ischemic heart produced again injury of cardiac tissue termed as IR injury (Collard and Gelman, 2001). Ischemic preconditioning (IPC) is a useful protective phenomenon of heart against the short and long event of myocardial ischemia following reperfusion which also produces the protection against prolonged ischemia (Murry et al., 1986). It is reported, that IPC exhibited a protective effect against IR injury by reducing myocar-dial infarct size, oxidative stress, accumulation of neutrophils (Sharma et al., 2010). However, cardioprotection produced by IPC gets weakened in speci ic pathological conditions including hypertension (Snoeckx et al., 1986(Snoeckx et al., , 1993, aging (Abete et al., 1996), heart failure (Ferdinandy et al., 1998(Ferdinandy et al., , 2007, diabetes mellitus (Ajmani et al., 2011;Yadav et al., 2010b) and hyperlipidemia (Yadav et al., 2010a). The estrogen de iciency is a big risk factor ischemic cardiovascular disease (Booth et al., 2008). Hence the accurate nature of the mechanism of attenuation of protection phenomenon in estrogen de iciency is still controversial.
The mPTP is the pore of an inner mitochondrial membrane that open on reperfusion damage mainly through Ca++ overload, oxidative stress, increased matrix pH and decrease ATP level. This opening causes decreases the mitochondrial ATP level and uncoupling of oxidative phosphorylation. Cyclosporine-A exhibit protection against I/R damage by inhibiting the mPTP opening (Yadav et al., 2010b). It has been proof that both erythropoietin and IPC show protection mediated through inactivation of GSK-3β and its phosphorylation (Nishihara et al., 2007).
Hence, the present study is planned to explore the role of 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDTZ-8) as GSK-3β inhibitors in the cardioprotection. The cardioprotection mediated through IPC would be investigated in the OVXchallenged rat heart. Finally, infract size, LDH, CKMB and coronary low would be estimated in the various experiments to ind signaling pathway (involved in cardioprotection) by low estrogen.

Animals
In the experiment, Wistar rats (female) weight about 160-200 gm were used in this study. These experimental rats were kept in the animal cages following a cycle of light and dark (12 hours each). This research work was approved by the authorities of Institutional Animal Ethics Committee using laboratory animals.

Chemicals and drugs
TDTZ-8 and atractyloside were procured from Helix Bioscience, India. These drugs were put into the Krebs-Henseleit (KH) buffer solution for the perfusion of rat heart. All the reagents of analytical grade were freshly prepared for the experiment before use.

Induction in ovariectomy rats
Bilateral ovariectomy produced an estrogen level expressed as pg/ml, as described previously (Vishwakarma et al., 2018).

Isolated rat heart preparation
The Heart was rapidly excised from heparinized rats and directly suspended to Langendorff's apparatus before starting the experiment. This isolated heart was now covered with the double-walled jacket by maintaining temperature to 37 • C using hot water circulation. The heart was retrospectively perfused at a coronary low rate of 7-9 mL/min by maintaining the pressure of 80 mmHg using KH buffer solution which comprised of composition (MgSO 4 .7H 2 O 1.2 mM; KCl 4.7 mM; NaCl 118 mM; glucose-11mM; KH 2 PO4 1.2 mM, NaHCO 3 25 mM; CaCl 2 2.5 mM to get pH 7.4). The temperature was maintained to 37 • C, and also passed the bubble of 5% CO 2 and 95% O2 (Hosseini et al., 2020).

Experimental protocol and induction of IPC
The study was performed on nine groups of female Wistar rats, and each group contained six rats (n=6). The detailed set of groups for an experiment is shown in Figure 1 and described here, 1. Sham Control, where n = 6: Isolated heart was subjected to 10 min of stabilization and then perfused with KH buffer for 190 min continuously. At this stage, there is no global ischemia.
2. IR Control; where n = 6: After 10 min of stabilization, the isolated heart was exposed to global ischemia for 30 min followed by reperfusion with KH buffer for 120 min continuously.
3. IPC Control; where n=6: The heart was kept for 4 cycles of IPC after 10 min of stabilization. Each cycle of IPC consists of 5 min global ischemia following reperfusion of 5 min with KH buffer solution, which was further continued to global ischemia of 30 min and 120 min reperfusion.
4. IPC in OVX rat; where n = 6: Isolated OVX rat heart was kept for 4 cycles of IPC as reported earlier in group-3. 5. IPC in pretreated with TDTZ-8 (1mg/kg), surgery operated OVX rats; n = 6: Preparation of isolated pretreated OVX rat heart with TDTZ-8 (1 mg/kg dose was given, in abdominal cavity 30 min prior) OVX rat was kept for 4 cycles of IPC and rest protocol as described in group-3.

Evaluation of myocardial infarct area
Isolated hearts were stored at −80 • C for 20 to 30 min. The slices were taken after cutting the frozen heart from apex to base. Each slide was measured by a thickness of 2 to 3 mm. The triphenyltetrazolium chloride solution (TTC; Sigma-Aldrich, USA) was used to stain prepared slices. The brick red color was stained for living myocardial tissues, while, the infarct area remained unstained. The per cent infarct area was measured using Image Jsoftware in relation to a total area of the heart (NIH, Bethesda, MD, USA) (Pachauri et al., 2017).

Measurement of cellular injury
In the coronary ef luent from heart preparation, the LDH and CKMB levels were determined for assessing the extent of cardiac injury in experimental. After an experiments, these levels have been estimated spectrophotometrically in the perfusate using commercial detection kits (Coral Clinical Systems Pvt. Ltd., India) (Pachauri et al., 2017). Generally, 10% buffered neutral formalin solution was used for tissue ixing in the histological experiment. After ixing, these tissues were placed in paraf in-wax, and then cut transverse midventricular sections (about 5 µm thickness) using a microtome. These sections were stained using haematoxylin and eosin (Srivastav et al., 2013).

Statistically analysis
All data values were expressed as mean±SD. The data of groups were statistically measured using one way ANOVA followed by Tukey's multiple comparisons test. The statistical signi icant value was considered as a p-value of less than 0.05.

Serum β-estradiol levels
The level of β-estradiol was signi icantly decreased in the OVX group. The level of β-estradiol did not show to zero because the ovary and adrenal gland secrete little amount female sex hormone ( Figure 2).

Effect of TDTZ-8 on the coronary low
Effect of TDTZ-8 (3 µM) on the coronary low in OVX rat heart mediating IPC has been observed which depicted in Figure 3. There was no signi icant difference in coronary low at a basal time among groups in all sets of experiments. IPC mediated decrease in the coronary low noted in OVX challenged rat heart after 1 min to ischemia exposure while TDTZ-8 produced IPC-mediated increase in the coronary low. Further, atractyloside was given with TDTZ-8, attenuates IPC-mediated and enhanced coronary low in OVX challenged heart. The effect carried on up to the end of the experiment. Figure 4 shows the effect of TDTZ-8 (3 µM) on IPCmediated changes, including LDH activity in OVXchallenged rats. In this study, IPC attenuated the LDH level of coronary ef luent in OVX-challenged rat heart noted at 1 min, while TDTZ-8 enhance IPC-mediated reduction in LDH level. Additionally, Atractyloside, along with TDTZ-8, diminish IPC mediated and decreased LDH level in coronary ef luent in OVX challenged rat heart. The effect carried on up to the end of the experiment.

Effect of TDTZ-8 on CKMB level
Effects of TDTZ-8 (3 µM) on IPC-mediated alterations in the CKMB activity of the coronary ef luent of OVX-challenged rats of the experimental design in Figure 5. In this study, IPC attenuated CKMB level of coronary ef luent in OVX-challenged rat heart noted at 1 min, while TDTZ-8 enhance IPC-mediated reduction in CKMB level. Additionally, Atractyloside, along with TDTZ-8, diminish IPC mediated reduction in CKMB level in the coronary ef luent of OVX challenged rat heart. These effects continued up to 120 min in the experiment.

Effect of TDTZ-8 on infarct size
Effect of TDTZ-8 (3 µM) on IPC-induced alterations in infarct size in OVX rats of the experimental design in Figure 6. It signi icantly reduces OVX rat heart infarct size in the all set of experiment. TDTZ-8 further increase the IPC mediated reduction in the infarct size in OVX rat heart. Additionally, Atractyloside, along with TDTZ-8 diminish IPC mediated decrease in infarct size in OVX heart.

Effect of TDTZ-8 on histological changes
In the Figure 7 a: Sham control rat heart shows the normal cytoarchitecture of the myocardium; Figure 7 b: IR control-treated heart shows the necrotic changes in cardiac tissue; Figure 7 c: IPC treated heart exhibit regenerative changes in cardiac tissue; Figure 7 d: IPC+OVX treated rat heart shows less regenerative changes in cardiac tissue as compared to IPC treated heart; Figure 7 e, IPC+OVX+TDTZ-8 treated heart shows more regenerative changes in cardiac tissue as compare to IPC+OVX treated rat heart; Figure 7 f, IPC+OVX+TDTZ-8+Atr treated rat heart shows less comparative same as group IPC+OVX+TDTZ-8.

DISCUSSION
In this study, four-episode of ischemia for 5 min following reperfusion of 5 min with KH buffer which was further continued to global ischemia of 30 min and reperfusion for 120 min in isolated Langendorff's perfusion with OVX heart. When it was compared with IR control group, it did not produce any signi icant effect. However, pretreated TDTZ-8 was given, produced signi icant cardioprotection against IR and IPC+OVX group. In addition, atractyloside, an mPTP opener, breaks the potentiating action of TDTZ-8 on IPC-mediated cardioprotection in OVXchallenged rats, in this research protocol. Atractyloside diminished IPC mediated cardioprotection in OVX challenged rat heart perfused with pretreated TDTZ-8 preconditioning as well as normal rat heart.
The cardiac injury was examined in terms of increased CK-MB, LDH, infarct size. The treatment with TDTZ-8 decreases the level of CK-MB, LDH enzyme in the coronary ef luent and also myocardial infarct size in OVX-challenged rats. The heart was perfused with TDTZ-8 along with atractyloside restricted the decrease in the level of CK-MB, LDH enzyme and myocardial infarct size in the OVXchallenged rats.
The regenerative changes in myocardial tissue were attenuated by IPC in OVX-challenged rat heart. When pretreatment with TDTZ-8, it increases the effect of IPC against OVX-induced increase in histological change. Moreover, the cardioprotective effect gets attenuated in OVX rat heart when TDTZ-8 along with atractyloside. These observations support the role of GSK-3β signalling protein, which potentiates the cardioprotective effect of IPC in OVXchallenged rats. This con irms the argument of scienti ic data (Oldenburg, 2002) in which, GSK-3β signalling pathways inhibit mPTP opening during reperfusion.
The experimental data represented that GSK-3β inhibitors TDTZ-8 giving IPC cycle produced the cardioprotective effects on myocardium against OVXinduced cardiac injury. So, these results may have a better opportunity to treat postmenopausal females, undergoing bypass surgery. In open-heart surgery, the controlled reperfusion of pretreatment of TDTZ-8 could be a potential adjuvant for the cardioprotection.

CONCLUSIONS
The current study suggested that estrogen de iciency may cause cardiovascular risk by activating GSK-3β signalling pathway. The role of TDTZ-8 inactivates GSK-3β signaling protein through impairment of mPTP opening, which potentiates IPC mediated cardioprotective action in OVX-challenged rat heart. These signaling pathways would be used in a variety of experimental conditions associating with estrogen de iciency. Furthermore, such protective mechanisms and signaling pathways would be useful in different clinical settings in open-heart surgery and undergoing cardiopulmonary bypass surgery.

Ethical Statement
This study was approved by the Institute Ethical Committee for Experimental Use of Animals (Permit Number: (PHAR/IAEC/18/01). The experiments were carried out in accordance with the principals and procedures of the Institute Ethical Committee for Experimental Use of Animals.