Ginsenoside-Mc1 reduces ischemia/reperfusion-induced cardiac arrhythmias through activating JAK2/STAT3 pathway and attenuating oxidative/endoplasmic reticulum stress in hyperlipidemic rats

Introduction

Despite recent advances in medicine and surgical interventions, ischemic heart disease is still the leading cause of death in the world [1]. Most patients suffering from myocardial ischemia-reperfusion (IR) injuries have varying degrees of other comorbidities such as obesity, hyperlipidemia, and diabetes. Hyperlipidemia and hypercholesterolemia are one of the main risk factors for myocardial diseases associated with IR injury and can exacerbate IR-induced heart damages [2]. Reperfusion-induced myocardial arrhythmias are of determining factors in the survival or death of patients with myocardial infarction (MI) whose severity and incidence increase in the presence of hyperlipidemia [2], [3], [4]. In animal models of hyperlipidemia, endogenous cardiac defense mechanisms are attenuated and lose their normal function, leading to the development of oxidative stress, apoptosis, and cell death [5]. Based on plenty of evidence, the Janus kinase-2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathway has also an important contribution in cardioprotection against myocardial IR injury [6, 7]. JAK2/STAT3 has antioxidative features and belongs to the survivor activating factor enhancement (SAFE) signaling pathway that its activity is considerably reduced in the setting of hyperlipidemia [5, 8]. These findings highlight the significance of activation of this pathway in improving the clinical outcomes of MI patients.

The detrimental effects of hyperlipidemia in cardioprotection can also be due to an overproduction of reactive oxygen species (ROS) such as superoxide free radicals and hydrogen peroxide [8, 9]. The exacerbation of oxidative stress caused by these reactions triggers apoptosis and negatively affects cell membranes, proteins, and cardiomyocytes’ organelles. The endoplasmic reticulum (ER) plays an important role in controlling numerous intracellular physiological functions (including calcium homeostasis, lipid biosynthesis, protein folding) [10]. Pathophysiological conditions of IR injury and hyperlipidemia disrupt ER homeostasis and activate the unfolded protein response (UPR) and ER stress, leading to the occurrence of arrhythmias [10, 11]. Prolonged ER stress in hyperlipidemic conditions induces oxidative stress, inflammation, and tissue apoptosis by activating ER transmembrane sensors and UPR initiation [12]. PERK and eIF2α proteins are among these ER stress sensors. With the onset of oxidative stress, the PERK signaling pathway inhibits protein synthesis by reducing the protein load in the ER lumen and induces phosphorylation of eIF2α [10]. Subsequently, these processes lead to cell death by activating the transcription factor ATE4 and CHOP. A few documents reported that JAK2/STAT3 signaling may prevent ER stress during myocardial IR injury [7, 13], leading to the reduction of cardiac arrhythmias incidence. However, the exact role of ER stress is not yet well understood not only in the pathophysiology of IR-induced cardiac arrhythmias but also in the effect of hyperlipidemia on the severity of IR injury.

Ginsenoside-compound Mc1 (GMC) is a de-glycosylated ginsenoside extracted from ginseng and has important effects in reducing oxidative stress, inflammatory and apoptotic reactions [14, 15]. A study has shown that this compound decreases oxidative stress in H9c2 cells and exerts promising effects in reducing the myocardial infarct size in obese mice [14]. It has recently been reported that ginsenoside compounds can lower hyperlipidemia in mice model [15]. GMC also improved liver steatosis and insulin resistance by attenuating ER stress [16]. Considering these beneficial potentials of GMC in reducing oxidative/ER stress, it seems to have a significant impact on reducing arrhythmias and blunting arrhythmogenic mechanisms of reperfusion injury in hyperlipidemic conditions. It would also be interesting to explore whether GMC cardioprotective action is associated with JAK2/STAT3 signaling. Therefore, in this study, we aimed to test this hypothesis and evaluate the effect of pretreatment of hyperlipidemic rats with GMC on the incidence and severity of ventricular arrhythmias as well as the expression of proteins involved in ER stress, oxidative stress, and histological damage in an in-vivo model of myocardial IR injury and further explore the role of JAK2/STAT3 activity in these effects.

Materials and methods

Recording and interpretation of myocardial arrhythmias

Electrocardiographs were recorded by connecting three electrodes to both right and left hands and right foot on the axis lead II during the IR experiments. Ventricular arrhythmias including premature ventricular complexes (PVC), ventricular tachycardia (VT), and ventricular fibrillation (VF) were evaluated during the reperfusion phase based on the Lambeth convention (Figure 1). The count and duration of PVC, VT, and VF, the incidence of VT and VF as well as the severity of arrhythmias were calculated. The following scoring system was employed to calculate the severity of arrhythmias: 0: no arrhythmia; 1: only PVC; 2: PVC plus 1–3 episode of VT; 3: PVC plus more episodes of VT; 4. PVC plus VT and reversible VF; 5 PVC plus VT and irreversible VF.

Figure 1:

Types of myocardial arrhythmias according to the Lambeth convention. PVC, premature ventricular complexes; VT, ventricular tachycardia; and VF, ventricular fibrillation. Single beats, bigeminy and salvos were considered as PVCs.

Results

GMC reduced IR-induced ventricular arrhythmias, whereas these effects were reversed by AG490 co-treatment

Induction of MI by IR injury in hyperlipidemic rats led to a significant increase in the number (Figure 2A) and duration (Figure 2B) of PVC, VT, and VF arrhythmias in reperfusion phase as compared with untreated hyperlipidemic rats (HL group) (p<0.05 to p<0.001). GMC administration for one month significantly reduced the number of PVC and VF (p<0.05) and the duration of PVC, VF (p<0.001), and VT (p<0.05) when compared to the HL + MI group. Inhibition of JAK2 by AG490, although did not have a significant effect on reperfusion-induced myocardial arrhythmias alone, inhibited the protective effect of GMC on the number and duration of all three types of arrhythmias compared with the HL + MI + GMC group (Figure 2).

Figure 2:

The effects of GMC administration on the number (A) and duration (B) of reperfusion-induced myocardial arrhythmias and their reversal by AG490 in hyperlipidemic (HL) rats.

Mean ± SD (n=7 in each group). MI, myocardial ischemia reperfusion injury; GMC, Ginsenoside-Mc1; AG490, JAK2 inhibitor; PVC, premature ventricular complexes; VT, ventricular tachycardia; and VF, ventricular fibrillation. *p<0.05, **p<0.01, ***p<0.001 vs. HL group; ⁺p<0.05, ⁺⁺⁺p<0.001 vs. HL + MI group; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. HL + MI + GMC group.

The incidence of VT and VF arrhythmias following IR induction in hyperlipidemic rats increased significantly (Figure 3A). However, pretreatment of hyperlipidemic rats with GMC was able to significantly reduce the incidence of VF (p<0.001), although its effect on reducing the incidence of VT was not statistically significant. Also, co-administration of AG490 abolished the effectiveness of GMC in reducing the incidence of these reperfusion-induced myocardial arrhythmias, especially VF (p<0.001), in comparison to the HL + MI + GM group (Figure 3A).

Figure 3:

The effects of GMC administration on the incidence of VT and VF (A) and severity (B) of reperfusion-induced myocardial arrhythmias and their reversal by AG490 in hyperlipidemic (HL) rats.

Mean ± SD (n=7 in each group). MI, myocardial ischemia reperfusion injury; GMC, Ginsenoside-Mc1; AG490, JAK2 inhibitor; VT, ventricular tachycardia; and VF, ventricular fibrillation. **p<0.01, ***p<0.001 vs. HL group; ⁺⁺⁺p<0.001 vs. HL + MI group; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs. HL + MI + GMC group.

Finally, the severity of arrhythmias in different groups is seen in Figure 3B. GMC administration significantly reduced the reperfusion-induced increased severity of arrhythmias in hyperlipidemic rats (p<0.01), and this effect was partially abolished by inhibition of JAK2 by AG490 co-administration (p<0.05) (Figure 3B).

GMC reduced myocardial IR-induced histological changes, whereas these effects were reversed by AG490 co-treatment

The results of the histological examination showed that although lesions such as intercellular edema and inflammatory cell infiltration were observed in the HL group, induction of IR injury exacerbated these lesions and caused necrotic damages in cardiomyocytes (Figure 4). On the other hand, GMC administration significantly diminished the severity of these lesions, and co-treatment with AG490 negatively affected GMC’s protective effect on histological changes.

Figure 4:

The effects of GMC administration on myocardial histological changes induced by ischemia-reperfusion injury and their reversal by AG490 in hyperlipidemic (HL) rats.

40× magnification. MI, myocardial ischemia reperfusion injury; GMC, Ginsenoside-Mc1; AG490, JAK2 inhibitor.

GMC reduced IR-induced myocardial oxidative stress, whereas these effects were reversed by AG490 co-treatment

To assess the status of oxidative stress, intracellular levels of MDA as an indicator of lipid peroxidation and SOD as an endogenous antioxidant enzyme were measured. The results showed that following the induction of IR injury in HL rats, the amount of MDA increased significantly (p<0.001), and the level of SOD decreased (p<0.05) (Figure 5A and B). Despite this, GMC administration significantly prevented the effect of IR on changes in these two factors; it decreased MDA (p<0.01) and increased SOD (p<0.001) when compared to the HL + MI group. However, inhibition of the JAK2 pathway significantly neutralized GMC effects on oxidative stress markers following IR injury in the heart of HL rats (p<0.01) (Figure 5).

Figure 5:

The effects of GMC administration on the levels of MDA (A) and SOD (B) following myocardial ischemia-reperfusion injury and their reversal by AG490 in hyperlipidemic (HL) rats.

Mean ± SD (n=7 in each group). MI, myocardial ischemia reperfusion injury; GMC, Ginsenoside-Mc1; AG490, JAK2 inhibitor. *p<0.05, ***p<0.001 vs. HL group; ⁺⁺p<0.01, ⁺⁺⁺p<0.001 vs. HL + MI group; ^##p<0.01 vs. HL + MI + GMC group.

GMC reduced IR-induced myocardial endoplasmic reticulum stress, whereas these effects were reversed by AG490 co-treatment

Following IR injury, phosphorylation levels of PERK and eIF-2α and protein expression of CHOP increased significantly compared to the HL group (Figure 6A–C, respectively). This finding indicates an increase in ER stress following myocardial IR injury in hyperlipidemic rats. Pretreatment of HL rats with GMC prevented the upregulation of the expression of these ER stress proteins following IR insult (p<0.01, and p<0.001). Also, administration of AG490 as the inhibitor of the JAK2/STAT3 pathway was able to increase again the level of phosphorylation and expression of these proteins in comparison with the HL + MI + GM group (p<0.05) and neutralized the effect of GMC on ER stress in HL rats (Figure 5).

Figure 6:

The effects of GMC administration on the expression of p-PERK (A), p-eIF2α (B), CHOP (C), and related representative bands (D) following myocardial ischemia-reperfusion injury and their reversal by AG490 in hyperlipidemic (HL) rats.

GAPDH was considered as an internal control of protein expression. Mean ± SD (n=7 in each group). MI, myocardial ischemia reperfusion injury; GMC, Ginsenoside-Mc1; AG490, JAK2 inhibitor. *p<0.05, **p<0.01, ***p<0.001 vs. HL group; ⁺⁺p<0.01, ⁺⁺⁺p<0.001 vs. HL + MI group; ^#p<0.05 vs. HL + MI + GMC group.

GMC upregulated IR-induced reduction in myocardial JAK2/STAT3 expression, whereas these effects were reversed by AG490 co-treatment

Phosphorylation of both JAK2 and STAT3 proteins was significantly reduced following induction of IR injury in hyperlipidemic rats as compared to the untreated HL group (Figure 7A and B). Treatment of HL rats with GMC resulted in increased phosphorylation of JAK2/STAT2 proteins in the IR setting (p<0.001). Finally, although AG490 alone did not have a significant effect on JAK2 and STAT3 expression compared to the HL + MI group, its concomitant usage partially abolished the increasing effect of GMC on JAK2/STAT3 phosphorylation (Figure 7).

Figure 7:

The effects of GMC administration on the expression of p-JAK (A), p-STAT3 (B), and related representative bands (C) following myocardial ischemia-reperfusion injury and their reversal by AG490 in hyperlipidemic (HL) rats.

GAPDH was considered as an internal control of protein expression. Mean ± SD (n=7 in each group). MI: myocardial ischemia reperfusion injury; GMC, Ginsenoside-Mc1; AG490, JAK2 inhibitor. *p<0.05, **p<0.01 vs. HL group; ⁺⁺⁺p<0.001 vs. HL + MI group; ^##p<0.01, ^###p<0.001 vs. HL + MI + GMC group.

Discussion

In the present study, pretreatment of hyperlipidemic rats with GMC decreased the severity, duration, and number of reperfusion-induced myocardial arrhythmias following IR injury, as well as decreased myocardial oxidative stress indices and endoplasmic reticulum stress protein expression and increased the activity of the JAK2/STAT3 pathway. Besides, the results showed that by inhibiting JAK2 via concomitant administration of AG490, the cardioprotective effects of GMC in HL rats were abolished. Therefore, GMC demonstrated anti-arrhythmic properties and reduced endoplasmic reticulum stress and cellular oxidative stress by activating the JAK2/STAT3 pathway in the setting of myocardial IR injury in hyperlipidemic rats.

Fatal reperfusion arrhythmias are the hallmark of myocardial IR injury [19, 20]. Sudden reperfusion to the ischemic tissue triggers a cascade of events such as oxidative stress, inflammatory cytokine production, imbalance in ionic homeostasis, and electrical activity which all finally may increase the occurrence of ventricular tachycardia and fibrillation leading to cardiac dysfunction and even death [3]. At the onset of reperfusion, marked inhomogeneity is seen in the electrical responses of the heart within the ischemic zone and areas at risk [21, 22]. Besides, hyperlipidemia also disturbs cardiac electrical activity directly, which may be related to myocardial ultrastructure changes, gradual accumulation of cardiac lipids, and consequent ER stress and oxidative stress [5]. This condition makes the heart vulnerable to IR injuries, leading to the induction of severe arrhythmias. In this line, our results showed that IR injury in hyperlipidemic rats considerably increased the number, duration, incidence, and scoring of ventricular arrhythmias, which are similar to the previous reports [9]. Arrhythmias usually occur in the early minutes of reperfusion, due to a variety of reasons, including the production of oxidative stress and the disruption of ionic and electrolyte balance in cardiomyocytes and dynamic organelles such as the mitochondria and ER. Here, the markers of ER and oxidative stress were greater and the activity of the JAK2/STAT3 pathway was lower in hyperlipidemic rats subjected to IR injury.

Accumulating evidence indicates that excessive ER stress may contribute to the occurrence of reperfusion-induced myocardial arrhythmias [10, 11]. PERK/eIF2α/ATF4/CHOP signaling pathway serves an essential role in ER stress and strongly causes lipid peroxidation, oxidative stress, and apoptotic cell death during IR injury, thereby reducing the ischemic tolerance of diabetic and hyperlipidemic hearts [12, 23]. When activated, PERK promotes phosphorylation of eIF2α and subsequent translation of ATF4 and activation of CHOP. Overexpression of CHOP, in turn, activates downstream targets of ER stress, resulting in oxidative stress, apoptosis, and mitochondrial dysfunction [18, 24]. Activation of unfolded protein response following ER stress may downregulate selective types of voltage gated sodium or potassium channels. This events increase the duration of ventricular action potential and reduce the rate of changes of action potential, contributing to electrical remodeling and ventricular arrhythmias [11, 25]. Pretreatment of hyperlipidemic rats with GMC (as a new de-glycosylated ginsenoside) significantly reduced the phosphorylation and activation of PERK/eIF2α/CHOP pathway in IR hearts. GMC also reversed IR-induced changes in the levels of lipid peroxidation marker MDA and exogenous antioxidative enzyme SOD. These effects of GMC were associated with the significant reduction in the counts, timing, incidence, and severity of ventricular arrhythmias including PVC, VT, and VF. In accordance with our findings, it has been recently reported that GMC attenuated oxidative stress and apoptosis in H9c2 cells cardiomyocytes through an AMPK dependent mechanism [14]. In addition, GMC reduced apoptosis and steatosis in the liver of high-fat diet-fed obese mice and prevented insulin resistance mostly by amelioration of the ER stress [16]. These findings demonstrated that the attenuation of ER stress and oxidative stress following IR injury by GMC pretreatment in hyperlipidemic rats are more likely involved in its antiarrhythmogenic potential.

Another feature of the cardioprotective effects of GMC in this study was the over-activation of the JAK2/STAT3 pathway along with suppression of IR-induced ER stress following GMC administration. More importantly, blockade of this pathway by AG490, as a specific inhibitor of JAK2, abolished the beneficial impacts of GMC on myocardial arrhythmias as well as oxidative and ER stress. It also impeded the positive impacts of GMC on IR-induced histopathological alterations. Therefore, these cardioprotective properties of GMC in hyperlipidemic rats are JAK2/STAT3 pathway-dependent. JAK2/STAT3 signaling is an important element of the SAFE pro-survival kinase signaling pathway in protecting the hearts against reperfusion injury and serves as a critical axis to mediate cardioprotective influences of ischemic and pharmacological conditionings [6, 8]. In this scenario, JAK2/STAT3 pathway offers antioxidative and antiapoptotic effects to the hearts subjected to IR injury. Also, as a multifaceted transcription factor, STAT3 enhances the expression of genes that are involved in antioxidative, anti-inflammatory, proangiogenic, and antifibrotic processes [7]. Interestingly, more evidence indicates that activation of this pathway may attenuate ER stress both in in-vivo and in-vitro settings, and inhibition of the JAK2/STAT3 signaling pathway may reduce the clinical cardiovascular outcomes of atherosclerosis and hyperlipidemia [13, 26, 27]. Increased expression and activity of JAK2/STAT3 pathway by GMC led to antiarrhythmic effects in the early reperfusion by affecting the abovementioned arrhythmogenic elements. Accordingly, in the present study, AG490 administration neutralized the effect of GMC on the phosphorylation of PERK/eIF2α and expression of CHOP. Also, GMC-induced JAK2 activation may improve cardiac function through correcting the histopathological changes following reperfusion phase. These findings postulate the immediate and gradual protective effects of GMC in IR injury which are JAK2/STAT3-dependent. It should be noted that although the use of AG490 alone did not have the significant impacts on the histopathological changes and biochemical parameters, it was able to considerably eliminate the effects of GMC to some extent. However, this inhibitory effect was not complete and the results suggest that there may be other parallel ways and mediators in the GMC’s efficacy pathway that need further study. Also, to fully understand the mechanisms of GMC effects here, it is necessary to show how GMC upregulates JAK2/STAT3 pathway activity.

In conclusion, the findings of this study revealed that administration of GMC to the hyperlipidemic rats exerted substantial antiarrhythmogenic and histological benefits following myocardial IR injury. JAK2/STAT3 pathway-dependent attenuation of ER stress and oxidative stress mediated the cardioprotective effects of GMC. These findings may provide new mechanistic insight into the GMC effects in the presence of comorbidities and propose the possible implication of GMC in the management of reperfusion-induced arrhythmias.