Empagliflozin attenuates ischemia and reperfusion injury through LKB1/AMPK signaling pathway

https://doi.org/10.1016/j.mce.2019.110642Get rights and content

Highlights

  • AMPK mediates the cardioprotection of Empagliflozin against ischemic insult.

  • Empagliflozin treatment augments cardiomyocytes contractile functions during ischemic insults.

  • The inflammatory response modulation by Empagliflozin is a critical factor.

Abstract

The beneficial effects of empagliflozin (EMPA) on cardiac functions during ischemia and reperfusion were characterized. The contractile functions of isolated cardiomyocytes from adult C57BL/6J mice were determined with IonOptix SoftEdgeMyoCam system. The mitochondrial superoxide production was measured by MitoSOX fluorescent probe. The ex vivo isolated heart perfusion system was used to determine the pharmacological effects of EMPA on heart's contractile functions under both physiological and pathological conditions. The in vivo regional myocardial ischemia and reperfusion by ligation of left artery coronary artery descending (LAD) was used to measure the myocardial infarction caused by ischemia and reperfusion with or without EMPA treatment. The results demonstrated that EMPA treatment significantly improves cardiomyocyte contractility under hypoxia conditions and augments the post-ischemic recovery in the ex vivo heart perfusion system. Furthermore, the in vivo myocardial infarction measurement shows that EMPA treatment significantly reduce myocardial infarct size caused by ischemia and reperfusion. The biochemical analysis demonstrated that EMPA can trigger cardiac AMPK signaling pathway and attenuate mitochondrial superoxide production under hypoxia and reoxygenation conditions. In conclusion, EMPA can trigger AMPK signaling pathways and modulate myocardial contractility and reduce myocardial infarct size caused by ischemia and reperfusion independent of hypoglycemic effect. The results for the first time demonstrate that the activation of AMPK by EMPA could one reason about EMPA's beneficial effects on heart disease.

Introduction

Diabetes is considered as a significant risk factor for cardiovascular disease (CVD), the leading cause of global mortality (Mazzone, 2010; Rawshani et al., 2018). Aims of diabetes treatment are multiple risk factors control, such as hyperglycemia, hyperlipidemia and hypertension, etc. At present, many kinds of hypoglycemic drugs are available for clinical use. As a request by the US Food and Drug Administration (FDA), clinical research evidence of cardiovascular (CV) safety must be submitted by the pharmaceutical industry before the approval of a novel antidiabetic agent.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are new class of antidiabetic agents which act by selectively inhibiting SGLT2 of the renal proximal tubule, with a consequent of increase in urinary excretion of glucose, reducing blood glucose concentration independent of insulin. Canagliflozin, dapagliflozin, empagliflozin (EMPA) and ertugliflozin of this family have been approved by FDA. Encouraging evidence of CV protection effects from several large clinical studies of SGLT2 inhibitors have been published (Zinman et al., 2015; Kosiborod et al., 2017; Neal et al., 2017, Wiviott et al., 2019). EMPA REG OUTCOME (Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes)is the first study for CV protection, which suggested that EMPA exerts a 38% risk reduction in death from CV causes, 32% risk reduction death from any cause, and 35% reduction on risk of hospitalization for HF (Zinman et al., 2015; Kosiborod et al., 2017; Neal et al., 2017,Wiviott et al., 2019). It is noteworthy that this effect of EMPA is independent of its hypoglycemic effect. A new indication has been approved by FDA for EMPA to reduce the risk of CVD death in adult patients with T2DM and CVD in 2016.

At present, many studies are trying to explore the mechanism of CV protection of SGLT2 inhibitors independent of hypoglycemia effect. Most of the research are focusing on energy metabolism, inflammation, oxidative stress, myocardial fibrosis and electrolyte homeostasis (Bertero et al., 2018). Multiple studies had been reported that EMPA could improve myocardial fibrosis in obese and diabetic mice (Lee et al., 2017; Ye et al., 2017) and also play the role of anti-oxidative stress and anti-apoptosis (Lin et al., 2014; Zhou and Wu, 2017). Meanwhile, EMPA was found to decrease the infarct area under ischemia/reperfusion (I/R) stress (Andreadou et al., 2017) and improve diastolic function of the left ventricle (LV) in diabetic mice (Hammoudi et al., 2017).

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is an important serine and threonine protein kinase, which plays an important role in cell energy balance (Hardie, 2004). Increasing the ratio between intracellular adenosine monophosphate (AMP) and adenosine triphosphate (ATP) will activate AMPK by phosphorylation (Hardie and Carling, 1997). In addition, liver kinase B1 (LKB1), calmodulin-dependent protein kinase kinase β (CaMKKβ), and AMPK kinase (AMPKK) are all upstream activators. AMPK also plays an important role in reducing oxidative stress, regulating autophagy, and anti-apoptosis of cardiomyocytes (Bertrand et al., 2006; He et al., 2013). It has been reported that EMPA can protect the heart by activating AMPK in cardiac microvascular endothelial cells (CMEC) of diabetic mice (Zhou et al., 2018). Canagliflozin can regulate energy metabolism via AMPK activation by inhibiting Complex I in the respiratory chain in HEK-293 cells and hepatocytes (Hawley et al., 2016). The basic research on SGLT2 inhibitors was mostly focused on animal models of diabetes and/or obesity. To date, it is unclear if SGLT2 inhibitors could offer protection against hypoxia/reoxygenation (H/R) or I/R injury in non-diabetic and non-obese model through AMPK signaling pathway. This study aims to explore whether short-term treatment of EMPA could protect against H/R or I/R injury in healthy young heart.

In this study, we selected healthy young male mice as research objects, explored whether short-term administration of EMPA displays protective effect on cardiomyocytes and cardiac function, and the influence of myocardial infarction (MI) area in vitro and in vivo under H/R and I/R stress conditions. At the same time, we tried to further explore whether AMPK related signaling pathway was involved in this mechanism.

Section snippets

Materials and methods

Young (12 weeks) male C57BL/6 J mice were obtained from Jackson Laboratory. EMPA and compound C were purchased from Sigma-Aldrich and ApexBio respectively. All animal activity protocols were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.

EMPA activates LKB1/AMPK signaling pathway in isolated mice cardiomyocytes under normoxia and H/R condition

We got the samples of 11 subgroups (including 0 min time point) with vehicle or EMPA in time gradient experiment under normoxia state. The immunoblotting data demonstrated that EMPA treatment triggered phosphorylated activation of LKB1 and AMPK in the isolated cardiomyocytes (Fig. 1A–C). Phosphorylated LKB1 (p-LKB1) and AMPK (p-AMPK) reached their peak value at 20–30 min of time gradient (Fig. 1B and C). Phosphorylated-ACC (p-ACC) and PGC1α level downstream also increased (Fig. 1D and E) (p

Discussion

Ischemic heart disease is the leading cause of death worldwide, and diabetes is an important triggering factor. Therefore, the treatment of diabetes is not simply to control blood glucose, but to control multiple risk factors such as blood pressure, serum lipid, uric acid, etc. in order to reduce the morbidity of CVD, other complications and mortality ultimately. At present, the safety requirements for hypoglycemic drugs is also increasing, at least not to increase the risk of CVD, it would be

Funding

The present study was supported by Sichuan Science and Technology Program [grant number 2019YJ0062], American Diabetes Association [grant number 1-17-IBS-296].

Declaration of competing interest

The authors declare that they have no conflict of interest.

References (43)

  • I. Andreadou et al.

    Empagliflozin limits myocardial infarction in vivo and cell death in vitro: role of STAT3, mitochondria, and redox aspects

    Front. Physiol.

    (2017)
  • A. Baartscheer et al.

    Empagliflozin decreases myocardial cytoplasmic Na(+) through inhibition of the cardiac Na(+)/H(+) exchanger in rats and rabbits

    Diabetologia

    (2017)
  • E. Bertero et al.

    Cardiac effects of SGLT2 inhibitors: the sodium hypothesis

    Cardiovasc. Res.

    (2018)
  • L. Bertrand et al.

    AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B

    Am. J. Physiol. Heart Circ. Physiol.

    (2006)
  • S.T. Cheng et al.

    The effects of empagliflozin, an SGLT2 inhibitor, on pancreatic beta-cell mass and glucose homeostasis in type 1 diabetes

    PLoS One

    (2016)
  • N. Hammoudi et al.

    Empagliflozin improves left ventricular diastolic dysfunction in a genetic model of type 2 diabetes

    Cardiovasc. Drugs Ther.

    (2017)
  • D.G. Hardie

    The AMP-activated protein kinase pathway--new players upstream and downstream

    J. Cell Sci.

    (2004)
  • D.G. Hardie et al.

    The AMP-activated protein kinase--fuel gauge of the mammalian cell?

    Eur. J. Biochem.

    (1997)
  • S.A. Hawley et al.

    The Na+/Glucose cotransporter inhibitor canagliflozin activates AMPK by inhibiting mitochondrial function and increasing cellular AMP levels

    Diabetes

    (2016)
  • C. He et al.

    Dissociation of Bcl-2-Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes

    Diabetes

    (2013)
  • Y. Huang et al.

    Resveratrol prevents sarcopenic obesity by reversing mitochondrial dysfunction and oxidative stress via the PKA/LKB1/AMPK pathway

    Aging

    (2019)
  • Cited by (65)

    View all citing articles on Scopus
    View full text