A SGLT2 inhibitor dapagliflozin comparison with insulinA SGLT2 inhibitor dapagliflozin comparison...

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A SGLT2 inhibitor dapagliflozin comparison with insulin exerts important effects on Zn2+-transporters in

cardiomyocytes from insulin-resistant metabolic syndrome rats through inhibition of oxidative stress

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2018-0466.R1

Manuscript Type: Article

Date Submitted by the Author: 24-Sep-2018

Complete List of Authors: Olgar, Yusuf; Ankara Universitesi Tip Fakultesi, bıophysıcsTuran, Belma; Ankara University,

Is the invited manuscript for consideration in a Special

Issue:2018 IACS Cuba

Keyword: SGLT2 inhibitors, diabetes, matrix metalloproteins, heart function, oxidative stress

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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A SGLT2 inhibitor dapagliflozin comparison with insulin exerts important

effects on Zn2+-transporters in cardiomyocytes from insulin-resistant metabolic

syndrome rats through inhibition of oxidative stress

Yusuf Olgar and Belma Turan*

Departments of Biophysics, Internal Medicine, Ankara University, Faculty of

Medicine, Ankara, Turkey.

*Correspondence: Belma Turan, PhD

Department of Biophysics, Faculty of Medicine, Ankara University, Ankara, Turkey.

Tel: 90 312 5958186

Fax: 90 312 3106370

Email: [email protected]

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mailto:[email protected]

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Abstract

Sodium-glucose cotransporter 2 (SGLT2)-inhibitors showed significant effect in

patients with diabetes or metabolic syndrome, MetS with high cardiovascular-risk.

Although the increased intracellular Zn2+ level ([Zn2+]i), oxidative stress and alterated

cardiac matrix metalloproteinases (MMPs) in diabetic cardiomyopathy can intersect

with different signaling pathways, the exact mechanisms are not known yet. Since

either MMPs or SGLT2 have important role in cardiac-fibrosis under hyperglycemia,

we aimed to examine the role of SGLT2-inhibitor dapagliflozin (DAP) on cardiac

Zn2+-transporters responsible from [Zn2+]i-regulation, comparison to insulin (INS),

together with MMP levels and systemic oxidative-stress-status in MetS-rats. High-

carbohydrated diet-induced MetS-rats received DAP or INS for two weeks. DAP but

not INS in MetS-rats significantly decreased high blood-glucose level, while both

treatments exerted benefits on increased total-oxidative-status and decreased total-

antioxidant-status in MetS-rat plasma as well as in heart tissue. Protein levels of Zn2+-

transporters, responsible from Zn2+-influx into cytosol, ZIP7 and ZIP14 were

decreased with no change in ZIP8 of MetS-rat cardiomyoctes, while Zn2+-

transporters, responsible from cytosolic Zn2+-efflux, ZnT7 was decreased. Both

treatments induced significant beneficial effects on altered ZIP14, ZIP8 and ZnT7

levels. Furthermore, both treatments exerted also benefits on depressed gelatin-

zymography and protein expression levels of MMP-2 and MMP-9 in MetS-rat

ventricular cardiomyocytes. The direct effect of DAP on heart was also confirmed

with measurement of left ventricular developed pressure. Overall, we showed that

DAP has important antioxidant-like cardio-protective effect in MetS-rats, similar to

INS-effect, affecting Zn2+-regulation via Zn2+-transporters, MMPs and oxidative-

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stress. Therefore one can suggest that SGLT2-inhibitors can be new therapeutic

agents for cardio-protection not only in hyperglycemia but also in failing heart.

Key words: diabetes, SGLT2 inhibitors, heart function, oxidative stress, matrix

metalloproteins, heart.

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Introduction

The global epidemic of the metabolic syndrome (MetS), known also as insulin

resistance syndrome, is a pathologic condition characterized by abdominal obesity,

hypertension, and hyperlipidemia besides insulin resistance, and is not a single disease

but a mixture of risk factors for cardiovascular diseases (Aguilar et al. 2010; Lauer et

al. 1992; Saklayen 2018). It is now well accepted that the MetS often is followed with

high incidence of obesity and type 2 diabetes (Genser et al. 2016). However,

regardless of the underlying genetic and environmental influences that mediate the

prevalence of the MetS, a higher prevalence will undoubtedly lead to undesirable

outcomes among humans worldwide. Clinical data documented that the risk of

cardiovascular diseases between diabetic and obese individuals is approximately two-

to four-fold higher than in those without them (Cameron et al. 2012; Moreira et al.

2014).

The pathogenesis of the MetS is multiple and still poorly understood. Experimental

evidence well documented the important role of oxidative stress in pathogenesis of

MetS, while oxidative stress is a common mediator in pathogenicity of established

cardiovascular risk factors (Mahjoub and Masrour-Roudsari 2012). In this regard, we

previously have shown that important increases in oxidative stress at cellular and

systemic levels in diabetic and MetS rats with marked depression in cardiac

contractile activity. Furthermore, experimental data have pointed out that there is

important contribution of increased cytosolic free Zn2+ level ([Zn2+]i) to these

alterations. Additionally, it was demonstared that the depressed level of matrix

metalloproteinases, MMPs (mainly MMP-2 and MMP-9) via a degradation in their

expression levels (Yaras et al. 2008). Moreover, the remodeling of cardiovascular

system in MetS individuals has a multifactorial pathogenesis that includes increased

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oxidative stress and also an altered MMPs expression, which, in turn, degrade

extracellular matrix proteins, such as collagen and fibronectin (Berg et al. 2014;

Hopps and Caimi 2012; Kadoglou et al. 2010). In addition, it is known that MMPs are

a family belonging to another family being structurally related Zn2+- and Ca2+-

dependent endopeptidases, involved in the cleavege of extracellular matrix proteins

(Shapiro 1998).

Identifying the oxidative stress markers in cells/tissues has been the focus of many

researchers as they have the potential to act as modulator of many signalling

pathways, driving cardiovascular pathobiology (Ilkun and Boudina 2013). Authors

pointed out the interrelationship between cardiac dysfunction and oxidative stress in

MetS individuals and beneficial effects antioxidant therapies on these relations in via

lipid accumulation, increased fibrosis and stiffness, altered Ca2+-homeostasis, altered

substrate utilization and mitochondrial dysfunction (Bonomini et al. 2015; Ilkun and

Boudina 2013).Functional significance of the oxidative modifications enhances their

validity as a proposed biological marker of cardiovascular disease, and is the strength

of the redox cysteine modifications. In this regard, in early studies, the experimental

data had implied that [Zn2+]i did increase rapidly in cardiomyocytes due to Zn2+

release from intracellular stores, at least, from metalloproteins via protein thiol

oxidation under increased oxidative stress as well as hyperglycemia (Tuncay et al.

2013; Tuncay and Turan 2016; Turan et al. 1997).

Recent studies emphasized the important role of sodium-glucose cotransporter 2

(SGLT2)-selective inhibitor dapagliflozin (DAP), for use as an antidiabetic agent in

normal and diabetic rats (Han et al. 2008; Hansen et al. 2014; Oku et al. 1999). In the

clinical studies, it has been demonstrated that SGLT2 inhibitors, in addition to

standard care of diabetics, provided important benefits against cardiovascular

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disorders (Ahmadieh et al. 2017; Lahnwong et al. 2018; Sato et al. 2017; Zinman et

al. 2015). Although these above data reported the important benefits of SGLT2

inhibitor trials in obesity and diabetes by clinicians for their benefits in prevention of

cardiovascular disease, rather than focusing on glycemic control, the mechanisms by

which SGLT2 inhibition improves cardiovascular outcomes are not fully understood.

Therefore, in the present study, we aimed to test whether the beneficial effect of a

SGLT2 inhibitor on cardiac dysfunction in insulin-resistant overweight MetS rats via

a way of affecting Zn2+-regulation via Zn2+-transporters, MMPs and oxidative-stress.

For this aim, we used isolated left ventricular cardiomyocytes from SGLT2 inhibitor

dapagliflozin (DAP) treated high-carbohydrated diet-induced metabolic syndrome

(MetS) rats comparison to those of insulin (INS) treated MetS rats. Our data indicate

that DAP, comparison with INS, has important an anti-oxidant like beneficial cardiac

effect, directly targeting heart, in MetS-rats affecting Zn2+-regulation via Zn2+-

transporters, MMPs and oxidative-stress, similar to INS-effects.

Materials and methods

Animals

Metabolic syndrome (MetS) was induced in male Wistar rats as described, previously

(Okatan et al. 2015). Shortly, 2-month old male rats were fed with drinking water

containing 32% sucrose besides their daily standard rat diet for 24 months. Following

confirmation the MetS induction in these animals (by measuring body weight, fasting

blood glucose level, insulin level, oral glucose tolerance test and and the development

of insulin resistance with HOMO-IR index), they were treated with either

dapagliflozin (DAP; 5 mg/kg, Bristo-myers Squibb Manufacturing Company,

Humacao, Porto Riko), insulin (INS; 0.15 mg/kg, Humalog Mix25 Kwikpen, Lilly) or

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vehicle (MetS) for 2 weeks. Experimental animals are exposed to a 12-h light–dark

cycle and had free access to tap water. All animals were fed standard chow ad libitum

and are housed in the standard rat cages.

Langendorff-perfused heart measurement

The left ventricular developed pressure changes (LVDP) are measured with a water-

filled latex balloon inserted into the left ventricle, as described previously (Durak et

al. 2017b). Briefly, hearts are electrically stimulated (DCS; Harward) at 300 beats/min

with 1.5 ms square waves (at twice the threshold voltage) and all data are recorded

online then stored and processed (Model 1050BP; BIOPAC Systems, Goleta,

California, USA). The results are given as percentage changes with respect to the

controls.

Isolation of left ventricular cardiomyocytes

The rats were anesthetized with pentobarbital sodium (30 mg/kg body weight, ip) and

the hearts were removed from the body immediately. Blood samples were collected

into heparinized tubes during the heart removal procedure.

Cardiomyocytes were isolated freshly from left ventricle of heart by enzymatic

method, as described previously (Turan et al. 1997). Briefly, isolated hearts were

initially perfused with a Ca2+-free, HEPES-buffered solution for 5 min at 37C and

then hearts were perfused (6-8 mL/min) with fresh buffer supplemented 1.3 mg/ml

collagenase (Type A, w) for 30 min. Following perfusion, we used the left ventricle

part to get lived cardiomyocytes into HEPES-buffered solution. Isolated

cardiomyocytes put into the HEPES buffer with 1 mM Ca2+ and 0.5 % bovine serum

albumin (pH 7.4) and then they were stored -80C for biochemical experiments.

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Total antioxidant status (TAS) and total oxidant status (TOS) measurement in

plasma and cardiomyocytes

TOS and TAS levels were measured in plasma as well as in cardiomyocytes by using

commercially available kit as described, previously (Okatan et al. 2015). The TAS

level in plasma was measured by using commercially available kit (RL0024, Rel

Assay Diagnostics, Turkey), as desribed previously (Erel 2004). Briefly, we used the

novel automated method, which is based on the bleaching of characteristic color of a

more stable ABTS (2,2′ - Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical

cation by antioxidants. The results are expressed as mmol Trolox equivalent/L. The

TOS level in plasma was measured using commercially available kits (RL0024, Rel

Assay Diagnostics, Turkey) as desribed previously (Erel 2004). Shortly, the oxidation

reaction is enhanced by glycerol molecules abundantly present in the reaction

medium. The ferric ion produced a colored complex with xylenol orange in an acidic

medium. The color intensity, measured spectrophotometrically, is related to the total

amount of oxidant molecules present in the sample. The assay is calibrated with H2O2

and the results are expressed in terms of µM H2O2 equivalent/L.

Western-blotting

The stored cardiomyocytes were used through all measurements. The experimental

procedure for Western-blot analysis were performed as described elsewhere (Tuncay

et al. 2017). Briefly, the cells following harvested in lysis buffer, were homogenated

and then centrifuged at 12,000×g for 15-min at 4°C. The protein concentration from

supernatants from was determined by using the Bradford assay. Equal amount of

protein preparations were run on SDS-polyacrylamide gels, electro-transferred to

polyvinylidine difluoride membranes, and blotted with a primary antibody against

ZIP7 (Santa Cruz, sc-83858, 1:500), ZIP8 (Protein Tech, 20459-1-AP; 1:500), ZIP14

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(Thermo, PA5-21077; 1:500), ZnT7 (Santa Cruz, sc-160948; 1:500), ZnT8 (Santa

Cruz, sc-98243, 1:500), MMP-2 (Santa Cruz, sc-6838, 1:500), MMP-9 (Santa Cruz

sc-6841, 1/500) and GAPDH (Cell Signalling, D16H11, 1/5000). Immunoreactive

bands were detected by a chemiluminescent reaction (ECL kit, Amersham Pharmacia,

USA).

Gelatin zymography

Gelatin zymography for MMP-2 and MMP-9 activities were performed as described

(Sawicki et al. 1998). Briefly, we loaded the non-reduced proteins onto an 8%

polyacrylamide gel containing gelatin and then gelatinolytic activities were detected

as transparent bands against the background of Coomassie blue-stained gelatin.

Gelatinolytic activity was identified using HT1080 cell culture medium as a standard.

The intensities of the bands were analysed using SigmaGel (Jandel).

Chemicals and statistics

Chemicals are obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

The results are expressed as means SEM. Statistical significance is tested with one-

way ANOVA followed by Tukey post-test and significance level is considered as

p<0.05.

Results

General status of animals

As presented previously, the mean (±SEM) body weight of MetS rats was

significantly high (460±10 g) comparasion with the aged-matched controls (400±12

g). Either DAP treatment of INS or treatment of MetS rats did not significantly

prevent the extra weight gain of the rats comparison to those of untreated MetS rats.

However, DAP treatment but not INS treatment of MetS rats modestly but

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significantly decreased blood glucose (95±3 mg/dL vs. 88±3 mg/dL in MetS vs.

MetS+DAP). Similarly, both treatment of MetS rats had no significant effect on the

OGTT during 60 min monitorization, as comparison with those of vehicle-treated

MetS rats.

DAP or INS treatment have beneficial effects on oxidative stress status in

systemic and organ levels of MetS rats

Similar to our previous study (Durak et al. 2017a), the total oxidative status (TOS)

level in plasma of MetS rats was significantly high comparison to those of aged-

matched controls (Fig.1A). Furthermore, the total antioxidant status (TAS) level was

significantly low in MetS rats comparison to aged-matched controls (Fig.1B). Either

DAP or INS treatment of MetS rats for 2 weeks similarly and significantly induced

positive effects. Interestingly, although it has been not shown any direct antioxidant

actions of either DAP or INS, the systemic recovery in increased oxidative stress

seems very important for MetS individuals.

We also measured the TOS and TAS levels in cardiomyocytes in order to detect the

direct effect of DAP treatment of MetS rats targeting of the heart. As can be seen in

Fig.1 C and D, the TOS level in isolated cardiomyocytes was markedly high while the

TAS level was significantly low comparison with those of controls. Either DAP or

INS treatment of MetS rats preserved the antioxidant defense of the heart,

significantly via targeting it directly.

Either DAP or INS treatment of Mets rats provided significant benefits on

cellular Zn2+-transporter protein expression levels

Taken into consideration our previously published data on the contribution of

increased [Zn2+]i ievel in depressed cardiac function under hyperglycemia (Olgar et al.

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2018b; Tuncay and Turan 2016), in here, we determined first the protein expression

levels of some Zn+2-transporters responsible from Zn+2–influx into cytosol of

cardiomyocytes, such as ZIP7, ZIP8 and ZIP14, which have been previously shown in

mammalian cardiomyocytes (Olgar et al. 2018a; Tuncay et al. 2018). As can be seen

in Fig. 2 (A to C), the protein expression level of ZIP7 significantly increased, similar

to in streptozotocin-induced diabetic rat heart (Tuncay et al. 2017). Similar to the our

previous data in the heart tissue from human heart failure, the ZIP8 level is found to

be markedly decreased with significantly increased ZIP14 level, similar to those of

heart homogenates from human heart failure. Either DAP or INS treatment of MetS

rats did not change the ZIP7 level, however, altered levels of both ZIP7 and ZIP14

were almost normalized with both types treatments.

The second groups Zn+2-transporters, ZIP7 and ZnT8 were also examined. As can be

seen in Fig. 3, the ZnT7 level (A) decreased with no change in the ZnT8 level (B) in

cardiomyocytes isolated from MetS rats. Interestingly, INS but not DAP treatment

induced fully recovery in ZnT7 level with no effect on the ZnT8 level.

Either DAP or INS treatment provided important protection against MMP

degradation in cardiomyocytes from MetS rats

First, in here, we demonstrated the already known relation between oxidative stress

and degradation of MMPs in the heart under hyperglycemia via determination of both

protein expression levels and activities of MMP-2 and MMP-9 (Yaras et al. 2008).

Fig. 4 shows the markedly decreased protein expression levels of both MMP-2 and

MMP-9 in cardiomyocytes from MetS rats. Either DAP or INS treatment of MetS rats

provided a similar significant and fully protection against MetS associated

degradation in these MMPs.

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DAP treatment of MetS rats provides benefits directly affecting the depressed

cardiac function in hyperglycemic and hyperinsulinemic rats

Previously, we have demonstrated that the left ventricular developed pressure (LVDP)

depressed significantly in MetS rats comparison to those of aged-matched controls

(about 35-40%) (Okatan et al. 2015). In order to demonstrate the direct beneficial

effect of DAP treatment on cardiac function via affecting the mechanical activity of

the heart, we used hyperglycemic and hyperinsulinemic condition for Langendorff-

perfusion of heart preparations. As can be seen in Fig. 5B, a direct exposure of DAP

(0.1 M; (Han et al. 2008)) or INS (0.1 M) induced significant increase (about 40%

w.r.t. control) in left ventricular developed pressure, LVDP in normal male rat heart

preparations. The original pressure changes are given in Fig.5A. When we exposed

heart preparations to DAP or INS following the MetS-mimicing via using

hyperglycemic and hyperinsulinemic perfussion (25 mM glucose and 0.1 nM insulin,

respectively), the contractile activity responses were about 20% compared with those

of controls. These data confirmed that DAP application has important cardio-

protective effect directly affecting the heart mechanical activity in insulin resistant

MetS rats.

Discussion

We previously have demonstrated that cellular free Zn2+-changes are primarily

coordinated by Zn2+-transporters and free Zn2+ releases during cardiac cycle (Tuncay

et al. 2011) results mostly in a cytosolic free Zn2+ increase, further triggering higher

production of pro-oxidant species and leading to cellular oxidative damage including

proteins and kinases from contractile machinary (Tuncay et al. 2013). Furthermore,

we have also been shown that either acute or chronic oxidant exposure induces

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marked increases in cytosolic free Zn2+ in cardiomyocytes (Ayaz and Turan 2006;

Turan et al. 1997) while either acute or chronic hyperglycemia causes oxidative stress

and increased levels of cytosolic free Zn2+, being responsible from cardiac

dysfunction (Ayaz and Turan 2006; Tuncay and Turan 2016). Having all these

previous documents in diabetic samples, in here, we examined the effect of a SGLT2

inhibitor dapagliflozin (DAP) on protein expression levels of Zn2+-transporters, which

have important roles in cellular Zn2+-regulation in cardiomyocytes from insulin-

resistant MetS rats. In order to confirm the DAP effect is associated with insulin

resistance, and insulin treatment is used in diabetic and/or insulin resistant individuals,

we also compared the DAP effects with those of insulin (INS) effects. In here, we

have shown that DAP treatment of MetS rats provided important antioxidant-like

benefits, in some aspects similar to INS effects, not only in systemic system of the rats

but also directly targeting the heart against increased oxidative stress. Therefore, our

present data provided important information related with how the SGLT2 inhibitors

exert cardiac benefits when they used in MetS rats as in vivo. We, in here, also

demonstrated that INS treatment could exert an anti-oxidant like action in MetS rats

via inducing significant increase in TAS levels with marked decrease in TOS levels

determined in both plasma and isolated cardiomyocytes from MetS rats. Supporing

our present data, recently, it has been demonstrated that treatment of Zucker diabetic

fatty (ZDF) rats with another SGLT2 inhibitor empagliflozin, an inhibitor of the renal

SGLT2, provided benefical effects on high blood glucose level as well as endothelial

dysfunction and increased oxidative stress in isolated aortic preparations of diabetic

rats (Steven et al. 2017). Interestingly, since there were persisting hyperlipidemia and

hyperinsulinemia in their animals, their data showed that empagliflozin could able to

reduce glucotoxicity and thereby might prevent the development of endothelial

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dysfunction together with an important reduction in oxidative stress of the

experimental animals. Therefore, our present, similar to the above data, can support

truely the previous pre-clinical observations with SGLT2 inhibitors. Most of pre-

clinical data (EMPA-REG trial and others) provide important insights about the

benefits of SGLT2 inhibitors on cardiovascular system in hyperglycemic and

hyperinsulinemic individuals via marked reduction in cardiovascular mortality among

the patients (Kashiwagi and Maegawa 2017; Kramer and Zinman 2016; Tanaka et al.

2017; Zinman et al. 2015). In this regard, experimental and clinical studies suggested

that those risk factors beyond glucose that can potentially be modulated positively

with SGLT2 inhibitors include blood pressure, weight, visceral adiposity,

hyperinsulinaemia, arterial stiffness, albuminuria, circulating uric acid levels and

oxidative stress (Kramer and Zinman 2016; Steven et al. 2017; Zinman et al. 2015). Other study results also provied important data on benefical effects of SGLT2

inhibitors on hyperglycemia, hyperlipidemia, hepatic steatosis, oxidative stress,

inflammation, and obesity in type 2 diabetic mice (Tahara et al. 2013) and prevention

of oxidative stress in the kidney of diabetic rats (Osorio et al. 2012). However, they

could not provide any data underlying the mechanisms of beneficial effects of SGLT2

inhibition on depressed cardiovascular function, independent of diuretic effect or

blood pressure lowering effect. Although SGLT2 inhibition seems to be a promising

therapeutic strategy for diabetic, obese or prediabetic MetS, further studies are also

needed.

The cardioprotective effect of SGLT2 inhibitors, particularly via effecting vascular

system have been demonstrated recently in diabetics as well as in prediabetics with

MetS (Hammoudi et al. 2017; Kusaka et al. 2016; Lee et al. 2018). Their data

demonstrated that empagliflozin significantly lowered HbA1c and slightly reduced

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body weight compared to vehicle treatment with no obvious changes in insulin levels,

however, it improved markedly left ventricular maximum pressure and in vivo indices

of diastolic function. In this aspect, Baartscheer A et al (2017) treated animals with

empagliflozin and measured cytoplasmic Na+ and Ca2+ concentrations, mitochondrial

Ca2+ concentration and Na+/H+ exchanger activity in isolated ventricular myocytes

(Baartscheer et al. 2017). Their data have demonstrated that extracellular high glucose

related changes in these above parameters could be preserved with SGLT2 inhibitor

treatment via implying its direct cardiac effects by lowering myocardial cytosolic

ionic mechanisms particularly associated with cardiac contractile activity.

Previous studies pointed out that enhanced oxidative stress or diabetes-induced

oxidative stress, under both in vitro and in vivo conditions, is known to increase the

basal levels of [Ca2+]i as well as [Zn2+]i, which is also known as a strong indicator of

the cellular level of oxidative stress in cardiomyocytes (Ayaz and Turan 2006; Tuncay

et al. 2013; Turan et al. 1997). In these aspects, we, previously, have shown that

exogenously application of oxidants could increase both [Ca2+]i and [Zn2+]i with

deleterious effects on proteins and kinases (RyR2, PKA, CaMKII, PKC, PLB) of

contractile machinary in cardiomyocytes, which, in turn, induce significant depression

effect on heart contractility. In addition, our previous data also demonstrated that

either acute or chronic hyperglycemia could induce significant increases in both

[Ca2+]i and [Zn2+]i as well as reactive oxygen and nitrogen species (Tuncay et al.

2011; Tuncay et al. 2013; Tuncay and Turan 2016). Taken into consideration all these

published data on increased [Ca2+]i and [Zn2+]i and increased oxidative stress under

hyperglycemia, the benefical effects of DAP treatment of MetS rats associated with

well-controlled systemic oxidative stress and antioxidant defense system together with

possible well-controlled [Zn2+]i through normalized protein expression levels of Zn2+-

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transporters will get much more interest for further clinical trials with SGLT2

inhibitors in diabetic and/or obese individuals or individuals with cardiac diseases.

Zn2+ is essential for numerous cellular functions although it is toxic for live cells

(Vallee and Falchuk 1993). Zn2+-homeostasis is therefore dynamically maintained by

a variety of transporters, stores, and other proteins distributed in distinct cellular

compartments.

In the present study, we have also shown that DAP treatment of MetS rats

significantly preserved the depressed expression levels of MMP-2 and MMP-9 in

isolated left ventricular cardiomyocytes. Similarly, we previously have demonstrated

the significantly depressed level of MMP-2 and MMP-9 via degradation in their

expression levels (Yaras et al. 2008). Moreover, the remodeling of cardiovascular

system in MetS individuals has a multifactorial pathogenesis that includes increased

oxidative stress and also an altered MMPs expression, which, in turn, degrade

extracellular matrix proteins, such as collagen and fibronectin (Berg et al. 2014;

Hopps and Caimi 2012; Kadoglou et al. 2010). In these aspects, it is known that

glomerular hypertrophy and extracellular matrix accumulation involve in the

pathogenesis of diabetic nephropathy through an increased matrix synthesis and

degradative pathway in diabetic kidney due to event series including dynamic process

involving synthesis as well as degradation in the regulation of extracellular matrix.

The latter is tightly regulated by a number of MMPs. In line with these events, Liu Bi-

cheng, Xu Yan, Ma Kun-ling et al. (2005) treated streptozotocin-induced diabetic rats

with a SGLT2 inhibitor and demonstrated its benefial effect on renal MMP-2 level

(Liu et al. 2005). Additionally, it is known that MMPs play an important role during

physiological tissue remodeling in pathophysiological conditions, including diabetes

and obesity while MMP expression and activity are regulated by different factors such

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as insulin resistance and obesity. The role of Zn2+ level and its binding-groups to

MMPs on the activities of MMPs and their roles in several pathological conditions is

geartly discussed by Jacobsen JA et al (2010) (Jacobsen et al. 2010). Therefore, in

here, we first demonstrated the degradation of MMPs in MetS rat cardiomyocytes and

their preservation with DAP treatment. At least, our data imply the possible cross-talk

between [Zn2+]i via Zn2+-transporters and MMPs (MMP-2 and MMP-9) in MetS

cardiac function and the benefical effect of DAP treatment on this cross-talk. Taken

into consideration the relation between this cross-talk and oxidative stress, the

importance of DAP treatment of individuals with hypergycemia raises much more

rapidly between clinical trials.

Taken into consideration of the direct effect of DAP on the heart, overall, we showed

that DAP has important antioxidant-like cardio-protective effect in MetS-rats, in some

aspects similar to INS-effect, affecting Zn2+-regulation via Zn2+-transporters, MMPs

and oxidative-stress. Therefore one can suggest that SGLT2-inhibitors can be new

therapeutic agents for cardio-protection not only in hyperglycemia but also in failing

heart.

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Acknowledgements

Authors present acknowledgements to Dr. Erkan Tuncay and Sinan Degirmenci for

their technical contribution to the study and Dr. Aysegul Durak for her helps to care

of experimental animals.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the

corresponding author on reasonable request.

Consent for publication

This study consists of animal data and is devoid of any human data.

Ethics approval and consent to participate

The experimental protocols with rats were performed in accordance with the the

standards of the European Community guidelines on the care and use of laboratory

animals" adhere to the to the Guide for the Care and Use of Laboratory Animals (8th

edition, National Academies Press) and had been approved by the local ethics

committee of Ankara University (No:2015-12-137).

Funding

This study was supported by TUBITAK SBAG-214S254 to BT.

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Tuncay, E., Bilginoglu, A., Sozmen, N.N., Zeydanli, E.N., Ugur, M., Vassort, G., and Turan, B. 2011. Intracellular free zinc during cardiac excitation-contraction cycle: calcium and redox dependencies. Cardiovasc Res 89(3): 634-642. doi: 10.1093/cvr/cvq352.Tuncay, E., Bitirim, C.V., Olgar, Y., Durak, A., Rutter, G.A., and Turan, B. 2018. Zn(2+)-transporters ZIP7 and ZnT7 play important role in progression of cardiac dysfunction via affecting sarco(endo)plasmic reticulum-mitochondria coupling in hyperglycemic cardiomyocytes. Mitochondrion. doi: 10.1016/j.mito.2017.12.011.Tuncay, E., Bitirim, V.C., Durak, A., Carrat, G.R.J., Taylor, K.M., Rutter, G.A., and Turan, B. 2017. Hyperglycemia-Induced Changes in ZIP7 and ZnT7 Expression Cause Zn2+ Release From the Sarco(endo)plasmic Reticulum and Mediate ER Stress in the Heart. Diabetes 66(5): 1346-1358. doi: 10.2337/db16-1099.Tuncay, E., Okatan, E.N., Vassort, G., and Turan, B. 2013. ss-blocker timolol prevents arrhythmogenic Ca(2)(+) release and normalizes Ca(2)(+) and Zn(2)(+) dyshomeostasis in hyperglycemic rat heart. PLoS One 8(7): e71014. doi: 10.1371/journal.pone.0071014.Tuncay, E., and Turan, B. 2016. Intracellular Zn(2+) Increase in Cardiomyocytes Induces both Electrical and Mechanical Dysfunction in Heart via Endogenous Generation of Reactive Nitrogen Species. Biol Trace Elem Res 169(2): 294-302. doi: 10.1007/s12011-015-0423-3.Turan, B., Fliss, H., and Desilets, M. 1997. Oxidants increase intracellular free Zn2+ concentration in rabbit ventricular myocytes. Am J Physiol 272(5 Pt 2): H2095-2106.Vallee, B.L., and Falchuk, K.H. 1993. The biochemical basis of zinc physiology. Physiol Rev 73(1): 79-118.Yaras, N., Sariahmetoglu, M., Bilginoglu, A., Aydemir-Koksoy, A., Onay-Besikci, A., Turan, B., and Schulz, R. 2008. Protective action of doxycycline against diabetic cardiomyopathy in rats. Br J Pharmacol 155(8): 1174-1184. doi: 10.1038/bjp.2008.373.Zinman, B., Wanner, C., Lachin, J.M., Fitchett, D., Bluhmki, E., Hantel, S., Mattheus, M., Devins, T., Johansen, O.E., Woerle, H.J., Broedl, U.C., and Inzucchi, S.E. 2015. Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N Engl J Med 373(22): 2117-2128. doi: 10.1056/NEJMoa1504720.

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Figure legends

Fig. 1. Assessment of in vivo DAP or INS treatment on systemic oxidative status

in plasma and cardiomyocytes of MetS rats. The total oxidant status (TOS) (A and

C) and the total antioxidant status (TAS) (B and D) measured in plasma and

cardiomyocytes of MetS rats comparison with aged-matched rats (Con). Data are

presenting as mean (±SEM) as percentage changes with respect to the corresponding

controls. The total number of rats in each group; n= 5-6 rats. *p<0.05 vs. Con; #p<0.05

vs. MetS.

Fig. 2. The protein expression levels of Zn2+-transporters responsible from Zn2+-

influx into cytosol in cardiomyocytes from either DAP or INS treated MetS rats.

Western-blot analysis of ZIP7, ZIP8 and ZIP 14 (at 51 kDa) protein expression levels

(with respect to GAPDH at 37 kDa) (A, B, C, respectively). Data are presenting as

mean (±SEM) as percentage changes with respect to the corresponding controls.

Number of animals /group; n= 5-7. *p<0.05 vs. Con; #p<0.05 vs. MetS.

Fig. 3. The protein expression levels of Zn2+-transporters responsible from

cytosolic Zn2+-efflux in cardiomyocytes from either DAP or INS treated MetS

rats. Western-blot analysis of ZnT7 (A) and ZnT8 (B) (at 41 kDa) protein expression

level (with respect to GAPDH at 37 kDa). Data are presenting as mean (±SEM) as

percentage changes with respect to the corresponding controls. Number of animals

/group; n= 5-7. *p<0.05 vs. Con; #p<0.05 vs. MetS.

Fig. 4. The protein expression levels and activities of matrix metalloproteinases

(MMPs) in cardiomyocytes from either DAP or INS treated MetS rats. The

protein expression levels and activities of MMP-2 at 72 kDa and MMP-9 at 92 kDa

determined by Western-blot (with respect to GAPDH at 37 kDa) and zymography (in

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A and C, respectively and in B and D, respectively). Data are presenting as mean

(±SEM) as percentage changes with respect to the corresponding controls. Number of

animals /group; n= 5-7. *p<0.05 vs. Con; #p<0.05 vs. MetS.

Fig. 5. The beneficial effect of DAP on hemodynamic parameters of the heart.

The original left ventricular developed (LVDP) traces with different protocols such as

either DAP (0.1 M) or INS (0.1 M) exposures (A). (B) To mimic MetS (MetSm) in

cardiac preparations (hyperglycemic and hyperinsulinemic), high glucose (25 mM)

and high circulating insulin (0.1 nM) were used for perfusion of the heart. Data are

presenting as mean (±SEM) as percentage changes with respect to the corresponding

controls. Number of animals; n=4-5/group, *p<0.05 vs. Con, #p<0.05 vs. MetSm.

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Assessment of in vivo DAP or INS treatment on systemic oxidative status in plasma and cardiomyocytes of MetS rats

254x190mm (300 x 300 DPI)

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The protein expression levels of Zn2+-transporters responsible from Zn2+-influx into cytosol in cardiomyocytes from either DAP or INS treated MetS rats.

254x190mm (300 x 300 DPI)

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The protein expression levels of Zn2+-transporters responsible from cytosolic Zn2+-efflux in cardiomyocytes from either DAP or INS treated MetS rats.

254x190mm (300 x 300 DPI)

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The protein expression levels and activities of matrix metalloproteinases (MMPs) in cardiomyocytes from either DAP or INS treated MetS rats.

254x190mm (300 x 300 DPI)

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The beneficial effect of DAP on hemodynamic parameters of the heart.

254x190mm (300 x 300 DPI)

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A SGLT2 inhibitor dapagliflozin comparison with insulinA SGLT2 inhibitor dapagliflozin comparison...

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