Cardioprotection by selective SGLT-2 inhibitors in a non-diabetic mouse model of myocardial ischemia/reperfusion injury: a class or a drug effect?

Major clinical trials with sodium glucose co-transporter-2 inhibitors (SGLT-2i) exhibit protective effects against heart failure events, whereas inconsistencies regarding the cardiovascular death outcomes are observed. Therefore, we aimed to compare the selective SGLT-2i empagliflozin (EMPA), dapagliflozin (DAPA) and ertugliflozin (ERTU) in terms of infarct size (IS) reduction and to reveal the cardioprotective mechanism in healthy non-diabetic mice. C57BL/6 mice randomly received vehicle, EMPA (10 mg/kg/day) and DAPA or ERTU orally at the stoichiometrically equivalent dose (SED) for 7 days. 24 h-glucose urinary excretion was determined to verify SGLT-2 inhibition. IS of the region at risk was measured after 30 min ischemia (I), and 120 min reperfusion (R). In a second series, the ischemic myocardium was collected (10th min of R) for shotgun proteomics and evaluation of the cardioprotective signaling. In a third series, we evaluated the oxidative phosphorylation capacity (OXPHOS) and the mitochondrial fatty acid oxidation capacity by measuring the respiratory rates. Finally, Stattic, the STAT-3 inhibitor and wortmannin were administered in both EMPA and DAPA groups to establish causal relationships in the mechanism of protection. EMPA, DAPA and ERTU at the SED led to similar SGLT-2 inhibition as inferred by the significant increase in glucose excretion. EMPA and DAPA but not ERTU reduced IS. EMPA preserved mitochondrial functionality in complex I&II linked oxidative phosphorylation. EMPA and DAPA treatment led to NF-kB, RISK, STAT-3 activation and the downstream apoptosis reduction coinciding with IS reduction. Stattic and wortmannin attenuated the cardioprotection afforded by EMPA and DAPA. Among several upstream mediators, fibroblast growth factor-2 (FGF-2) and caveolin-3 were increased by EMPA and DAPA treatment. ERTU reduced IS only when given at the double dose of the SED (20 mg/kg/day). Short-term EMPA and DAPA, but not ERTU administration at the SED reduce IS in healthy non-diabetic mice. Cardioprotection is not correlated to SGLT-2 inhibition, is STAT-3 and PI3K dependent and associated with increased FGF-2 and Cav-3 expression.

Novel Evidence-Based Combination of Plant Extracts with Multitarget Mechanisms of Action for the Elimination of Hot Flashes during Menopause.

Hot flashes are considered the most bothersome complaint during menopause. Although hormone therapy is an effective option to relieve hot flashes, it has been associated with significant side effects. The aim of our study is to suggest a novel combination of different plant extracts with distinct mechanisms of action against hot flashes. We selected the rhizome of Glycyrrhiza glabra L. (Fabaceae), the rhizome of Actaea racemosa L. (Ranunculaceae), the aerial parts of Hypericum perforatum L. (Hypericaceae) to produce extracts rich in bioactive phytochemicals and the seed oil of Oenothera biennis L. (Onagraceae). We investigated their estrogenic and antioxidant potential and their inhibitory effect against prostaglandin D2 receptor 1 (DP1) as a novel mechanistic pathway for vasodilation in hot flashes, alone or in combination. The phytochemical footprint of the extracts was analyzed using HPLC-PDA and UPLC-HRMS. We observed that the tested extracts possess different mechanisms of action. A. racemosa exerts a beneficial activation of the estrogen receptor, H. perforatum possesses the highest antioxidant capacity and the seed oil of O. biennis inhibits the DP1 receptor. The triple combination in the optimal doses pertains to efficacy against all three mechanisms of action, serves as a multitarget plant-based therapy and could serve as a novel strategy for the alleviation of hot flashes in postmenopausal women.

Molecular mechanisms of carfilzomib-induced cardiotoxicity in mice and the emerging cardioprotective role of metformin.

Carfilzomib (Cfz), an irreversible proteasome inhibitor licensed for relapsed/refractory myeloma, is associated with cardiotoxicity in humans. We sought to establish the optimal protocol of Cfz-induced cardiac dysfunction, to investigate the underlying molecular-signaling and, based on the findings, to evaluate the cardioprotective potency of metformin (Met). Mice were randomized into protocols 1 and 2 (control and Cfz for 1 and 2 consecutive days, respectively); protocols 3 and 4 (control and alternate doses of Cfz for 6 and 14 days, respectively); protocols 5A and 5B (control and Cfz, intermittent doses on days 0, 1 [5A] and 0, 1, 7, and 8 [5B] for 13 days); protocols 6A and 6B (pharmacological intervention; control, Cfz, Cfz+Met and Met for 2 and 6 days, respectively); and protocol 7 (bortezomib). Cfz was administered at 8 mg/kg (IP) and Met at 140 mg/kg (per os). Cfz resulted in significant reduction of proteasomal activity in heart and peripheral blood mononuclear cells in all protocols except protocols 5A and 5B. Echocardiography demonstrated that Cfz led to a significant fractional shortening (FS) depression in protocols 2 and 3, a borderline dysfunction in protocols 1 and 4, and had no detrimental effect on protocols 5A and 5B. Molecular analysis revealed that Cfz inhibited AMPKα/mTORC1 pathways derived from increased PP2A activity in protocol 2, whereas it additionally inhibited phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase pathway in protocol 3. Coadministration of Met prevented Cfz-induced FS reduction and restored AMPKα phosphorylation and autophagic signaling. Conclusively, Cfz decreased left ventricular function through increased PP2A activity and inhibition of AMPKα and its downstream autophagic targets, whereas Met represents a novel promising intervention against Cfz-induced cardiotoxicity.

Reactive Vasodilation Predicts Mortality in Primary Systemic Light-Chain Amyloidosis.

Rationale: Cardiac involvement and hypotension dominate the prognosis of light-chain amyloidosis (AL). Evidence suggests that there is also peripheral vascular involvement in AL but its prognostic significance is unknown. Objective: To evaluate vascular dysfunction in patients with AL as a potential future area of intervention, we assessed the prognostic utility of flow-mediated dilatation (FMD), a marker of vascular reactivity, which is augmented under conditions of hypotension and autonomic dysfunction. Methods and Results: We prospectively evaluated 115 newly diagnosed untreated AL patients in whom FMD was measured. FMD in AL patients was significantly higher than age-, sex- and risk factors-matched controls (4.0% versus 2.32%; P=0.006) and comparable with control groups at lower cardiovascular risk (P>0.1). Amyloidosis patients presented increased plasma and exhaled markers of the NO pathway while their FMD significantly correlated with augmented sustained vasodilatation after sympathetic stimulation. Increased FMD (≥4.5%) was associated with early mortality (hazard ratio, 4.36; 95% CI, 1.41-13.5; P=0.010) and worse survival (hazard ratio, 2.11; 95% CI, 1.17-3.82; P=0.013), even after adjustment for Mayo stage, nerve involvement and low systolic blood pressure. This finding was confirmed in a temporal validation AL cohort (n=55; hazard ratio, 4.2; 95% CI, 1.45-12.3; P=0.008). FMD provided significant reclassification value over the best prognostic model (continuous Net Reclassification Index, 0.61; P=0.001). Finally, better hematologic response was associated with lower posttreatment FMD. Conclusions: FMD is relatively increased in AL and independently associated with inferior survival with substantial reclassification value. Reactive vasodilation merits further investigation as a novel risk biomarker in AL.Visual Overview: An online visual overview is available for this article.

Elucidating Carfilzomib's Induced Cardiotoxicity in an In Vivo Model of Aging: Prophylactic Potential of Metformin.

BACKGROUND: Carfilzomib is a first-line proteasome inhibitor indicated for relapsed/refractory multiple myeloma (MM), with its clinical use being hampered by cardiotoxic phenomena. We have previously established a translational model of carfilzomib cardiotoxicity in young adult mice, in which metformin emerged as a prophylactic therapy. Considering that MM is an elderly disease and that age is an independent risk factor for cardiotoxicity, herein, we sought to validate carfilzomib's cardiotoxicity in an in vivo model of aging. METHODS: Aged mice underwent the translational two- and four-dose protocols without and with metformin. Mice underwent echocardiography and were subsequently sacrificed for molecular analyses in the blood and cardiac tissue. RESULTS: Carfilzomib decreased proteasomal activity both in PBMCs and myocardium in both protocols. Carfilzomib induced mild cardiotoxicity after two doses and more pronounced cardiomyopathy in the four-dose protocol, while metformin maintained cardiac function. Carfilzomib led to an increased Bip expression and decreased AMPKα phosphorylation, while metformin coadministration partially decreased Bip expression and induced AMPKα phosphorylation, leading to enhanced myocardial LC3B-dependent autophagy. CONCLUSION: Carfilzomib induced cardiotoxicity in aged mice, an effect significantly reversed by metformin. The latter possesses translational importance as it further supports the clinical use of metformin as a potent prophylactic therapy.

Exploitation of Vitis vinifera, Foeniculum vulgare, Cannabis sativa and Punica granatum By-Product Seeds as Dermo-Cosmetic Agents.

In the current study, by-product seed pastes (VSPs) from Vitis vinifera, Foeniculum vulgare, Cannabis sativa and Punica granatum, generated during the oil production process, were investigated for their potential exploitation as dermo-cosmetic agent. The extraction pipeline of all the raw materials was developed with emphasis on green methodologies and employed on laboratory scale based on industry-adopted techniques. Two different protocols were applied, Supercritical Fluid Extraction (SFE) and Ultrasound Assisted Extraction (UAE); the by-product pastes were defatted with supercritical CO2 and n-Hexane, respectively. Then, two SFE extracts (CO2 with 10% and 20% of ethanol as co-solvent) and two UAE extracts (with ethanol and ethanol/water 1:1 v/v) were obtained from each raw material. The providing yield range was between 2.6 to 76.3 mg/g raw material. The extracts were analyzed with High-Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD) and Liquid Chromatography coupled with High-Resolution Mass Spectrometer (LC-HRMS), and the major compounds, were identified. All the extracts were evaluated for their antioxidant and inhibition activity against collagenase, elastase and tyrosinase enzymes. Grapevine by-product extracts found rich in proanthocyanidins and presented the higher inhibition activity. A holistic green experimental methodology is proposed for the obtainment of extracts from significant medicinal plants by-products that provides us with promising results concerning dermo-cosmetic properties, especially for grape seeds extracts.

Investigating the Vascular Toxicity Outcomes of the Irreversible Proteasome Inhibitor Carfilzomib.

BACKGROUND: Carfilzomib's (Cfz) adverse events in myeloma patients include cardiovascular toxicity. Since carfilzomib's vascular effects are elusive, we investigated the vascular outcomes of carfilzomib and metformin (Met) coadministration. METHODS: Mice received: (i) saline; (ii) Cfz; (iii) Met; (iv) Cfz+Met for two consecutive (acute) or six alternate days (subacute protocol). Leucocyte-derived reactive oxygen species (ROS) and serum NOx levels were determined and aortas underwent vascular and molecular analyses. Mechanistic experiments were recapitulated in aged mice who received similar treatment to young animals. Primary murine (prmVSMCs) and aged human aortic smooth muscle cells (HAoSMCs) underwent Cfz, Met and Cfz+Met treatment and viability, metabolic flux and p53-LC3-B expression were measured. Experiments were recapitulated in AngII, CoCl2 and high-glucose stimulated HAoSMCs. RESULTS: Acutely, carfilzomib alone led to vascular hypo-contraction and increased ROS release. Subacutely, carfilzomib increased ROS release without vascular manifestations. Cfz+Met increased PGF2α-vasoconstriction and LC3-B-dependent autophagy in both young and aged mice. In vitro, Cfz+Met led to cytotoxicity and autophagy, while Met and Cfz+Met shifted cellular metabolism. CONCLUSION: Carfilzomib induces a transient vascular impairment and oxidative burst. Cfz+Met increased vascular contractility and synergistically induced autophagy in all settings. Therefore, carfilzomib cannot be accredited for a permanent vascular dysfunction, while Cfz+Met exert vasoprotective potency.

Hydrolytic Activity of Mitochondrial F1FO-ATP Synthase as a Target for Myocardial Ischemia-Reperfusion Injury: Discovery and In Vitro and In Vivo Evaluation of Novel Inhibitors.

F1FO-ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, it reverses its activity, hydrolyzing ATP and leading to energetic deficit and cardiac injury. We aimed to discover novel inhibitors of ATP hydrolysis, accessing the druggability of the target within ischemia(I)/reperfusion(R) injury. New molecular scaffolds were revealed using ligand-based virtual screening methods. Fifty-five compounds were tested on isolated murine heart mitochondria and H9c2 cells for their inhibitory activity. A pyrazolo[3,4-c]pyridine hit structure was identified and optimized in a hit-to-lead process synthesizing nine novel derivatives. Three derivatives significantly inhibited ATP hydrolysis in vitro, while in vivo, they reduced myocardial infarct size (IS). The novel compound 31 was the most effective in reducing IS, validating that inhibition of F1FO-ATP hydrolytic activity can serve as a target for cardioprotection during ischemia. Further examination of signaling pathways revealed that the cardioprotection mechanism is related to the increased ATP content in the ischemic myocardium and increased phosphorylation of PKA and phospholamban, leading to the reduction of apoptosis.

Mineralocorticoid Receptor Pathway Is a Key Mediator of Carfilzomib-induced Nephrotoxicity: Preventive Role of Eplerenone.

Carfilzomib is an irreversible proteasome inhibitor indicated for relapsed/refractory multiple myeloma. Carfilzomib toxicity includes renal adverse effects (RAEs) of obscure pathobiology. Therefore, we investigated the mechanisms of nephrotoxicity developed by Carfilzomib. In a first experimental series, we used our previously established in vivo mouse models of Carfilzomib cardiotoxicity, that incorporated 2 and 4 doses of Carfilzomib, to identify whether Carfilzomib affects renal pathways. Hematology and biochemical analyses were performed, while kidneys underwent histological and molecular analyses. In a second and third experimental series, the 4 doses protocol was repeated for 24 hours urine collection and proteomic/metabolomic analyses. To test an experimental intervention, primary murine collecting duct tubular epithelial cells were treated with Carfilzomib and/or Eplerenone and Metformin. Finally, Eplerenone was orally co-administered with Carfilzomib daily (165 mg/kg) in the 4 doses protocol. We additionally used material from 7 patients to validate our findings and patients underwent biochemical analysis and assessment of renal mineralocorticoid receptor (MR) axis activation. In vivo screening showed that Carfilzomib-induced renal histological deficits and increased serum creatinine, urea, NGAL levels, and proteinuria only in the 4 doses protocol. Carfilzomib decreased diuresis, altered renal metabolism, and activated MR axis. This was consistent with the cytotoxicity found in primary murine collecting duct tubular epithelial cells, whereas Carfilzomib + Eplerenone co-administration abrogated Carfilzomib-related nephrotoxic effects in vitro and in vivo. Renal SGK-1, a marker of MR activation, increased in patients with Carfilzomib-related RAEs. Conclusively, Carfilzomib-induced renal MR/SGK-1 activation orchestrates RAEs and water retention both in vivo and in the clinical setting. MR blockade emerges as a potential therapeutic approach against Carfilzomib-related nephrotoxicity.

Chronic Empagliflozin Treatment Reduces Myocardial Infarct Size in Nondiabetic Mice Through STAT-3-Mediated Protection on Microvascular Endothelial Cells and Reduction of Oxidative Stress.

Aims: Empagliflozin (EMPA) demonstrates cardioprotective effects on diabetic myocardium but its infarct-sparing effects in normoglycemia remain unspecified. We investigated the acute and chronic effect of EMPA on infarct size after ischemia-reperfusion (I/R) injury and the mechanisms of cardioprotection in nondiabetic mice. Results: Chronic oral administration of EMPA (6 weeks) reduced myocardial infarct size after 30 min/2 h I/R (26.5% ± 3.9% vs 45.8% ± 3.3% in the control group, p < 0.01). Body weight, blood pressure, glucose levels, and cardiac function remained unchanged between groups. Acute administration of EMPA 24 or 4 h before I/R did not affect infarct size. Chronic EMPA treatment led to a significant reduction of oxidative stress biomarkers. STAT-3 (signal transducer and activator of transcription 3) was activated by Y(705) phosphorylation at the 10th minute of R, but it remained unchanged at 2 h of R and in the acute administration protocols. Proteomic analysis was employed to investigate signaling intermediates and revealed that chronic EMPA treatment regulates several pathways at reperfusion, including oxidative stress and integrin-related proteins that were further evaluated. Superoxide dismutase and vascular endothelial growth factor were increased throughout reperfusion. EMPA pretreatment (24 h) increased the viability of human microvascular endothelial cells in normoxia and on 3 h hypoxia/1 h reoxygenation and reduced reactive oxygen species production. In EMPA-treated murine hearts, CD31-/VEGFR2-positive endothelial cells and the pSTAT-3(Y705) signal derived from endothelial cells were boosted at early reperfusion. Innovation: Chronic EMPA administration reduces infarct size in healthy mice via the STAT-3 pathway and increases the survival of endothelial cells. Conclusion: Chronic but not acute administration of EMPA reduces infarct size through STAT-3 activation independently of diabetes mellitus.

Acute administration of the olive constituent, oleuropein, combined with ischemic postconditioning increases myocardial protection by modulating oxidative defense.

Oleuropein, one of the main polyphenolic constituents of olive, is cardioprotective against ischemia reperfusion injury (IRI). We aimed to assess the cardioprotection afforded by acute administration of oleuropein and to evaluate the underlying mechanism. Importantly, since antioxidant therapies have yielded inconclusive results in attenuating IRI-induced damage on top of conditioning strategies, we investigated whether oleuropein could enhance or imbed the cardioprotective manifestation of ischemic postconditioning (PostC). Oleuropein, given during ischemia as a single intravenous bolus dose reduced the infarct size compared to the control group both in rabbits and mice subjected to myocardial IRI. None of the inhibitors of the cardioprotective pathways, l-NAME, wortmannin and AG490, influence its infarct size limiting effects. Combined oleuropein and PostC cause further limitation of infarct size in comparison with PostC alone in both animal models. Oleuropein did not inhibit the calcium induced mitochondrial permeability transition pore opening in isolated mitochondria and did not increase cGMP production. To provide further insights to the different cardioprotective mechanism of oleuropein, we sought to characterize its anti-inflammatory potential in vivo. Oleuropein, PostC and their combination reduce inflammatory monocytes infiltration into the heart and the circulating monocyte cell population. Oleuropein's mechanism of action involves a direct protective effect on cardiomyocytes since it significantly increased their viability following simulated IRI as compared to non-treated cells. Οleuropein confers additive cardioprotection on top of PostC, via increasing the expression of the transcription factor Nrf-2 and its downstream targets in vivo. In conclusion, acute oleuropein administration during ischemia in combination with PostC provides robust and synergistic cardioprotection in experimental models of IRI by inducing antioxidant defense genes through Nrf-2 axis and independently of the classic cardioprotective signaling pathways (RISK, cGMP/PKG, SAFE).

Phytochemical analysis of olive flowers' hydroalcoholic extract and in vitro evaluation of tyrosinase, elastase and collagenase inhibition activity.

Olea europaea L. is historically one of the most important trees of the Mediterranean countries. Increasing scientific interest regarding its fruits, leaves and olive oil has led to the elucidation of several phytochemical and biological characteristics. However, the phytochemical and biological studies regarding olive flowers remain limited. The aim of the present study was the phytochemical characterization of olive flowers' hydroalcoholic extract from Greek variety Lianolia, the effective isolation of the major secondary metabolites and evaluation of their inhibition activity against tyrosinase, elastase and collagenase. UPLC-HRMS/MS analysis was used to investigate the chemical composition of hydroalcoholic extract resulting in the identification of sixty-three secondary metabolites witch mainly belong to phenilethanoids, triterpenoids, flavonoids and secoiridoids. The orthogonial combination of Centrifugal Partition Chromatography and preparative HPLC in the same purification process led to the isolation of nine major compounds of the extract including two triterpenic acids, two flavonoid glycosides and five secoiridoid derivatives. From them, oleofloside A and oleofloside B are new natural products. Although, the hydroalcoholic extract and isolated secoiridoids exhibited weak or no inhibition activity towards tyrosinase and elastase, they exhibit remarkable anti-collagenase activity with 2΄-ethoxyoleuropein being the most active compound.

Hyperlipidaemia and cardioprotection: Animal models for translational studies.

Hyperlipidaemia is a well-established risk factor for cardiovascular diseases and therefore, many animal model have been developed to mimic the human abnormal elevation of blood lipid levels. In parallel, extensive research for the alleviation of ischaemia/reperfusion injury has revealed that hyperlipidaemia is a major co-morbidity that attenuates the cardioprotective effect of conditioning strategies (preconditioning, postconditioning and remote conditioning) and that of pharmacological interventions by interfering with cardioprotective signalling pathways. In the present review article, we summarize the existing data on animal models of hypercholesterolaemia (total, low density and HDL abnormalities) and hypertriglyceridaemia used in ischaemia/reperfusion injury and protection from it. We also provide recommendations on preclinical animal models to be used for translations of the cardioprotective strategies into clinical practice. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

Investigating and re-evaluating the role of glycogen synthase kinase 3 beta kinase as a molecular target for cardioprotection by using novel pharmacological inhibitors.

AIMS: Glycogen synthase kinase 3 beta (GSK3ß) link with the mitochondrial Permeability Transition Pore (mPTP) in cardioprotection is debated. We investigated the role of GSK3ß in ischaemia (I)/reperfusion (R) injury using pharmacological tools. METHODS AND RESULTS: Infarct size using the GSK3ß inhibitor BIO (6-bromoindirubin-3'-oxime) and several novel analogues (MLS2776-MLS2779) was determined in anaesthetized rabbits and mice. In myocardial tissue GSK3ß inhibition and the specificity of the compounds was tested. The mechanism of protection focused on autophagy-related proteins. GSK3ß localization was determined in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from Langendorff-perfused murine hearts (30'I/10'R or normoxic conditions). Calcium retention capacity (CRC) was determined in mitochondria after administration of the inhibitors in mice and in vitro. The effects of the inhibitors on mitochondrial respiration, reactive oxygen species (ROS) formation, ATP production, or hydrolysis were measured in SSM at baseline. Cyclosporine A (CsA) was co-administered with the inhibitors to address putative additive cardioprotective effects. Rabbits and mice treated with MLS compounds had smaller infarct size compared with control. In rabbits, MLS2776 and MLS2778 possessed greater infarct-sparing effects than BIO. GSK3ß inhibition was confirmed at the 10th min and 2 h of reperfusion, while up-regulation of autophagy-related proteins was evident at late reperfusion. The mitochondrial amount of GSK3ß was similar in normoxic SSM and IFM and was not altered by I/R. The inhibitors did not affect CRC or respiration, ROS and ATP production/hydrolysis at baseline. The co-administration of CsA ensured that cardioprotection was CypD-independent. CONCLUSION: Pharmacological inhibition of GSK3ß attenuates infarct size beyond mPTP inhibition.

Empagliflozin Limits Myocardial Infarction in Vivo and Cell Death in Vitro: Role of STAT3, Mitochondria, and Redox Aspects.

Empagliflozin (EMPA), a drug approved for type 2 diabetes management, reduced cardiovascular death but is unknown if it reduces myocardial infarction. We sought to investigate: (i) the effect of EMPA on myocardial function and infarct size after ischemia/reperfusion in mice fed with western diet (WD), (ii) the underlying signaling pathways, (iii) its effects on cell survival in rat embryonic-heart-derived cardiomyoblasts (H9C2) and endothelial cells (ECs). To facilitate the aforementioned aims, mice were initially randomized in Control and EMPA groups and were subjected to 30 min ischemia and 2 h reperfusion. EMPA reduced body weight, blood glucose levels, and mean arterial pressure. Cholesterol, triglyceride, and AGEs remained unchanged. Left ventricular fractional shortening was improved (43.97 ± 0.92 vs. 40.75 ± 0.61%) and infarct size reduced (33.2 ± 0.01 vs. 17.6 ± 0.02%). In a second series of experiments, mice were subjected to the above interventions up to the 10th min of reperfusion and myocardial biopsies were obtained for assessment of the signaling cascade. STAT3 was increased in parallel with reduced levels of malondialdehyde (MDA) and reduced expression of myocardial iNOS and interleukin-6. Cell viability and ATP content were increased in H9C2 and in ECs. While, STAT3 phosphorylation is known to bestow infarct sparing properties through interaction with mitochondria, we observed that EMPA did not directly alter the mitochondrial calcium retention capacity (CRC); therefore, its effect in reducing myocardial infarction is STAT3 dependent. In conclusion, EMPA improves myocardial function and reduces infarct size as well as improves redox regulation by decreasing iNOS expression and subsequently lipid peroxidation as shown by its surrogate marker MDA. The mechanisms of action implicate the activation of STAT3 anti-oxidant and anti-inflammatory properties.