Cardioprotective Function of Green Rooibos (Aspalathus linearis) Extract Supplementation in Ex Vivo Ischemic Prediabetic Rat Hearts.

Diabetic patients develop ischemic heart disease and strokes more readily. Following an ischemic event, restoration of blood flow increases oxidative stress resulting in myocardial damage, termed ischemia/reperfusion injury. Aspalathus linearis (rooibos), rich in the antioxidant phenolic compound aspalathin, has been implicated as cardioprotective against ischemia/reperfusion injury with undefined mechanism in control rats. Primarily, the therapeutic potential of Afriplex green rooibos extract to prevent ischemia/reperfusion injury in cardiovascular disease-compromised rats was investigated. Additionally, Afriplex Green rooibos extract's cardioprotective signaling on metabolic markers and stress markers was determined using western blotting. Three hundred male Wistar rats received either 16-wk standard diet or high-caloric diet. During the final 6 wk, half received 60 mg/kg/day Afriplex green rooibos extract, containing 12.48% aspalathin. High-caloric diet increased body weight, body fat, fasting serum triglycerides, and homeostatic model assessment of insulin resistance - indicative of prediabetes. High-caloric diet rats had increased heart mass, infarct size, and decreased heart function. Afriplex green rooibos extract treatment for 6 wk lowered pre-ischemic heart rate, reduced infarct size, and improved heart function pre- and post-ischemia, without significantly affecting biometric parameters. Stabilized high-caloric diet hearts had decreased insulin independence via adenosine monophosphate activated kinase and increased inflammation (p38 mitogen-activated protein kinase), whereas Afriplex green rooibos extract treatment decreased insulin dependence (protein kinase B) and conferred anti-inflammatory effect. After 20 min ischemia, high-caloric diet hearts had upregulated ataxia-telangiectasia mutated kinase decreased insulin independence, and downregulated insulin dependence and glycogen synthase kinase 3 ß inhibition. In contrast, Afriplex green rooibos extract supplementation downregulated insulin independence and inhibited extracellular signal-regulated kinase 1 and 2. During reperfusion, all protective signaling was decreased in high-caloric diet, while Afriplex green rooibos extract supplementation reduced oxidative stress (c-Jun N-terminal kinases 1 and 2) and inflammation. Taken together, Afriplex green rooibos extract supplementation for 6 wk preconditioned cardiovascular disease-compromised rat hearts against ischemia/reperfusion injury by lowering inflammation, oxidative stress, and heart rate.

Long-chain free fatty acids inhibit ischaemic preconditioning of the isolated rat heart.

We recently reported that non-preconditioned hearts from diet-induced obese rats showed, compared to controls, a significant reduction in infarct size after ischaemia/reperfusion, whilst ischaemic preconditioning was without effect. In view of the high circulating FFA concentration in diet rats, the aims of the present study were to: (i) compare the effect of palmitate on the preconditioning potential of hearts from age-matched controls and diet rats (ii) elucidate the effects of substrate manipulation on ischaemic preconditioning. Substrate manipulation was done with dichloroacetate (DCA), which enhances glucose oxidation and decreases fatty acid oxidation. Isolated hearts from diet rats, age-matched controls or young rats, were perfused in the working mode using the following substrates: glucose (10 mM); palmitate (1.2 mM)/3% albumin) + glucose (10 mM) (HiFA + G); palmitate (1.2 mM/3% albumin) (HiFA); palmitate (0.4 mM/3% albumin) + glucose(10 mM) (LoFA + G); palmitate (0.4 mM/3% albumin) (LoFA). Hearts were preconditioned with 3 × 5 min ischaemia/reperfusion, followed by 35 min coronary ligation and 60 min reperfusion for infarct size determination (tetrazolium method) or 20 min global ischaemia/10 or 30 min reperfusion for Western blotting (ERKp44/42, PKB/Akt). Preconditioning of glucose-perfused hearts from age-matched control (but not diet) rats reduced infarct size, activated ERKp44/42 and PKB/Akt and improved functional recovery during reperfusion (ii) perfusion with HiFA + G abolished preconditioning and activation of ERKp44/42 (iii) DCA pretreatment largely reversed the harmful effects of HiFA. Hearts from non-preconditioned diet rats exhibited smaller infarcts, but could not be preconditioned, regardless of the substrate. Similar results were obtained upon substrate manipulation of hearts from young rats. Abolishment of preconditioning in diet rats may be due to altered myocardial metabolic patterns resulting from changes in circulating FA. The harmful effects of HiFA were attenuated by stimulation of glycolysis and inhibition of FA oxidation.

Cardioprotective Effects of Beta3-Adrenergic Receptor (ß3-AR) Pre-, Per-, and Post-treatment in Ischemia-Reperfusion.

The ß3-AR (beta3-adrenergic receptor) is resistant to short-term agonist-promoted desensitization and delivers a constant intracellular signal, making this receptor a potential target in acute myocardial infarction (AMI). AIM: To investigate whether selective modulation of ß3-AR prior to or during ischemia and/or reperfusion may be cardioprotective. METHODS: Isolated perfused rat hearts were exposed to 35-min regional ischemia (RI) and 60-min reperfusion. The ß3-AR agonist (BRL37344, 1 µM) or antagonist (SR59230A, 0.1 µM) was applied: (i) before RI (PreT) or (ii) last 10 min of RI (PerT) or (iii) onset of reperfusion (PostT) or (iv) during both PerT+PostT. Nitric oxide (NO) involvement was assessed, using the NOS inhibitor, L-NAME (50 µM). Endpoints were functional recovery, infarct size (IS), cGMP levels, and Western blot analysis of eNOS, ERKp44/p42, PKB/Akt, and glycogen synthase kinase-3ß (GSK-3ß). RESULTS: Selective treatment with BRL significantly reduced IS. L-NAME abolished BRL-mediated cardioprotection. BRL (PreT) and BRL (PerT) significantly increased cGMP levels (which were reduced by L-NAME) and PKB/Akt phosphorylation. BRL (PostT) produced significantly increased cGMP levels, PKB/Akt, and ERKp44/p42 phosphorylation. BRL (PerT+PostT) caused significant eNOS, PKB/Akt, ERKp44/p42, and GSK-3ß phosphorylation. CONCLUSION: ß3-AR activation by BRL37344 induced significant cardioprotection regardless of the experimental protocol. However, the pattern of intracellular signaling with each BRL treatment differed to some degree and suggests the involvement of cGMP, eNOS, ERK, GSK-3ß, and particularly PKB/Akt activation. The data also suggest that clinical application of ß3-AR stimulation should preferably be incorporated during late ischemia or/and early reperfusion.

Melatonin improves cardiac and mitochondrial function during doxorubicin-induced cardiotoxicity: A possible role for peroxisome proliferator-activated receptor gamma coactivator 1-alpha and sirtuin activity?

Mitochondrial dysfunction is a central element in the development of doxorubicin (DXR)-induced cardiotoxicity. In this context, melatonin is known to influence mitochondrial homeostasis and function. This study aimed to investigate the effects of melatonin on cardiac function, tumor growth, mitochondrial fission and fusion, PGC1-α and sirtuin activity in an acute model of DXR-induced cardiotoxicity. During the in vitro study, H9c2 rat cardiomyoblasts were pre-treated with melatonin (10 µM, 24 h) followed by DXR exposure (3 µM, 24 h). Following treatment, cellular ATP levels and mitochondrial morphology were assessed. In the in vivo study, female Sprague Dawley rats (16 weeks old), were inoculated with a LA7 rat mammary tumor cell line and tumors were measure daily. Animals were injected with DXR (3 × 4 mg/kg) and/or received melatonin (6 mg/kg) for 14 days in their drinking water. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. DXR treatment increased cell death and mitochondrial fission which were reduced with melatonin treatment. Cardiac output increased in rats treated with DXR + melatonin compared to DXR-treated rats. Tumor volumes was significantly reduced in DXR + melatonin-treated rats on Day 8 in comparison to DXR-treated rats. Furthermore, DXR + melatonin treatment increased cellular ATP levels, PGC1-α and SIRT1 expression which was attenuated by DXR treatment. These results indicate that melatonin treatment confers a dual cardio-protective and oncostatic effect by improving mitochondrial function and cardiac function whilst simultaneously retarding tumor growth during DXR-induced cardiotoxicity.

Melatonin and cardioprotection against ischaemia/reperfusion injury: What's new? A review.

Melatonin is a pleiotropic hormone with several functions. It binds to specific receptors and to a number of cytosolic proteins, activating a vast array of signalling pathways. Its potential to protect the heart against ischaemia/reperfusion damage has attracted much attention, particularly in view of its possible clinical applications. This review will focus mainly on the possible signalling pathways involved in melatonin-induced cardioprotection. In particular, the role of the melatonin receptors and events downstream of receptor activation, for example, the reperfusion injury salvage kinase (RISK), survivor activating factor enhancement (SAFE) and Notch pathways, the sirtuins, nuclear factor E2-related factor 2 (Nrf2) and translocases in the outer membrane (TOM70) will be discussed. Particular attention is given to the role of the mitochondrion in melatonin-induced cardioprotection. In addition, a brief overview will be given regarding the status quo of the clinical application of melatonin in humans.

The differential effects of FTY720 on functional recovery and infarct size following myocardial ischaemia/reperfusion.

AIM: The aim of this study was to evaluate the effects of the sphingosine analogue, FTY720 (Fingolimod), on the outcomes of myocardial ischaemia/reperfusion (I/R) injury. METHODS: Two concentrations of FTY720 (1 or 2.5 µM were administered either prior to (PreFTY), or following (PostFTY) 20 minutes' global (GI) or 35 minutes' regional ischaemia (RI) in the isolated, perfused, working rat heart. Functional recovery during reperfusion was assessed following both models of ischaemia, while infarct size (IFS) was determined following RI. RESULTS: FTY720 at 1 µM exerted no effect on functional recovery, while 2.5 µM significantly impaired aortic output (AO) recovery when administered prior to GI (% recovery: control: 33.88 ± 6.12% vs PreFTY: 0%, n = 6-10; p < 0.001), as well as before and after RI ( % recovery: control: 27.86 ± 13.22% vs PreFTY: 0.62% ; p < 0.05; and PostFTY: 2.08%; p = 0.0585, n = 6). FTY720 at 1 µM administered during reperfusion reduced IFS (% of area at risk (AAR): control: 39.89 ± 3.93% vs PostFTY: 26.56 ± 4.32%, n = 6-8; p < 0.05), while 2.5 µM FTY720 reduced IFS irrespective of the time of administration ( % of AAR: control: 39.89 ± 3.93% vs PreFTY: 29.97 ± 1.03% ; and PostFTY: 30.45 ± 2.16%, n = 6; p < 0.05). CONCLUSION: FTY720 exerted divergent outcomes on function and tissue survival depending on the concentration administered, as well as the timing of administration.

Mitochondrial catastrophe during doxorubicin-induced cardiotoxicity: a review of the protective role of melatonin.

Anthracyclines, such as doxorubicin, are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side effects such as cardiotoxicity. Doxorubicin-induced cardiotoxicity is emerging as a critical issue among cancer survivors and is an area of much significance to the field of cardio-oncology. Abnormalities in mitochondrial functions such as defects in the respiratory chain, decreased adenosine triphosphate production, mitochondrial DNA damage, modulation of mitochondrial sirtuin activity and free radical formation have all been suggested as the primary causative factors in the pathogenesis of doxorubicin-induced cardiotoxicity. Melatonin is a potent antioxidant, is nontoxic, and has been shown to influence mitochondrial homeostasis and function. Although a number of studies support the mitochondrial protective role of melatonin, the exact mechanisms by which melatonin confers mitochondrial protection in the context of doxorubicin-induced cardiotoxicity remain to be elucidated. This review focuses on the role of melatonin on doxorubicin-induced bioenergetic failure, free radical generation, and cell death. A further aim is to highlight other mitochondrial parameters such as mitophagy, autophagy, mitochondrial fission and fusion, and mitochondrial sirtuin activity, which lack evidence to support the role of melatonin in the context of cardiotoxicity.

Effect of chronic CPT-1 inhibition on myocardial ischemia-reperfusion injury (I/R) in a model of diet-induced obesity.

PURPOSE: By increasing circulating free fatty acids and the rate of fatty acid oxidation, obesity decreases glucose oxidation and myocardial tolerance to ischemia. Partial inhibition of fatty acid oxidation may improve myocardial tolerance to ischemia/reperfusion (I/R) in obesity. We assessed the effects of oxfenicine treatment on post ischemic cardiac function and myocardial infarct size in obese rats. METHODS: Male Wistar rats were fed a control diet or a high calorie diet which resulted in diet induced obesity (DIO) for 16 weeks. Oxfenicine (200 mg/kg/day) was administered to control and DIO rats for the last 8 weeks. Isolated hearts were perfused and infarct size and post ischemic cardiac function was assessed after regional or global ischemia and reperfusion. Cardiac mitochondrial function was assessed and myocardial expression and activity of CPT-1 (carnitine palmitoyl transferase-1) and IRS-1 (insulin receptor substrate-1) was assessed using Western blot analysis. RESULTS: In the DIO rats, chronic oxfenicine treatment improved post ischemic cardiac function and reduced myocardial infarct size after I/R but had no effect on the cardiac mitochondrial respiration. Chronic oxfenicine treatment worsened post ischemic cardiac function, myocardial infarct size and basal mitochondrial respiration in control rat hearts. Basal respiratory control index (RCI) values, state 2 and state 4 respiration rates and ADP phosphorylation rates were compromised by oxfenicine treatment. CONCLUSION: Chronic oxfenicine treatment improved myocardial tolerance to I/R in the obese rat hearts but decreased myocardial tolerance to I/R in control rat hearts. This decreased tolerance to ischemia of oxfenicine treated controls was associated with adverse changes in basal and reoxygenation mitochondrial function. These changes were absent in oxfenicine treated hearts from obese rats.

Pre-treatment with a DPP-4 inhibitor is infarct sparing in hearts from obese, pre-diabetic rats.

Cardiovascular risk is closely associated with insulin resistance and type 2 diabetes. Therapy based on the actions of GLP-1 is currently seen as a novel approach to treat this disease. The aims of this study was therefore to use an animal model to determine whether (i) pre-treatment of obese, insulin resistant but pre-diabetic rats with a DPP4 inhibitor, PFK275-055, could protect the heart from ischaemia/reperfusion injury and (ii) the possible mechanisms involved in such protection. Obese, pre-diabetic rats (DIO) were treated for 4 weeks with 10 mg/kg/day of the DPP4 inhibitor PFK275-055. Ex vivo perfusion was used to subject hearts to ischaemia/reperfusion to determine infarct size, functional recovery and post-ischaemic activation of proteins associated with cardiac protection. Adult ventricular cardiomyocytes were isolated to determine insulin sensitivity. Other assessments included body weight, intra-peritoneal fat weight, insulin and GLP-1 levels as well as histological evaluation of the pancreata. Results showed that DIO animals had higher body mass and intra-peritoneal fat mass than chow-fed animals. They presented with elevated plasma insulin levels and lower GLP-1 levels. Treatment with the DPP4 inhibitor resulted in smaller infarct size development in hearts from DIO rats after ischaemia/reperfusion accompanied by activation of cardioprotective kinases. GLP-1 levels were elevated and plasma insulin levels lower after treatment. In addition, the beta-cell to alpha-cell ratio of the pancreas was improved. We conclude that treatment with PFK275-055 for 4 weeks protected the heart against ischaemia/reperfusion injury, elevated GLP-1 levels and improved metabolic control in obese, pre-diabetic rats.

Inhibition of myocardial apoptosis by ischaemic and beta-adrenergic preconditioning is dependent on p38 MAPK.

INTRODUCTION: Apoptosis occurring during ischaemia /reperfusion contributes independently to tissue damage, and involves activation of the stress-kinase, p38 MAPK during reperfusion. Ischaemic preconditioning (IPC) protects against ischaemia/reperfusion mediated necrosis and apoptosis. The role of p38 MAPK in the protective effect of preconditioning against apoptosis is unknown. Pharmacologic preconditioning with isoproterenol (beta-PC) also protects against necrosis, but it is not known whether it protects against apoptosis. AIM: The aim of the study was to investigate whether the protective effect of IPC against apoptosis is related to activation of p38 MAPK and whether beta-PC also protects against apoptosis. MATERIALS AND METHODS: Isolated perfused rat hearts were used to study the effect of ischaemia and reperfusion on apoptosis and infarct size. Ischaemic preconditioning was elicited by 3 x 5 min global ischaemia, and beta-PC by 5 min isoproterenol 10(-7) M. For infarct size hearts were subjected to regional ischaemia for 35 min followed by 120 min reperfusion. Infarct size was determined by the tetrazolium staining technique, and expressed as percentage of area at risk. For markers of apoptosis hearts were subjected to global ischaemia of 25 min plus 30 min reperfusion. Apoptosis was determined by Western blot using antibodies against caspase-3 and PARP. p38 MAPK activation was inhibited by SB203580 (1 microM) administration 10 min prior to commencing ischaemia, and bracketing the IPC and beta-PC preconditioning protocols. p38 MAPK was activated by administration of anisomycin (5 microM) 10 min prior to index ischaemia in one protocol, and 10 min during reperfusion in non-preconditioned as well as IPC and beta-PC hearts. Results were analysed using ANOVA and a Newman-Keuls post-hoc test. RESULTS: In the apoptosis model using global ischaemia, IPC and beta-PC both resulted in a significant decrease in p38 MAPK activation at the end of reperfusion when compared to non-preconditioned hearts. This was accompanied by a significant decrease in apoptosis as measured with both caspase-3 activation and PARP cleavage. Inhibiting p38 MAPK by administration of SB203580 10 min prior to ischaemia resulted in a significant reduction in both markers of apoptosis. Bracketing the triggering phase of either IPC or beta-PC with SB203580 resulted in attenuated p38 MAPK activation during reperfusion and did not abolish the protective effect of IPC or beta-PC against apoptosis. Activating p38 MAPK with anisomycin prior to ischaemia resulted in a reduction of markers of apoptosis, whereas activation of p38 MAPK with anisomycin during reperfusion did not exacerbate apoptosis in any groups, exept for an increase in PARP cleavage in IPC hearts. In the model of regional ischaemia, IPC and beta-PC reduced infarct size significantly, and to the same extent as inhibition of p38 MAPK by administration of SB203580 10 min prior to ischaemia. Bracketing the triggering phase of either IPC or beta-PC did not abolish the reduction in infarct size. Activating p38 MAPK during reperfusion was accompanied by an increase in infarct size only in IPC hearts, but not in beta-PC hearts. CONCLUSION: These results indicate that (1) Both IPC and beta-PC elicit protection against apoptosis and necrosis, (2) activation of p38 MAPK is not a trigger of preconditioning against apoptosis and necrosis and (3) activation of p38 MAPK during reperfusion increases necrosis only if ischaemia is used to precondition hearts, but not with pharmacologic preconditioning with isoproterenol.

The temporal relationship between p38 MAPK and HSP27 activation in ischaemic and pharmacological preconditioning.

An ischaemic preconditioning protocol and subsequent sustained ischaemia were characterized by activation and attenuation of p38 MAPK phosphorylation, respectively. However, the significance of events downstream of p38 MAPK needs investigation. Therefore the temporal relationship between phosphorylation of p38 MAPK and its downstream substrate HSP27 was studied during either an ischaemic or beta-adrenergic preconditioning protocol and during sustained ischaemia. Isolated rat hearts were preconditioned (with or without a p38 MAPK inhibitor, SB203580) with 1 x 5 min or 3 x 5 min global ischaemia or 5 min beta-adrenergic stimulation (10(-7) M isoproterenol), followed by 25 min sustained ischaemia and 30 min reperfusion. Hearts were freeze-clamped at different time intervals and fractionated to determine p38 MAPK and HSP27 phosphorylation, via Western blotting. Significant phosphorylation of cytosolic p38 MAPK and membrane (myo-fibrillar) HSP27 occurred at the end of the first preconditioning episode. However, p38 MAPK phosphorylation disappeared during subsequent preconditioning episodes, while HSP27 phosphorylation was maintained for the duration of the protocol. Similar changes in p38 MAPK and HSP27 occurred with 5 min beta-adrenergic preconditioning. After 25 min ischaemia, significant phosphorylation of cytosolic and membrane HSP27 was observed, while p38 MAPK phosphorylation was attenuated in ischaemic and beta-adrenergic preconditioned compared to non-preconditioned hearts. SB203580-induced abolishment of p38 MAPK and HSP27 phosphorylation during the triggering phase of both preconditioning protocols reversed the changes in these parameters seen after sustained ischaemia. The results suggest that p38 MAPK activation triggers HSP27 phosphorylation during both the preconditioning protocols and during sustained ischaemia. Protection of preconditioned hearts during sustained ischaemia was characterized by phosphorylation of both cytosolic and myofibrillar HSP27.

Nitric oxide triggers classic ischemic preconditioning.

The role of NO in the classic ischemic preconditioning phenomenon of the myocardium is not well defined, and was investigated by using the isolated perfused rat heart as a model. Hearts were preconditioned with 3 x 5 minute ischemia in the presence and absence of the NOS inhibitors L-NAME (50 microM) and L-NNA (50 microM), and the guanylyl cyclase inhibitor ODQ (20 microM). These inhibitors significantly attenuated the protective effect of preconditioning against 25-min global ischemia (as measured by functional recovery), specifically if administered during the triggering phase. Cyclic infusions (3 x 5 min) of the NO-donors SNAP (50 microM) and SNP (100 microM) elicited protection against both 25-min global or low-flow ischemia. Hearts preconditioned with NO donors displayed significantly superior functional reserve, if stimulated with adrenaline, compared to hearts preconditioned with ischemia. Although the NO donors SNAP and SNP both activated p38 MAPK during the preconditioning protocol, protection was accompanied by significantly decreased p38 MAPK activity during sustained ischemia, as was the case in ischemic preconditioning. We conclude that (1) NO is a trigger for classic preconditioning, (2) cGMP generation plays an important role in its protection, (3) attenuation of p38 MAPK during sustained ischemia accompanies NO preconditioning and may mediate cardiac protection, and (4) preconditioning with NO may be more advantageous than using ischemia.