Coronary microvascular injury in myocardial infarction: perception and knowledge for mitochondrial quality control.

Endothelial cells (ECs) constitute the innermost layer in all blood vessels to maintain the structural integrity and microcirculation function for coronary microvasculature. Impaired endothelial function is demonstrated in various cardiovascular diseases including myocardial infarction (MI), which is featured by reduced myocardial blood flow as a result of epicardial coronary obstruction, thrombogenesis, and inflammation. In this context, understanding the cellular and molecular mechanisms governing the function of coronary ECs is essential for the early diagnosis and optimal treatment of MI. Although ECs contain relatively fewer mitochondria compared with cardiomyocytes, they function as key sensors of environmental and cellular stress, in the regulation of EC viability, structural integrity and function. Mitochondrial quality control (MQC) machineries respond to a broad array of stress stimuli to regulate fission, fusion, mitophagy and biogenesis in mitochondria. Impaired MQC is a cardinal feature of EC injury and dysfunction. Hence, medications modulating MQC mechanisms are considered as promising novel therapeutic options in MI. Here in this review, we provide updated insights into the key role of MQC mechanisms in coronary ECs and microvascular dysfunction in MI. We also discussed the option of MQC as a novel therapeutic target to delay, reverse or repair coronary microvascular damage in MI. Contemporary available MQC-targeted therapies with potential clinical benefits to alleviate coronary microvascular injury during MI are also summarized.

Ataxia Telangiectasia Mutated Protein Kinase: A Potential Master Puppeteer of Oxidative Stress-Induced Metabolic Recycling.

Ataxia Telangiectasia Mutated protein kinase (ATM) has recently come to the fore as a regulatory protein fulfilling many roles in the fine balancing act of metabolic homeostasis. Best known for its role as a transducer of DNA damage repair, the activity of ATM in the cytosol is enjoying increasing attention, where it plays a central role in general cellular recycling (macroautophagy) as well as the targeted clearance (selective autophagy) of damaged mitochondria and peroxisomes in response to oxidative stress, independently of the DNA damage response. The importance of ATM activation by oxidative stress has also recently been highlighted in the clearance of protein aggregates, where the expression of a functional ATM construct that cannot be activated by oxidative stress resulted in widespread accumulation of protein aggregates. This review will discuss the role of ATM in general autophagy, mitophagy, and pexophagy as well as aggrephagy and crosstalk between oxidative stress as an activator of ATM and its potential role as a master regulator of these processes.

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.

Mitochondrial Oxidative Phosphorylation Function and Mitophagy in Ischaemic/Reperfused Hearts from Control and High-Fat Diet Rats: Effects of Long-Term Melatonin Treatment.

PURPOSE: Oxidative stress causes mitochondrial dysfunction in myocardial ischaemia/reperfusion (I/R) as well as in obesity. Mitochondrial depolarization triggers mitophagy to degrade damaged mitochondria, a process important for quality control. The aims of this study were to evaluate (i) the effect of I/R on mitochondrial oxidative phosphorylation and its temporal relationship with mitophagy in hearts from obese rats and their age-matched controls, and (ii) the role of oxidative stress in these processes using melatonin, a free radical scavenger. METHODS: Male Wistar rats were divided into 4 groups: control (normal diet ± melatonin) and high-fat sucrose diet (HFSD ± melatonin). Rats received melatonin orally (10 mg/kg/day). After 16 weeks, hearts were removed and subjected to 40-min stabilization, and 25-min global ischaemia/10-min reperfusion for preparation of mitochondria. Mitochondrial oxidative phosphorylation was measured polarographically. Western blotting was used for evaluation of PINK1, Parkin, p62/SQSTM1 (p62) and TOM 70. Infarct size was measured using tetrazolium staining. RESULTS: Ischaemia and reperfusion respectively reduced and increased mitochondrial QO2 (state 3) and the ox-phos rate in both control and HFSD mitochondria, showing no major changes between the groups, while melatonin pretreatment had little effect. p62 as indicator of mitophagic flux showed up- and downregulation of mitophagy by ischaemia and reperfusion respectively, with melatonin having no significant effect. Melatonin treatment caused a significant reduction in infarct size in hearts from both control and diet groups. CONCLUSIONS: The results suggest that I/R (i) affects mitochondria from control and HFSD hearts similarly and (ii) melatonin-induced cardioprotection is not associated with reversal of mitochondrial dysfunction or changes in the PINK1/Parkin pathway.

Mitochondrial oxidative phosphorylation and mitophagy in myocardial ischaemia/reperfusion: effects of chloroquine.

AIM: The aim of this study was to evaluate the temporal relationship between mitochondrial oxidative phosphorylation and mitophagy in rat hearts subjected to ischaemia/reperfusion. Measurements were made at specific points during the experimental protocol (snapshot approach) and by assessments of mitophagic flux, using chloroquine pre-treatment. METHODS: Isolated working rat hearts were subjected to 25 or 30 minutes of global ischaemia/10 minutes of reperfusion. Half of each group received chloroquine (10 mg/kg, intraperitoneally) one hour before experimentation. Mitochondria were isolated after stabilisation, ischaemia and reperfusion, and oxidative phosphorylation was measured polarographically. Mitochondrial mitophagy markers were detected by Western blot analysis. RESULTS: Mitochondrial oxygen uptake (state 3) and oxidative phosphorylation rate were reduced by ischaemia and increased by reperfusion. Chloroquine pre-treatment increased both parameters. Using a snapshot approach, exposure to ischaemia ± reperfusion had little effect on mitochondrial PINK1, Parkin and p62/SQSTM1 expression. Ischaemia reduced Rab9 expression, and reperfusion upregulated the phosphor DRP1, phosphor/total DRP1 ratio and Rab9 levels. Chloroquine significantly reduced PINK1, p62/SQSTM1, Rab9 and particularly Parkin expression during reperfusion, without an effect on mitochondrial total and phospho DRP1 levels. CONCLUSIONS: Ischaemia/reperfusion-induced changes in mitochondrial oxidative phosphorylation function occurred concomitantly with changes in mitophagic flux. Pre-treatment with chloroquine profoundly affected mitochondrial function as well as the pattern of mitophagy during ischaemia/reperfusion.

Corrigendum to "The impact of sugar-sweetened beverage intake on rat cardiac function" [Heliyon 5 (3) (March 2019) e01357].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e01357.].

The impact of sugar-sweetened beverage intake on rat cardiac function.

AIMS: Although there is evidence linking sugar-sweetened beverage (SSB) intake with the development of cardio-metabolic diseases, the underlying mechanisms remain unclear. The current study therefore evaluated the effects of SSB consumption by establishing a unique in-house in vivo experimental model. MAIN METHODS: Male Wistar rats were divided into two groups: a) one consuming a popular local SSB (SSB- Jive), and b) a control group (Control-water) for a period of three and six months (n = 6 per group), respectively. Rats were gavaged on a daily basis with an experimental dosage amounting to half a glass per day (in human terms) (SSB vs. water). Cardiac function was assessed at baseline (echocardiography) and following ex vivo ischemia-reperfusion of the isolated perfused working rat heart. Oral glucose tolerance tests and mitochondrial respiratory analyses were also performed. In addition, the role of non-oxidative glucose pathways (NOGPs), i.e. the polyol pathway, hexosamine biosynthetic pathway (HBP) and PKC were assessed. KEY FINDINGS: These data show that SSB intake: a) resulted in increased weight gain, but did not elicit major effects in terms of insulin resistance and cardiac function after three and six months, respectively; b) triggered myocardial NOGP activation after three months with a reversion after six months; and c) resulted in some impairment in mitochondrial respiratory capacity in response to fatty acid substrate supply after six months. SIGNIFICANCE: SSB intake did not result in cardiac dysfunction or insulin resistance. However, early changes at the molecular level may increase risk in the longer term.

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.

Treatment with a fixed dose combination antiretroviral therapy drug containing tenofovir, emtricitabine and efavirenz is associated with cardioprotection in high calorie diet-induced obese rats.

HIV-infection, certain antiretroviral drug classes, especially protease inhibitors (PI), and obesity are associated with increased ischaemic heart disease (IHD) risk. However, the effect of PI-free fixed dose combination (FDC) antiretroviral therapy (ART) on hearts exposed to ischaemia-reperfusion injury (I/R) is unknown, particularly in obesity. This is becoming relevant as World Health Organisation guidelines recommend a FDC ART containing (non-) nucleoside reverse transcriptase inhibitors (tenofovir (TDF), emtricitabine (FTC) and efavirenz (EFV)) as first-line HIV treatment. Additionally, obesity rates are rising in HIV-infected populations, not only in ART-experienced individuals, but also at the time of ART initiation, which may further increase the risk of IHD. Therefore, we investigated the effects of PI-free FDC ART in myocardial I/R-exposed hearts from obese rats. Obesity was induced in male wistar rats via a 16-week high calorie diet. At week 10, treatment with a FDC ART drug containing TDF/FTC/EFV was initiated. Biometric and metabolic parameters, as well as myocardial functional recovery and infract size (IS), and myocardial signalling proteins following I/R were assessed after 16 weeks. Obese rats presented with increased body and intraperitoneal fat mass, elevated triglyceride and TBARS levels, whilst the hearts responded to I/R with impaired functional performance and increased IS. The FDC ART treatment did not alter biometric and metabolic parameters in obese rats. In a novel finding, ART protected obese hearts against I/R as shown by improved functional performance and smaller IS vs. untreated obese hearts. Cardioprotection was underscored by increased myocardial phosphorylated endothelial nitric oxide synthase (eNOS) and reduced AMP-kinase levels. In conclusion, these results demonstrate for the first time, that 6-weeks treatment of obese rats with a FDC ART drug specifically containing TDF/FTC/EFV conferred cardioprotection against I/R. The FDC ART-induced cardioprotection was seemingly unrelated to metabolic changes, but rather due to direct cardiac mechanisms including the up-regulation of myocardial eNOS.

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.

Role of melatonin in glucose uptake by cardiomyocytes from insulin-resistant Wistar rats.

AIM: Melatonin supplementation reduces insulin resistance and protects the heart in obese rats. However, its role in myocardial glucose uptake remains unknown. This study investigated the effect of short-term melatonin treatment on glucose uptake by cardiomyocytes isolated from obese and insulin-resistant rats. METHODS: Cardiomyocytes were isolated from obese rats fed a high-calorie diet for 16 to 23 weeks, their age-matched controls, as well as young control rats aged four to eight weeks. After incubation with melatonin with or without insulin, glucose uptake was initiated by the addition of 2-deoxy-D- [3H] glucose and measured after 30 minutes. Additional control and obese rats received melatonin in the drinking water (4 mg/kg/day) for the last six weeks of feeding (20 weeks) and glucose uptake was determined in isolated cardiomyocytes after incubation with insulin. Intraperitoneal glucose tolerance and biometric parameters were also measured. RESULTS: Obese rats (fed for more than 20 weeks) developed glucose intolerance. Cardiomyocytes isolated from these obese rats had a reduced response to insulin-stimulated glucose uptake (ISGU) (p < 0.05). Melatonin administration in vitro had no effect on glucose uptake per se. However, it increased ISGU by cardiomyocytes from the young rats (p < 0.05), while having no effect on ISGU by cardiomyocytes from the older control and obese groups. Melatonin in vivo had no significant effect on glucose tolerance, but it increased basal (p < 0.05) and ISGU by cardiomyocytes from the obese rats (50.1 ± 1.7 vs 32.1 ± 5.1 pmol/mg protein/30 min, p < 0.01). CONCLUSION: These data suggest that short-term melatonin treatment in vivo but not in vitro improved glucose uptake and insulin responsiveness of cardiomyocytes in obesity and insulin-resistance states.

The Role of MKP-1 in Insulin-Induced Cardioprotection.

PURPOSE: The mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases that inactivate MAPKs by dephosphorylation. Impairment of MKP-1 expression in insulin resistance has been suggested to affect the cardioprotective properties of insulin. We hypothesized that manipulation of its activity during myocardial ischaemia/reperfusion of control as well as insulin-resistant rats may affect the outcome. METHODS: Hearts from 16 week dietary induced obese Wistar rats and their age matched controls were isolated, perfused in the working mode and subjected to 15 min global ischaemia / 30 min reperfusion or 35 min coronary artery ligation/ 60 min reperfusion. Hearts received insulin (1mIU/ml), a MKP-1 inhibitor (sanguinarine 2.5uM), or insulin + sanguinarine for 15 min pre- and 10 min post-ischaemia. Endpoints were functional recovery and infarct size. Hearts from control and experimental groups were freeze-clamped either immediately after removal from the animal (baseline) or at 10 min reperfusion after global ischaemia and Western blot analysis done for total and phosphorylated MKP-1. RESULTS: Insulin treatment significantly increased total work recovery while sanguinarine abolished the insulin-mediated protection. Insulin had no effect on infarct size while sanguinarine reduced infarct size. Insulin increased while sanguinarine attenuated phosphorylation of MKP-1 at 10 min reperfusion. CONCLUSION: Inhibition of MKP-1 with sanguinarine abolished the insulin-induced improvement in functional recovery, but reduced infarct size. Although the data suggest a role for this phosphatase in insulin-induced cardioprotection, the multiple downstream effects of insulin hamper interpretation of the data obtained. In addition, the effects of sanguinarine per se in myocardial ischaemia/reperfusion need to be further elucidated.

The significance of the washout period in preconditioning.

AIMS: Exposure of the heart to 5 min global ischaemia (I) followed by 5 min reperfusion (R) (ischaemic preconditioning, IPC) or transient Beta 2-adrenergic receptor (B2-AR) stimulation with formoterol (B2PC), followed by 5 min washout before index ischaemia, elicits cardioprotection against subsequent sustained ischaemia. As the washout period during preconditioning is essential for subsequent cardioprotection, the aim of this study was to investigate the involvement of protein kinase A (PKA), reactive oxygen species (ROS), extracellular signal-regulated kinase (ERK), PKB/Akt, p38 MAPK and c-jun N-terminal kinase (JNK) during this period. METHODS: Isolated perfused rat hearts were exposed to IPC (1x5min I / 5min R) or B2PC (1x5min Formoterol / 5min R) followed by 35 min regional ischaemia and reperfusion. Inhibitors for PKA (Rp-8CPT-cAMP)(16µM), ROS (NAC)(300µM), PKB (A-6730)(2.5µM), ERKp44/p42 (PD98,059)(10µM), p38MAPK (SB239063)(1µM) or JNK (SP600125)(10µM) were administered for 5 minutes before 5 minutes global ischaemia / 5 min reperfusion (IPC) or for 5 minutes before and during administration of formoterol (B2PC) prior to regional ischaemia, reperfusion and infarct size (IS) determination. Hearts exposed to B2PC or IPC were freeze-clamped during the washout period for Western blots analysis of PKB, ERKp44/p42, p38MAPK and JNK. RESULTS: The PKA blocker abolished both B2PC and IPC, while NAC significantly increased IS of IPC but not of B2PC. Western blot analysis showed that ERKp44/p42 and PKB activation during washout after B2PC compared to IPC was significantly increased. IPC compared to B2PC showed significant p38MAPK and JNKp54/p46 activation. PKB and ERK inhibition or p38MAPK and JNK inhibition during the washout period of B2PC and IPC respectively, significantly increased IS. CONCLUSION: PKA activation before regional ischaemia is a prerequisite for cardioprotection in both B2PC and IPC. However, ROS was crucial only in IPC. Kinase activation during the washout phase of IPC and B2PC, albeit different, affords the same cardioprotective response.

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.

Dependence of Cardiac Systolic Function on Elevated Fatty Acid Availability in Obese, Insulin-Resistant Rats.

BACKGROUND: Clinical data advocating an adverse effect of obesity on left ventricular (LV) systolic function independent of comorbidities is controversial. We hypothesized that in obesity with prediabetic insulin resistance, circulating fatty acids (FAs) become a valuable fuel source in the maintenance of normal systolic function. METHODS: Male Wistar rats were fed a high caloric diet for 32 weeks to induce obesity. Myocardial LV systolic function was assessed using echocardiography and isolated heart preparations. RESULTS: Aortic output was reduced in obese rat hearts over a range of filling pressures (for example: 15 cmH2O, obese: 32.6 ± 1.2 ml/min vs control: 46.2 ± 0.9 ml/min, P < .05) when perfused with glucose alone. Similarly, the slope of the LV end-systolic pressure-volume relationship decreased, and there was a right shift in the LV end-systolic stress-strain relationship as determined in Langendorff perfused, isovolumic rat heart preparations in the presence of isoproterenol (10(-8)M) (LV systolic stress-strain relationship and a reduced load-independent intrinsic systolic myocardial function, obese: 791 ± 62 g/cm(2) vs control: 1186 ± 74 g/cm(2), P < .01). The addition of insulin to the perfusion buffer improved aortic output, whereas the addition of FAs completely normalized aortic output. LV function was maintained in obese animals in vivo during an inotropic challenge. CONCLUSIONS: Elevated circulating FA levels may be important to maintain myocardial systolic function in the initial stages of obesity and insulin resistance.

ATM protein kinase signaling, type 2 diabetes and cardiovascular disease.

The ataxia-telangiectasia mutated (ATM) protein kinase is well known to play a significant role in the response to double stranded DNA breaks in the nucleus. Recently, it has become apparent that ATM is also involved in a large number of cytoplasmic processes and responses, some of which may contribute to metabolic and cardiovascular complications when disrupted. Due to its involvement in these processes, therapeutic activation of ATM could potentially be a novel approach for the prevention or treatment of cardiovascular disease. However, relatively little is currently known about the cardiovascular role of ATM. In this review, we highlight studies that have shed some light on the role of ATM in the cardiovascular context, namely in oxidative stress, atherosclerosis and metabolism, insulin resistance and cardiac remodeling.

High carbohydrate and high fat diets protect the heart against ischaemia/reperfusion injury.

BACKGROUND: Although obesity is still considered a risk factor in the development of cardiovascular disorders, recent studies suggested that it may also be associated with reduced morbidity and mortality, the so-called "obesity paradox". Experimental data on the impact of diabetes, obesity and insulin resistance on myocardial ischaemia/reperfusion injury are controversial. Similar conflicting data have been reported regarding the effects of ischaemic preconditioning on ischaemia/reperfusion injury in hearts from such animals. The aim of the present study was to evaluate the susceptibility to myocardial ischaemia/reperfusion damage in two models of diet-induced obesity as well as the effect of ischaemic and pharmacological preconditioning on such hearts. METHODS: Three groups of rats were fed with: (i) normal rat chow (controls) (ii) a sucrose-supplemented diet (DIO) (iii) a high fat diet (HFD). After 16 weeks, rats were sacrificed and isolated hearts perfused in the working mode and subjected to 35 min regional ischaemia/60 min reperfusion. Endpoints were infarct size and functional recovery. Infarct size was determined, using tetrazolium staining. Activation of PKB/Akt and ERKp44/p42 (RISK pathway) during early reperfusion was determined using Western blot. Statistical evaluation was done using ANOVA and the Bonferroni correction. RESULTS: Infarct sizes of non-preconditioned hearts from the two obese groups were significantly smaller than those of the age-matched controls. Ischaemic as well as pharmacological (beta-adrenergic) preconditioning with a beta2-adrenergic receptor agonist, formoterol, caused a significant reduction in infarct size of the controls, but were without effect on infarct size of hearts from the obese groups. However, ischaemic as well as beta-preconditioning caused an improvement in functional performance during reperfusion in all three groups. A clear-cut correlation between the reduction in infarct size and activation of ERKp44/p42 and PKB/Akt was not observed: The reduction in infarct size observed in the non-preconditioned hearts from the obese groups was not associated with activation of the RISK pathway. However, beta-adrenergic preconditioning caused a significant activation of ERKp44/p42, but not PKB/Akt, in all three groups. CONCLUSIONS: Relatively long-term administration of the two obesity-inducing diets resulted in cardioprotection against ischaemia/reperfusion damage. Further protection by preconditioning was, however, without effect on infarct size, while an improvement in functional recovery was observed.

Short-term melatonin consumption protects the heart of obese rats independent of body weight change and visceral adiposity.

Chronic melatonin treatment has been shown to prevent the harmful effects of diet-induced obesity and reduce myocardial susceptibility to ischaemia-reperfusion injury (IRI). However, the exact mechanism whereby it exerts its beneficial actions on the heart in obesity/insulin resistance remains unknown. Herein, we investigated the effects of relatively short-term melatonin treatment on the heart in a rat model of diet-induced obesity. Control and diet-induced obese Wistar rats (fed a high calorie diet for 20 wk) were each subdivided into three groups receiving drinking water with or without melatonin (4 mg/kg/day) for the last 6 or 3 wk of experimentation. A number of isolated hearts were perfused in the working mode, subjected to regional or global ischaemia-reperfusion; others were nonperfused. Metabolic parameters, myocardial infarct sizes (IFS), baseline and postischaemic activation of PKB/Akt, ERK42/44, GSK-3ß and STAT-3 were determined. Diet-induced obesity caused increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin and triglyceride (TG) levels with a concomitant cardiac hypertrophy, large postischaemic myocardial IFSs and a reduced cardiac output. Melatonin treatment (3 and 6 wk) decreased serum insulin levels and the HOMA index (P < 0.05) with no effect on weight gain (after 3 wk), visceral adiposity, serum TG and glucose levels. It increased serum adiponectin levels, reduced myocardial IFSs in both groups and activated baseline myocardial STAT-3 and PKB/Akt, ERK42/44 and GSK-3ß during reperfusion. Overall, short-term melatonin administration to obese/insulin resistant rats reduced insulin resistance and protected the heart against ex vivo myocardial IRI independently of body weight change and visceral adiposity.