Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice.

Atrial fibrillation (AF) is prevalent in patients with obesity and diabetes, and such patients often exhibit cardiac steatosis. Since the role of cardiac steatosis per se in the induction of AF has not been elucidated, the present study was designed to explore the relation between cardiac steatosis and AF. Transgenic (Tg) mice with cardiac-specific overexpression of perilipin 2 (PLIN2) were housed in the laboratory for more than 12 mo before the study. Electron microscopy of the atria of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and five- to ninefold greater atrial triacylglycerol (TAG) content compared with wild-type (WT) mice. Electrocardiography showed significantly longer RR intervals in PLIN2-Tg mice than in WT mice. Transesophageal electrical burst pacing resulted in significantly higher prevalence of sustained (>5 min) AF (69%) in PLIN2-Tg mice than in WT mice (24%), although it was comparable in younger (4-mo-old) mice. Connexin 43 (Cx43), a gap junction protein, was localized at the intercalated disks in WT atria but was heterogeneously distributed on the lateral side of cardiomyocytes in PLIN2-Tg atria. Langendorff-perfused hearts using the optical mapping technique showed slower and heterogeneous impulse propagation in PLIN2-Tg atria compared with WT atria. Cardiac overexpression of hormone-sensitive lipase in PLIN2-Tg mice resulted in atrial TAG depletion and amelioration of AF susceptibility. The results suggest that PLIN2-induced steatosis is associated with Cx43 remodeling, impaired conduction propagation, and higher incidence of AF in aged mice. Therapies targeting cardiac steatosis could be potentially beneficial against AF in patients with obesity or diabetes.

Molecular Mechanism of the Association Between Atrial Fibrillation and Heart Failure Includes Energy Metabolic Dysregulation Due to Mitochondrial Dysfunction.

BACKGROUND: Atrial fibrillation (AF) and heart failure (HF) commonly coexist, yet the molecular mechanisms of this association have not been determined. We hypothesized that an energy deficit due to mitochondrial dysfunction plays a significant role in pathogenic link between AF and HF. METHODS AND RESULTS: Myocardial energy metabolism and mitochondria were examined in atrial tissue samples from patients and mice (cardiac-specific LKB1 knock-out) with HF and/or AF. There was significant atrial energy (ATP) deficit in patients with HF (11.5±1.3 nmol/mg, n=10; vs without HF 17±3.8 nmol/mg, n=5, P = .032). AF was associated with further energy depletion (ATP 5.4±1.2 nmol/mg, n=9) in HF (P = .001) and metabolic stress (AMP/ATP 1.6±0.1 vs 0.7±0.2 in HF alone; P = .043). The left atrium demonstrated lower ATP than the right (P = .004). Mitochondrial dysfunction and remodeling caused ATP depletion with impaired oxidative phosphorylation complexes (succinate dehydrogenase and cytochrome c oxidase), increased reactive oxygen species, and mtDNA damage in mice and human atria with AF and HF. CONCLUSIONS: Molecular mechanisms of the association between HF and AF include an energy deficit due to mitochondrial dysfunction in atrial myocardium. Mitochondrial functional and structural remodeling in human and mouse atria is associated with energy metabolic dysregulation and oxidative stress that promote AF in HF and vice versa.

Chronic consumption of a high-fat diet rich in corn oil activates intrinsic cell death pathway and induces several ultrastructural changes in the atria of healthy and type 1 diabetic rat.

This study investigates the effect of chronic consumption of a high-fat diet rich in corn oil (CO-HFD) on atrial cells ultrastructure, antioxidant levels and markers of intrinsic cell death of both control and type 1 diabetes mellitus (T1DM)-induced rats. Adult male rats (10 rats/group) were divided into four groups: control fed standard diet (STD) (3.82 kcal/g, 9.4% fat), CO-HFD (5.4 kcal/g, 40% fat), T1DM fed STD, and T1DM + CO-HFD. CO-HFD and T1DM alone or in combination impaired systolic and diastolic functions of rats and significantly reduced levels of GSH and the activity of SOD, enhanced lipid peroxidation, increased protein levels of P53, Bax, cleaved caspase-3, and ANF and decreased levels of Bcl-2 in their atria. Concomitantly, atrial cells exhibited fragmentation of the myofibrils, disorganized mitochondria, decreased number of atrionatriuretic factor (ANF) granules, and loss of gap junctions accompanied by changes in capillary walls. Among all treatments, the severity of all these findings was more severe in T1DM and most profound in the atria of T1DM + CO-HFD. In conclusion, chronic consumption of CO-HFD by T1DM-induced rats elicits significant biochemical and ultrastructural damage to rat atrial cells accompanied by elevated oxidative stress and mitochondria-mediated cell death.

Does Levetiracetam Administration Prevent Cardiac Damage in Adulthood Rats Following Neonatal Hypoxia/Ischemia-Induced Brain Injury?

Cardiovascular abnormalities are widespread when a newborn is exposed to a hypoxic-ischemic injury in the neonatal period. Although the neuroprotective effects of levetiracetam (LEV) have been reported after hypoxia, the cardioprotective effects of LEV have not been documented. Therefore, we aimed to investigate whether levetiracetam (LEV) has a protective effect on cardiac-contractility and ultrastructure of heart muscle in rats exposed to hypoxia-ischemia (HI) during the neonatal period. A total of 49 seven-day-old rat pups were separated into four groups. For HI induction, a combination of right common carotid artery ligation with 8% oxygen in seven-day-old rat pups for 2 h was performed for saline, LEV100, and LEV200 groups. Just after hypoxia, LEV100 and LEV200 groups were administered with 100 mg/kg and 200 mg/kg of LEV, respectively. The arteries of rats in the control group were only detected; no ligation or hypoxia was performed. At the end of the 16th week after HI, cardiac mechanograms were recorded, and samples of tissue were explored by electronmicroscopy.While ventricular contractility in the control group was similar to LEV100, there were significant decreases in both saline and LEV200 groups (p < 0.05). Although ventricular contractile duration of the control and saline groups was found to be similar, durations in the LEV100 and LEV200 groups were significantly higher (p < 0.05). After HI, mitochondrial damage and ultrastructural deteriorative alterations in ventricles and atriums of the LEV-administered groups were significantly less severe than the saline group. The present study showed that neonatal HI caused long-term cardiac dysfunction and ultrastructural deteriorations in cardiac muscles. LEV administration just after HI might possess some protective effects against myocardial damage and contractility.

Taurine Reverses Atrial Structural Remodeling in Ach-Cacl2 Induced Atrial Fibrillation Rats.

Taurine has been reported to have anti-arrhythmia effects, but the anti-atrial fibrillation (AF) effects and its mechanism remain incompletely understood. In the present study, the therapy effects and partly mechanisms were investigated. AF animal model was established by intravenous administered with the mixture of acetylcholine (Ach) and CaCl2 (66 µg/mL + 10 mg/mL) (i.v.) for 7 days. The actions of taurine (99 mg/kg∙d, introgastric administration) on the levels of Hs-CRP, IL-6, TNF-α, MMP-9, AngII, the extent of the fibrosis and ultrastructural changes in left atrial were studied. The data showed that the serum levels of TNF-α, IL-6, AngII and the plasma levels of Hs-CRP and MMP-9 were significantly elevated in automatic recovery group relative to the control group (p < 0.01), which were all decreased by taurine administration (p < 0.01) similar to Verapamil treatment. Masson's trichrome staining of the left atrial tissue showed an obvious interstitial fibrosis in rats of automatic recovery group. The alteration could be reversed by additional taurine. Electron microscopy revealed that taurine administration could significantly alleviate the ultrastructural damage of atrial cells, and the effects were similar to the Verapamil treatment. In conclusion, the results suggested that taurine could inhibit the structural remodeling of AF in rats partly by decreasing the levels of inflammatory factors and profibrotic molecules, attenuating the extent of myocardial fibrosis and protecting the integrity of myocardial ultrastructure.

Ultrastructural Morphological Characterization of Right Atrial and Left Ventricular Rat Cardiomyocytes during Postreperfusion Period.

Immunocytochemistry and transmission electron microscopy were employed to examine the ultrastructural morphometric parameters of the left ventricular cardiomyocytes and right atrial secretory myocytes in rats during early and delayed postreperfusion periods. The revealed alterations in these cells are stereotypical, but differed by their severity, probably due to specific morphofunctional peculiarities of these heart structures.

Telocytes and putative stem cells in ageing human heart.

Tradition considers that mammalian heart consists of about 70% non-myocytes (interstitial cells) and 30% cardiomyocytes (CMs). Anyway, the presence of telocytes (TCs) has been overlooked, since they were described in 2010 (visit www.telocytes.com). Also, the number of cardiac stem cells (CSCs) has not accurately estimated in humans during ageing. We used electron microscopy to identify and estimate the number of cells in human atrial myocardium (appendages). Three age-related groups were studied: newborns (17 days-1 year), children (6-17 years) and adults (34-60 years). Morphometry was performed on low-magnification electron microscope images using computer-assisted technology. We found that interstitial area gradually increases with age from 31.3 ± 4.9% in newborns to 41 ± 5.2% in adults. Also, the number of blood capillaries (per mm(2) ) increased with several hundreds in children and adults versus newborns. CMs are the most numerous cells, representing 76% in newborns, 88% in children and 86% in adults. Images of CMs mitoses were seen in the 17-day newborns. Interestingly, no lipofuscin granules were found in CMs of human newborns and children. The percentage of cells that occupy interstitium were (depending on age): endothelial cells 52-62%; vascular smooth muscle cells and pericytes 22-28%, Schwann cells with nerve endings 6-7%, fibroblasts 3-10%, macrophages 1-8%, TCs about 1% and stem cells less than 1%. We cannot confirm the popular belief that cardiac fibroblasts are the most prevalent cell type in the heart and account for about 20% of myocardial volume. Numerically, TCs represent a small fraction of human cardiac interstitial cells, but because of their extensive telopodes, they achieve a 3D network that, for instance, supports CSCs. The myocardial (very) low capability to regenerate may be explained by the number of CSCs, which decreases fivefold by age (from 0.5% to 0.1% in newborns versus adults).

[QUANTITATIVE ANALYSIS OF BRAIN NATRIURETIC PEPTIDE OF CARDIAC MUSCLE CELLS IN EARLY POSTREPERFUSION PERIOD IN RATS].

The intensity of accumulation and release of brain natriuretic peptide (BNP) in right atrial cardiac muscle cells was investigated in rats after 5 and 60 min from the reperfusion start. Total ischemia was simulated by cardiovascular bundle compression according to V. G. Korpachev. Immunocytochemical identification of BNP in atrial myocyte granules was investigated in ultrathin cuts. We applied polyclonal antibodies to BNP. The calculation of granules (A- and B-types) with BNP was carried out in (38 x 38 µm) visual fields in transmission electronic microscope. The results were assessed using Mann-Whitney U-test (p < 0.05). After 5 min from the reperfusion start, the amount of the granules with BNP did not change compared to intact animals rate. On the 60th min of the post-reperfusion period was shown active accumulation and release of BNP; the amount of A-type granules increased by 134%, B-type granules increased by 210 % in comparison with intacy level. The results showed stimulating effect of ischemic and reperfusion factors on the processes of the brain natriuretic peptide synthesis and secretion in the early post-reperfusion period.

Innervation of sinoatrial nodal cardiomyocytes in mouse. A combined approach using immunofluorescent and electron microscopy.

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart preparations demonstrates a particularly dense and complex network of nerve fibres and cardiomyocytes which are positive to the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4-positive cardiomyocytes) in the sinoatrial node region and adjacent areas around the root of right cranial vein. The present study was designed to investigate the morphologic and histochemical pattern of nerve fibres and HCN4-positive cardiomyocytes using fluorescent techniques and/or electron microscopy. Adrenergic and cholinergic nerve fibres together with HCN4-positive cardiomyocytes were identified using primary antibodies for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and the HCN4 channel respectively. Amid HCN4-positive cardiomyocytes, fluorescence and electron microscopy data demonstrated a dense distribution of nerve fibres immunoreactive for ChAT and TH. In addition, novel electron microscopy data revealed that the mouse sinoatrial node contained exclusively unmyelinated nerve fibres, in which the majority of axons possess varicosities with clear mediatory vesicles that can be classified as cholinergic. Synapses occurred without any clear terminal connection with the effector cell, i.e. these synapes were of "en passant" type. In general, the morphologic pattern of innervation of mouse HCN4-positive cardiomyocytes identified using electron microscopy corresponds well to the dense network of nerve fibres demonstrated by fluorescent immunohistochemistry in mouse sinoatrial node and adjacent areas. The complex and extraordinarily dense innervation of HCN4-positive cardiomyocytes in mouse sinoatrial node underpins the importance of neural regulation for the cardiac conduction system. Based on the present observations, it is concluded that the occurrence of numerous nerve fibres nearby atrial cardiomyocytes serves as a novel reliable extracellular criterion for discrimination of SA nodal cardiomyocytes using electron microscopy.

Morphological and mechanical examination of the atrial 'intima'.

AIMS: To examine morphology and mechanical properties of the atrial 'intima', which we defined as the tissue interposed between atrial endocardium and myocardium, in patients without known cardiovascular disease. METHODS AND RESULTS: Post-mortem right and left atrial tissue was obtained from male infants (<1 year, n = 4), children (10-19 years, n = 4), and adults (58-69 years, n = 7). Using light microscopy and an ocular micrometer, atrial intimal (AIT) thickness was measured. Intimal collagen bundle thickness was measured using electron microscopy. Passive atrial wall stiffness was measured using a planar biaxial testing device. Among infants, left AIT (0.2 ± 0.2 mm) and right (0.2 ± 0.1 mm) AIT were not significantly different (P = 0.84). Among children, left AIT (0.6 ± 0.2 mm) was significantly greater than right (0.2 ± 0.1 mm) AIT (P = 0.03), and left AIT was marginally greater than in infants (P = 0.07). Among adults, with the exception of the appendage region, left AIT (1.0 ± 0.2 mm) was markedly greater than right AIT (0.3 ± 0.1 mm; P < 0.05), and left AIT was significantly greater than that in other age groups (P < 0.05). There were no differences in right AIT among age groups. Left intimal collagen bundle thickness was greater in adults (0.0512 ± 0.0056 µm) than infants (0.0432 ± 0.0071 µm) or children (0.0435 ± 0.0013 µm), and bundles were less organized. Wall stiffness was attributable primarily to the intima (1245 ± 132, vs. 260 ± 45 N/m(2) for the remaining atrial wall). CONCLUSION: The left atrial intima, but not the right, thickens with age, becomes more disorganized ultrastructurally, and is responsible for the majority of atrial wall stiffness.

[Ultrastructural estimation facilities of atrial cardiomyocyte secretory activity].

The wide experience in the ultrastructural study of myoendocrine cells of different animal species in normal and experimental conditions allows us to choose the optimal methodology that gives the most complete information about the state of intracellular secretory apparatus. It is revealed that the combined set of atrial myoendocrine cell qualitative and quantitative parameters allows defining the natriuretic regulatory system status, as well as it's acute and chronic responses to hemodynamic changes. The information value of such approach is illustrated by examples of the ontogenetic investigation in two rat lines: with normal arterial blood pressure and with inherited stress-induced arterial hypertension. The proposed methodology is quite sensitive and descriptive; so it is of high importance due to insufficiency of other universal, specific, and accurate methods for cardiac natriuretic peptides investigation.

[Cardiac natriuretic peptides and hypertension: experimental study].

The peculiarities of cardiac natriuretic peptide secretion have been investigated during ontogenesis using the hypertonic disease model in ISIAH rat line (with inherited stress induced arterial hypertension). The qualitative and quantitative ultrastructural investigations of right atrium myocardium revealed that the cardiac hormonal synthetic and secretory activities in ISIAH rats are higher as compared with normotensive even-aged rats from control group. Secretory hyperactivity of atrial myocytes in ISIAH rats during early ontogeny precedes the manifestations of hereditary hypertension. Natriuretic peptides present the hypotensive circuit of hemodynamic regulation during the whole ontogeny and the complementary chain in hypertension development.

[Age-related structural and functional characteristics of cardiac myoendocrine cells of rats in a normal state and with hereditary hypertension].

A qualitative and quantitative ultrastructural study of right atrial cardiomyocytes in WAG (normotensive control) and ISIAH (inherited stress-induced arterial hypertension) rats of different age (on day 18 of embryogenesis, on days 12 and 21 after birth, and at an age of 6 and 13 months) was performed. It was shown that, in embryos with an as yet incomplete atrial morphogenesis, secretory granules containing natriuretic peptides are actively formed, accumulated, and dissolved. In postnatal ontogeny, the secretory product is accumulated in atrial cells. In all ontogeny stages studied, the numerical density of secretory granules in the myoendocrine cells of hypertensive rats is greater and the qualitative composition of these granules is more diverse than in the control. It was established that, in atrial myocytes of ISIAH rats, the morphological signs of natriuretic peptide hypersecretion precede the development of genetically programmed high blood pressure. In adult hypertensive rats, hypertrophic and degenerative changes in myocytes are accompanied by excessive accumulation of secretory granules, some of which undergo intracellular degradation.

Upregulation of ß3-adrenergic receptors contributes to atrial structural remodeling in rapid pacing induced atrial fibrillation canines.

Accumulating evidence suggests that the adrenergic receptors (ARs) play an important role in cardiac diseases. The expression of ß3-AR has been recently demonstrated in atria, however, its role in atrial structural remodeling of atrial fibrillation (AF) is unclear. Therefore, the present study was designed to investigate the role of ß3-AR in atrial structural remodeling in AF and to clarify its possible mechanisms. Twenty-eight dogs were randomly divided into sham, pacing, ß3-AR agonist (BRL37344) and ß3-AR antagonist (L748337) groups. AF was induced by rapid atrial pacing at 600 beats per minute for 3 weeks and evaluated by determining the ultrastructure and function of atria. The expression of ß3-AR and p38 mitogen-activated protein kinase (MAPK) was examined by western blot, immunohistochemistry and real-time RT-PCR. Additionally, the extent of oxidative stress was tested. We found the atrial enlargement and dysfunction in pacing group. Moreover, atrial interstitial fibrosis, apoptosis and oxidative stress were increased and the levels of ß3-AR and phosphorylated p38 MAPK were increased after pacing. Activation of ß3-AR exacerbated the pathologic changes and oxidative stress, which were effectively inhibited by L748337. We concluded that ß3-AR was upregulated in paced atria, which contributed to oxidative stress and exacerbated atrial structural remodeling by regulating p38 MAPK. Our study provides novel insights into the pharmacological role of ß3-AR in AF.

Development of the venous pole of the heart in the frog Xenopus laevis: a morphological study with special focus on the development of the venoatrial connections.

The heart of lung-breathing vertebrates normally shows an asymmetric arrangement of its venoatrial connections along the left-right (L-R) body axis. The systemic venous tributaries empty into the right atrium while the pulmonary venous tributaries empty into the left atrium. The ways by which this asymmetry evolves from the originally symmetrically arranged embryonic venous heart pole are poorly defined. Here we document the development of the venous heart pole in Xenopus laevis (stages 40-46). We show that, prior to the appearance of the mouth of the common pulmonary vein (MCPV), the systemic venous tributaries empty into a bilaterally symmetric chamber (sinus venosus) that is demarcated from the developing atriums by a circular ridge of tissue (sinu-atrial ridge). A solitary MCPV appears during stage 41. From the time point of its first appearance onwards, the MCPV lies cranial to the sinu-atrial ridge and to the left of the developing interatrial septum and body midline. L-R lineage analysis shows that the interatrial septum and MCPV both derive from the left body half. The CPV, therefore, opens from the beginning into the future left atrium. The definitive venoatrial connections are established by the formation of a septal complex that divides the lumen of the venous heart pole into systemic and pulmonary venous flow pathways. This complex arises from the anlage of the interatrial septum and the left half of the sinu-atrial ridge.

[Morphogenesis and reaction to hypoxia of atrial myoendocrine cells in chick embryos (Gallus gallus)].

The ultrastructural and stereomorphometrical study of the right atrium of chick embryos at the 14th day of incubation has shown the cardiomyocytes to divide by mitosis and to be at different stages of differentiation. In the cytoplasm of some muscle cells we detected secretory granules that by their sizes and morphology can be classified into the forming, mature, and dissolved forms. By the 18th day of incubation most cardiomyocytes are already differentiated, and the number of secretory granules in them rises. Under conditions of hypoxia, after 3 days, in myoendocrine cells there are noted signs of accelerated release of the peptides synthesized earlier and accumulated in granules, while after one week - of enhancement of their synthesis. It can be concluded that in chick embryos, beginning from at least the 14th incubation day, the system of natriuretic heart peptides takes part in regulation of hemodynamics and of water-salt balance and responds to hypoxia.

Transverse tubules are a common feature in large mammalian atrial myocytes including human.

Transverse (t) tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca(2+) channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca(2+). T tubules are virtually absent in the atria of small mammals, and therefore Ca(2+) release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca(2+) release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 ± 0.03, 0.77 ± 0.03, 0.84 ± 0.03, and 1.56 ± 0.19 µm of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca(2+) transient and how this is perturbed in disease.

Molecular mechanism of atrial remodeling in patients with aging atrial fibrillation under the expression of microRNA-1 and microRNA-21.

We investigated the expression levels of microRNA-1 (miRNA-1) and microRNA-21 (miRNA-21) in the atrial tissues of patients with atrial fibrillation (AF) and the molecular mechanism of action in atrial remodeling. Patients with valvular heart disease were selected as the subjects. The ultrastructure, degree of myocardial fibrosis, apoptosis index (AI), expression of microRNA-1, expression of microRNA-21, and mRNA of TIMP-1, MMP-9, BCL-2, and Bax of patients were compared and analyzed in each group. The results showed that the degree of myocardial fibrosis and AI in patients with AF of the same age were extremely higher than those of patients with sinus rhythm (SR) (P < 0.01). Patients with AF showed much higher messenger RNA (mRNA) levels of mini-mental Parkinson 9 (MMP9) and Bax and obvious lover mRNA levels of tissue inhibitors of metalloproteinase 1 (TIMP-1) and Bcl-2 compared with patients with sinus rhythm (SR) (P < 0.05). It indicated that the expression of miRNA-1 in the AF patients was markedly down-regulated, and that miRNA-21 was up-regulated. This showed that microRNA-1 and microRNA-21 were involved in the molecular remodeling of aging AF through the regulation of primers, which would provide a critical basis for diagnosis and treatment of aging AF.

Role of AMPK in the protective effects exerted by triiodothyronine in ischemic-reperfused myocardium.

Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular 'fuel-gauge' enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3ß phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.