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The aim of the research is to study the structure, innervation, and state of epicardial adipose tissue of the aortic-pulmonary region of the heart in rats at the age of 3–4 and 18–23 months using neural immunohistochemical markers. Using a complex of histological and immunohistochemical methods, different nervous apparatuses (ganglia, clusters of chromaffin cells, nerve trunks, nerve-fiber bundles, nerve plexuses, synaptic endings) with different mediators are detected in lobules of white and brown adipose tissue of the base of the rat heart. It is established that parasympathetic and sympathetic postganglionic nerve fibers are involved in the innervation of white and brown adipose tissue. They penetrate into the lobules as a part of Remak's cords of varicose axons along arterial vessels, form terminal synaptic plexuses of the en-passant type, and are involved in the innervation of adipocytes of both types of epicardial adipose tissue. It is established that PGP 9.5+ cholinergic terminal nerve fibers prevail over catecholaminergic ones in mature rats. In the process of aging, common neurodegenerative, involutive (desimatization), and destructive pathological changes are noted in the epicardial adipose tissue of the studied animals. [ABSTRACT FROM AUTHOR]

Cellular changes in different parts of the heart of Wistar rats at the age of 18–23 months are studied using histological methods of staining with toluidine blue, hematoxylin-eosin, and immunohistochemical reactions for PGP 9.5 protein, tyrosine hydroxylase (TH), and Iba-1 protein. Focal inflammatory infiltrates are found in the connective tissue of the heart base, near which PGP 9.5+ and TH+ plexuses consisting of parasympathetic and sympathetic nerve fibers are detected. In the area of the valvular heart apparatus and at the border between the fibrous ring and myocardium of the right atrium, the following pathological changes in nerve structures are observed: degeneration of nerve fibers and granular destruction of varicose axons of the terminal plexus. A close relationship is established between the axons of the terminal neural network and cells of inflammatory infiltrates and blood capillaries. The features of the localization of neurocellular inflammatory complexes consisting of nerve fibers, blood capillaries, and cells participating in the local inflammatory process (mast cells, macrophages, fibroblasts, and plasma cells) are described in various parts of the myocardium in old animals. The chronic nature of neurogenic inflammation is determined for the heart during aging. [ABSTRACT FROM AUTHOR]

Natural and chemically modified N-terminal galanin fragments (WTLNSAGYLLGPHA-OH (G1) and WTLNSAGYLLGPβAH-OH (G2), respectively) reduce functional and metabolic disturbances in the heart during experimental ischemia and reperfusion. The aim of this work was to examine whether these peptides and full-length rat galanin (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2, G3) decrease the formation of reactive oxygen species (ROS) and lipid peroxidation products in the heart with ischemia/reperfusion (I/R) injury. The peptides were synthesized by the automatic solid phase method using Fmoc technology. Their structure was identified by 1H-NMR spectroscopy and MALDI-TOF mass spectrometry. Experiments were performed on anaesthetized open-chest rats subjected to myocardial regional ischemia and reperfusion. The microdialysis method and the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap were used to monitor ROS content in the area at risk (AAR). Intravenous administration of G1, G2 or G3 after occlusion of coronary artery significantly decreased the content of DMPO-OH spin adduct in interstitium of the AAR during reperfusion compared to the control. These effects were accompanied by limitation of infarct size, reduction of the plasma activity of creatine kinase-MB and lactate dehydrogenase, and better preservation of the energy state of the AAR. In addition, peptides G2 and G3 significantly reduced the formation of thiobarbituric acid reactive substances in the AAR. The overall protective action of the peptides decreased in the order G2 > G3 > G1. The results suggest that pharmacological agonists of galanin receptors can be considered as promising agents to reduce oxidative stress in myocardial I/R injury. [ABSTRACT FROM AUTHOR]

Exposure to fine particulate matter (PM2.5) increased the risks of cardiovascular diseases. PM2.5-bound 1-nitropyrene (1-NP) and 9-nitroanthracene (9-NA) are released from the incomplete combustion of fossil fuels and derived from polycyclic aromatic hydrocarbons (PAHs). The toxicities of 1-NP and 9-NA are mainly reflected in their carcinogenicity and mutagenicity. However, studies of PM2.5-bound 1-NP and 9-NA on the cardiac genotoxicity are limited so far. In this study, histopathology, DNA damage, DNA repair-related gene expression, and oxidative stress were investigated in the hearts of male Wistar rats exposed to PM2.5 [1.5 mg/kg body weight (b.w.)] or three different dosages of 1-NP (1.0 × 10− 5, 4.0 × 10− 5, and 1.6 × 10− 4 mg/kg b.w.) or 9-NA (1.3 × 10− 5, 4.0 × 10− 5, and 1.2 × 10− 4 mg/kg b.w.). The results revealed that (1) PM2.5, higher dosages of 1-NP (4.0 × 10− 5 and 1.6 × 10− 4 mg/kg b.w.) and 9-NA (4.0 × 10− 5 and 1.2 × 10− 4 mg/kg b.w.) caused obvious pathological responses and DNA damage (DNA strand breaks, 8-OHdG formation and DNA–protein cross-link), accompanied by increasing OGG1 and GADD153 expression while inhibiting MTH1 and XRCC1 expression in rat hearts. Also, they elevated the hemeoxygenase-1 (HO-1), glutathione S-transferase (GST), and malondialdehyde (MDA) levels and decreased superoxide dismutase (SOD) activity compared with the control. (2) The lowest dosages 1-NP or 9-NA could not cause DNA damage and oxidative stress. (3) At the approximately equivalent dose level, PM2.5-induced DNA damage effects were more obvious than 1-NP or 9-NA along with positive correlation. Taken together, heart DNA damage caused by PM2.5, 1-NP and 9-NA may be mediated partially through influencing the DNA repair capacity and causing oxidative stress, and such negative effects might be related to the genotoxicity PM2.5, 1-NP, and 9-NA. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a major global health disorder and, currently, over 450 million people have diabetes with 90% suffering from type 2 diabetes. Left untreated, diabetes may lead to cardiovascular diseases which are a leading cause of death in diabetic patients. Calcium is the trigger and regulator of cardiac muscle contraction and derangement in cellular Ca2+ homeostasis, which can result in heart failure and sudden cardiac death. It is of paramount importance to investigate the regional involvement of Ca2+ in diabetes-induced cardiomyopathy. Therefore, the aim of this study was to investigate the voltage dependence of the Ca2+ transients in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of the Goto-Kakizaki (GK) rats, an experimental model of type 2 diabetes mellitus. Simultaneous measurement of L-type Ca2+ currents and Ca2+ transients was performed by whole-cell patch clamp techniques. GK rats displayed significantly increased heart weight, heart weight/body weight ratio, and non-fasting and fasting blood glucose compared to controls (CON). Although the voltage dependence of L-type Ca2+ current was unaltered, the voltage dependence of the Ca2+ transients was reduced to similar extents in EPI-GK and ENDO-GK compared to EPI-CON and ENDO-CON myocytes. TPK L-type Ca2+ current and Ca2+ transient were unaltered. THALF decay of L-type Ca2+ current was unaltered; however, THALF decay of the Ca2+ transient was shortened in ENDO and EPI myocytes from GK compared to CON rat hearts. In conclusion, the amplitude of L-type Ca2+ current was unaltered; however, the voltage dependence of the Ca2+ transient was reduced to similar extents in EPI and ENDO myocytes from GK rats compared to their respective controls, suggesting the possibility of dysfunctional sarcoplasmic reticulum Ca2+ transport in the GK diabetic rat hearts. [ABSTRACT FROM AUTHOR]

Purpose: The contribution of extra virgin olive oil (EVOO) macro- and micro-constituents in heart oxidative and inflammatory status in a hypercholesterolemic rat model was evaluated. Fatty acid profile as well as α-tocopherol, sterol, and squalene content was identified directly in rat hearts to distinguish the effect of individual components or to enlighten the potential synergisms. Methods: Oils and oil-products with discernible lipid and polar phenolic content were used. Wistar rats were fed a high-cholesterol diet solely, or supplemented with one of the following oils, i.e., EVOO, sunflower oil (SO), and high-oleic sunflower oil (HOSO) or oil-products, i.e., phenolics-deprived EVOO [EVOO(−)], SO enriched with the EVOO phenolics [SO(+)], and HOSO enriched with the EVOO phenolics [HOSO(+)]. Dietary treatment lasted 9 weeks; at the end of the intervention blood and heart samples were collected. Results: High-cholesterol-diet-induced dyslipidemia was shown by increase in serum total cholesterol, low-density lipoprotein cholesterol, and triacylglycerols. Dyslipidemia resulted in increased malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α) levels, while glutathione and interleukin 6 levels remained unaffected in all intervention groups. Augmentation observed in MDA and TNF-α was attenuated in EVOO, SO(+), and HOSO(+) groups. Heart squalene and cholesterol content remained unaffected among all groups studied. Heart α-tocopherol was determined by oil α-tocopherol content. Variations were observed for heart β-sitosterol, while heterogeneity was reported with respect to heart fatty acid profile in all intervention groups. Conclusions: Overall, we suggest that the EVOO-polar phenolic compounds decreased MDA and TNF-α in hearts of cholesterol-fed rats. [ABSTRACT FROM AUTHOR]

β-Adrenoceptor antagonists are important drugs for the treatment of cardiovascular diseases and some of those drugs also block the so-called low-affinity site of β-adrenoceptors although at much higher concentrations. This low-affinity site, also identified in vivo and in human tissue, may come into play under certain pathophysiological situations including arrhythmias. The aim of our study was to determine the potency of 14 compounds chemically related to bupranolol or bevantolol and two xanthone derivatives at the low-affinity site of the β-adrenoceptor. The potency of the compounds at the low- and high-affinity site of β-adrenoceptors (β and β; both increasing heart rate) was compared in the pithed rat. One compound was also studied in the isolated rat heart and its α-adrenolytic effect determined in the isolated rat mesenteric artery. In the pithed rat, four compounds blocked the β-adrenoceptor at a ≥10-fold lower potency than the β-adrenoceptor whereas the xanthone derivative (−)-MH-3 was equipotent. In the spontaneously beating right atrium (−)-MH-3 was a non-competitive antagonist of comparable potency at either receptor; its apparent pD′ value for the β-adrenoceptor ranged from 5.6 to 6.4 under various conditions, including the Langendorff preparation. Its apparent pA at the α-adrenoceptor in the mesenteric artery was 8.4. (−)-MH-3 is the first compound with virtually the same potency at the low- and high-affinity site of β-adrenoceptors in vivo; it appears to be a non-competitive antagonist at either site in vitro. [ABSTRACT FROM AUTHOR]

This study investigated the effects of peptide apelin-12 (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A12) and its novel structural analog (H-(NMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, AI) on myocardial antioxidant enzyme activities, lipid peroxidation, and reactive oxygen species formation in ex vivo and in vivo models of myocardial ischemia/reperfusion (I/R) injury. Isolated working rat hearts were subjected to global ischemia and reperfusion. Infusion of 140 μM A12 or AI before global ischemia improved cardiac function recovery; increased the activity of Cu,Zn superoxide dismutase (Cu,Zn SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); decreased malondialdehyde (MDA) content in reperfused heart; and reduced the formation of hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline- N-oxide in the myocardial effluent during early reperfusion compared with these indices in control. Anesthetized open-chest rats were subjected to the left anterior descending coronary artery occlusion and coronary reperfusion. Peptide A12 or its analog AI was injected intravenously at the onset of reperfusion at a dose of 0.35 μmol/kg. Treatment with A12 or AI significantly limited infarct size and reduced the activity of lactate dehydrogenase and creatine kinase MB isoenzyme in blood plasma at the end of reperfusion compared with control. These effects were accompanied by complete recovery of Cu,Zn SOD, CAT, and GSH-Px activities; and decrease in MDA content in the area at risk by the end of reperfusion. The study concluded that C-terminal fragment of native peptide apelin-12 and its synthesized analog is involved in the upregulation of cardiac antioxidant defense systems and attenuation of lipid peroxidation in myocardial I/R injury. [ABSTRACT FROM AUTHOR]

A partially purified Na,K-ATPase preparation from rat heart containing α1- and α2-isoforms of the enzyme was shown to include both subunits in S-glutathionylated state. Glutathionylation of the α1-subunit (but not of the α2-subunit) was partially removed when the preparation was isolated in the presence of dithiothreitol. The addition of oxidized glutathione irreversibly inhibited both isoforms. Inhibition of the enzyme containing the α1-subunit was biphasic, and the rate constants of the inhibition were 3745 ± 360 and 246 ± 18 M·min. ATP, ADP, and AMP protected the Na,K-ATPase against inactivation by oxidized glutathione. [ABSTRACT FROM AUTHOR]

Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O consumption of rat heart (1 mm tissue cubes) by 33% (from 4.69 to 3.11 μmol O/min. g tissue). Mitochondrial O consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O and HO due to complex I inhibition. The increased NO production, as shown by 38% increased mtNOS biochemical activity and 31% increased mtNOS functional activity, is expected to fuel an increased ONOO generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia. [ABSTRACT FROM AUTHOR]

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of HO, we studied its role during ontogenic development of rat heart. HO was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2′-7′-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, HO was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the HO-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of HO in the heart vessel wall during development and HO oxidatively modifies elastin producing protein blue autofluorescence. [ABSTRACT FROM AUTHOR]

Injection of dinitrosyl iron complexes with glutathione at the onset of 40-min regional myocardial ischemia in rat was shown to exert a clear cardioprotective action by decreasing the infarct size and suppressing the cardiac rhythm disturbance. After introducing the preparation, its effective accumulation with protein thiol-containing ligands in the myocardial tissue was registered be the EPR method. It was also found that in postischemic reperfusion, the rate of decrease in the content of these complexes in the ischemic area increases, which reflects effective scavenging of short-lived reactive oxygen species by the dinitrosyl iron complexes. [ABSTRACT FROM AUTHOR]

Cruciferous vegetables are known for antioxidant and anti-carcinogenic effects. In the current study we asked whether dietary broccoli sprouts can protect the heart from ischemia-reperfusion. Rats were fed either control diet (sham and control groups) or a diet mixed with 2% dried broccoli sprouts for 10 days. After 10 days the isolated hearts were subjected to ischemia for 20 min and reperfusion for 2 h, and evaluated for cell death, oxidative damage, and Nrf2-regulated phase 2 enzyme activities. Broccoli sprouts feeding inhibited markers of necrosis (lactate dehydrogenase release) and apoptosis (caspase-3 activity) by 78-86%, and decreased indices of oxidative stress (thiobarbituric acid reactive substances and aconitase inactivation) by 82-116%. While broccoli sprouts increased total glutathione and activities of the phase 2 enzymes glutamate cysteine ligase and quinone reductase in liver, they did not affect these in ischemic-reperfused heart. While the mechanism is not clear, the results show that a relatively short dietary treatment with broccoli sprouts can strongly protect the heart against oxidative stress and cell death caused by ischemia-reperfusion. [ABSTRACT FROM AUTHOR]

It is known that adenosine 5′-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X1–7 and P2Y1–14 receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X5 was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y1, P2Y2, and P2Y14 mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y2 and P2Y14 levels were highest, respectively. We extended these studies to investigate P2X4 receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X4 receptor mRNA was upregulated by 93% in SAN ( P < 0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X4-mediated effects might be modulated in heart failure. mRNA for P2X4–7 and P2Y1,2,4,6,12–14, but not P2X2,3 and P2Y11, was detected in human right atrium and SAN. In addition, mRNA for P2X1 was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X4 and P2X7 mRNA was the highest for P2X receptors. P2Y1 and P2Y2 mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y1, P2Y2, and P2Y14 were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the mechanism of effect of 3-nitropropionic acid-(3-NP) induced late preconditioning in rat heart. For this purpose 20–30 min before 3-NP (20 mg/kg, i.p.) injection, the rats were treated intraperitoneally with 5-hydroxydecanoate (40 mg/kg, 5-HD, mitochondrial KATP-channel blocker), L-NAME (100 mg/kg, NOS inhibitor), N-2-mercaptopropionylglycine (100 mg/kg, MPG, free radical scavenger), or superoxide dismutase+catalase (10000+10000 IU/kg, SOD+CAT). Control rats received saline only without 3-NP pretreatment. After two days, hearts were isolated and perfused at a constant pressure in a Langendorff apparatus. 15-min global ischemia followed by 30-min reperfusion was applied to all hearts. Pretreatment of 3-NP significantly reduced infarct size, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) levels, and incidence of ventricular tachycardia (VT) compared with the control group receiving saline only. 5-HD, L-NAME, MPG, or SOD+CAT treatment statistically reversed 3-NP-induced reduction in infarct size. Although CK-MB, LDH levels, and incidence of VT were also reduced by L-NAME, MPG, or SOD+CAT treatment, only 5-HD significantly inhibited beneficial effects of 3-NP on all of the parameters above. These results showed that mito-KATP channels play a pivotal role in late preconditioning effect of 3-NP in the isolated rat heart. However, other mediators such as reactive oxygen species and NO may be, at least in part, involved in mechanisms of this effect. [ABSTRACT FROM AUTHOR]

The cardioprotective effects of KR-31762, a newly synthesized K+ ATP opener, were evaluated in rat models of ischemia/reperfusion (I/R) heart injury. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, KR-31762 (3 and 10 μM) significantly increased the left ventricular developed pressure (LVDP) and double product (heart rate × LVDP) after 30-min referfusion in a concentration-dependent manner, while decreasing the left ventricular end-diastolic pressure (LVEDP). KR-31762 also significantly increased the time to contracture (TTC) during ischemic period (20.0, 22.4 and 26.4 min for control, 3 and 10 μM, respectively), while decreasing the release of lactate dehydrogenase (LDH) from the heart during 30 min reperfusion (30.4, 14.3 and 19.7 U/g heart weight, respectively). All these parameters except LDH release were reversed by glyburide (1 μM), a nonselective blocker of K+ ATP channel, but not by 5-hydroxydecanoate, a selective blocker of mitoK+ ATP channel. In anesthetized rats subjected to 45-min occlusion of left anterior descending coronary artery followed by 90-min reperfusion, KR-31762 significantly decreased the infarct size (60.8, 40.5 and 37.8% for control, 0.3 and 1.0 mg/kg, iv, respectively). KR-31762 slightly relaxed the isolated rat aorta precontracted with methoxamine (IC50: 23.5 μM). These results suggest that KR-31762 exerts potent cardioprotective effects through the opening of sarcolemmal KATP channel in rat hearts with the minimal vasorelaxant effects. [ABSTRACT FROM AUTHOR]

To investigate the involvement of reperfusion-induced salvage kinases (RISK) as possible signaling molecules for the cardioprotective effects of BMS-180448, a prototype mitochondrial ATP-sensitive K+ (mitoKATP) channel opener, we measured its cardioprotective effects in a rat model of ischemia/reperfusion (I/R) heart injury, together with western blotting analysis of five different signaling proteins. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, BMS-180448 (1, 3 and 10 μM) significantly increased reperfusion left ventricular developed pressure (LVDP) and 30-min reperfusion double product (heart rate x LVDP) in a concentration-dependent manner, while decreasing left ventricular end-diastolic pressure (LVEDP) throughout reperfusion period in a concentration-dependent manner. SDS-PAGE/western blotting analysis of left ventricle reperfused for 30 min revealed that BMS-180448 significantly decreased phospho-GSK3β at high concentration, whereas it tended to increase slightly phospho-eNOS and phospho-p70S6K with concentration. However, BMS-180448 had no effect on phospho-Akt and phospho-Bad. These results suggest that the cardioprotective effects of BMS-180448 against I/R heart injury may result from direct activation of mitoKATP channel in cardiomyocytes, with the minimal role of RISK pathway in the activation of this channel and the cardioprotective effects of BMS-180448. [ABSTRACT FROM AUTHOR]

Resveratrol, a natural phytoalexin found in wine, has been suggested to have benefits in preventing cardiovascular diseases. However, the direct effects of resveratrol on the activity of cardiac tissues and its mechanism of action have not been determined. This study examined the effects of resveratrol on the right and left atrium and left papillary muscle isolated from the rat heart. The contractile responses of the right atrium and papillary muscle and the action potential from the left atrium were recorded and the effects of resveratrol on these responses were observed. The resting force of the isolated right atrium and the peak developed force of the left papillary muscle were depressed by resveratrol (0.1 nM – 0.1 mM). Exposure to the KATP channel blocker glyburide (3 μM) prevented significantly the resveratrol-induced decrease. Resveratrol (0.1 mM) shortened the repolarization phase of action potential recorded from the left atrium and this effect of resveratrol was reversed by glyburide (3 μM). These results indicated that resveratrol depressed cardiac muscle contraction and shortened action potential duration probably due to the activation of KATP channels in the rat heart. [ABSTRACT FROM AUTHOR]

To improve current knowledge of the molecular mechanisms underlying exercise-induced cardioprotection in a rat model of mild exercise training, Sprague–Dawley rats were trained to run on a treadmill up to 55% of their maximal oxygen uptake for 1 h/day, 3 days/week, 14 weeks, with age-matched sedentary controls ( n = 20/group). Rats were sacrificed 48 h after the last training session. Despite lack of cardiac hypertrophy, training decreased blood hemoglobin (7.94 ± 0.21 mM vs. 8.78 ± 0.23 mM, mean ± SE, P = 0.01) and increased both plasma malondialdehyde (0.139 ± 0.005 mM vs. 0.085 ± 0.009 mM, P = 0.05) and the activity of Mn-superoxide dismutase (11.6 ± 0.6 vs. 16.5 ± 1.6 mU/μg, P = 0.01), whereas total superoxide dismutase activity was unaffected. When subjected to 30-min ischemia followed by 90-min reperfusion, hearts from trained rats ( n = 5) displayed reduced infarct size as compared to controls (37.26 ± 0.92% vs. 49.09 ± 2.11% of risk area, P = 0.04). The biochemical analyses in the myocardium, which included gene expression profiles, real-time PCR, Western blot and determination of enzymatic activity, showed training-induced upregulation of the following mRNAs and/or proteins: growth-arrest and DNA-damage induced 153 (GADD153/CHOP), heme-oxygenase-1 (HO-1), cyclooxygenase-2 (Cox-2), heat-shock protein 70/72 (HSP70/72), whereas heat-shock protein 60 (HSP60) and glucose-regulated protein 75 (GRP75) were decreased. As a whole, these data indicate that mild exercise training activates a second window of myocardial protection against ischemia/reperfusion by upregulating a number of protective genes, thereby warranting further investigation in man. [ABSTRACT FROM AUTHOR]

Both hypoxia and aging affect the morphology and the function of rat myocardial tissue. Moreover the heart tries to counteract the impaired function by activating specific signalling cascades. Here we report the involvement of CREB protein in “in vivo” response to hypoxic challenge and during aging in rat hearts. CREB is activated in parallel to HIF-1α nuclear translocation in the young after hypoxia exposure followed by reoxygenation, while this kind of response is not so dramatic in the old, neither in terms of CREB activation, neither in terms of HIF-1α expression and translocation, suggesting in the old the existence of an impaired oxygen-sensing mechanism or an adaptation of the cells to hypoxia. Moreover in the young a PKC α/Erk pathway seems to be involved in the activation of HIF-1α along with CREB, suggesting an attempt of the young to counteract the damage evoked by hypoxia, while in the old a PKC α/p38 MAPK/CREB pathway could determine the occurrence of both aging and aged cell hypoxia response. [ABSTRACT FROM AUTHOR]

Angiotensin converting enzyme (ACE) inhibitors have cardioprotective effects in different species including human. This cardioprotective effect is mainly due to the inhibition of bradykinin (BK) degradation rather than inhibition of the conversion of angiotensin I to angiotensin II. Bradykinin, a nonapeptide, has been considered to be the potential target for various enzymes including ACE, neutral endopeptidase 24.11, carboxypeptidase M, carboxypeptidase N, proline aminopeptidase, endopeptidase 24.15, and meprin. In the present study, the coronary vascular beds of Sprague Dawley rat isolated hearts were perfused (single passage) with Krebs solution alone or with different concentrations of BK i.e. 2.75×10−10, 10−7, 10−6 and 10−5 M solution. Percent degradation of BK was determined by radioimmunoassay. The degradation products of BK after passing through the isolated rat-hearts were determined using RP-HPLC and mass spectroscopy. All the four doses of BK significantly decreased the perfusion pressure during their passage through the hearts. The percentage degradation of all four doses was decreased as the concentration of drug was increased, implying saturation of a fixed number of active sites involved in BK degradation. Bradykinin during a single passage through the hearts degraded to give [1–7]-BK as the major metabolite, and [1–8]-BK as a minor metabolite, detected on HPLC. Mass spectroscopy not only confirmed the presence of these two metabolites but also detected traces of [1–5]-BK and arginine. These findings showed that primarily ACE is the major cardiac enzyme involved in the degradation of bradykinin during a single passage through the coronary vascular of bed the healthy rat heart, while carboxypeptidase M may have a minor role. [ABSTRACT FROM AUTHOR]

It has been recently shown that long-term thyroxine administration increases the tolerance of the heart to ischaemia. The present study investigated whether thyroxine induced cardioprotection involves alterations in the pattern of p38 mitogen activated protein kinase (p38MAPK) and c-Jun NH2-terminal kinases (JNKs) activation during ischaemia-reperfusion. L-thyroxine (T4) was administered in Wistar rats (25 μg/100 g/day, subcutaneously) for 2 weeks (THYR), while normal animals served as controls (NORM). NORM and THYR isolated rat hearts were perfused in Langendorff mode and subjected to 10 or 20 min of zero-flow global ischaemia only and also to 20 min of ischaemia followed by 10, 20 or 45 min of reperfusion. Postischaemic recovery of left ventricular developed pressure at 45 min of reperfusion was expressed as % of the initial value. Activation of p38 MAPK and JNKs was assessed at the different times of the experimental setting by standard Western blotting techniques using a dual phospho p38MAPK and phospho JNKs (p46/p54) antibodies. Activation of p38 MAPK was significantly attenuated during ischaemia and reperfusion in thyroxine treated hearts compared to normal hearts. JNKs were found to be activated only during the reperfusion period. The levels of phospho JNKs were found to be lower in thyroxine treated hearts as compared to untreated hearts, though not at a statistically significant level. Postischaemic functional recovery was higher in THYR as compared to NORM, p < 0.05. In summary, in hearts pretreated with thyroxine, p38 MAPK was attenuated during ischaemia and at reperfusion and this was associated with improved postischaemic recovery of function. [ABSTRACT FROM AUTHOR]

Modulation of the sinus rate and contractile force by taurine at different extracellular Ca2+ concentrations ([Ca2+]o) was examined using rat right atria loaded with forced swimming stress. Serum concentration of corticosterone profoundly increased in stress-loaded rats as compared with native rats. The taurine level in serum also increased in stress-loaded rats, but was not changed in the different heart tissues and aorta. Heat-shock protein (HSP72) was detectable in cardiac muscles and in the lumen of cardiac blood vessels of stress-loaded rats using a monoclonal antibody. Increasing [Ca2+]o (from 0.9 to 3.6 m M) enhanced the sinus rate and contractile force in a [Ca2+]o-dependent fashion in native rats, but not in stress-loaded rats. Taurine (1–20 m M) caused a negative chronotropic and inotropic effect in a concentration-dependent manner. At 1.8 m M [Ca2+]o, the negative chronotropic effect of taurine (10–20 m M) was attenuated in stress-loaded rats as compared with native rats. These results indicate that swimming stress causes a release of taurine into the serum and reduces the sensitivity to [Ca2+]o. Taurine administration might, in part, exhibit the protective actions on acute stress-induced responses. [ABSTRACT FROM AUTHOR]

The intensity of free radical processes and the regulation of NADP-isocitrate dehydrogenase (EC 1.1.1.42; NADP-IDH) activity have been studied in the cytoplasmic fraction of normal and ischemized rat myocardium. Chemiluminescence parameters, such as the light sum (S) of slow flash and the tangent of the kinetic curve slope angle (tanα1), which characterize the intensity of free radical processes, were increased in ischemia 2.1- and 20.0-fold, respectively. The slow flash intensity (Imax) was increased 22-fold. The contents of lipid peroxidation products–diene conjugates and malonic dialdehyde–were increased 11.9- and 4.7-fold, respectively, suggesting pronounced oxidative stress. Using homogenous enzyme preparations of NADP-IDH isolated from the normal and experimentally ischemized rat myocardium, a number of catalytic properties of the enzyme were characterized for normal and pathologic conditions. NADP-IDH from the normal and ischemized myocardium had the same electrophoretic mobility and was regulated similarly by Fe2+, Cu2+, Zn2+, and also with succinate and fumarate. However, under normal and pathologic conditions NADP-IDH was different in the affinity for substrates and in the sensitivity to inhibitory effects of hydrogen peroxide, reduced glutathione, and of Ca2+. The degree of synergy in the enzyme inhibition with Fe2+ and H2O2 was less pronounced in ischemia. The inhibitory effect of the reaction product 2-oxoglutarate was higher under normal conditions than in ischemia (the Ki values were 0.22 and 0.75 mM, respectively). The specific features of the NADP-IDH regulation in ischemia are suggested to promote the stimulation of the enzyme functioning during increased level of free radical processes, and this seems to be important for NADPH supplying for the glutathione reductase/glutathione peroxidase antioxidant system of cardiomyocytes. [ABSTRACT FROM AUTHOR]

Oxygen-derived free radicals and oxidants (reactive oxygen intermediates, ROI) have been implicated in cardiovascular diseases. The protective role of nitric oxide (NO) against ROI-mediated tissue injury is not resolved. We tested the effects of exogenous NO, L- and D-arginine and a NO synthase inhibitor on electrolysis-induced cardiac injury and the generation of ROI by electrolysis. Superoxide dismutase (SOD) and catalase were used for comparison. Hearts (n=7) from male rats (350±30 g) were perfused in vitro at 10 ml min–1 g–1, ROI generated by electrolysis of the perfusion medium (15 mA, 10 s), and cardiac function and the level of isoluminol-derived chemiluminescence in electrolysed perfusion medium documented for 15 min (n=4). The ROI-induced maximal reduction of left ventricular developed pressure to 55±5% of baseline, and a 2.2±0.1-fold rise in coronary perfusion pressure 3 min after electrolysis, were prevented by SOD (50 U ml–1), catalase (100 U ml–1), S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 nmol l–1); L-arginine (1 mmol l–1), NG-nitro-L-arginine (L-NNA, 200 µmol l–1) or D-arginine (1 mmol l–1). The effect of L-arginine was concentration dependent. In all cases, the beneficial effects were closely matched by a near-total reduction of ROI in the perfusion medium. We conclude that, besides mimicking or enhancing NO activity, L-arginine and donor-derived exogenous NO are cardioprotective by reducing ROI-mediated tissue injury. The protective effect of L-NNA and D-arginine implies that the protection results from a direct chemical interaction between the drug and the oxidizing species. [ABSTRACT FROM AUTHOR]

Adult male Wistar rats were exposed to intermittent high altitude hypoxia of 7000 m simulated in a hypobaric chamber for 8 h/day, 5 days a week; the total number of exposures was 25. The concentration of individual phospholipids and their fatty acid (FA) profile was determined in right (RV) and left (LV) ventricles. Adaptation to hypoxia decreased the concentration of diphosphatidylglycerol (DPG) in hypertrophied RV by 19% and in non-hypertrophied LV by 12% in comparison with normoxic controls. Chronically hypoxic hearts exhibited lower phospholipid n-6 polyunsaturated FA (PUFA) content mainly due to decreased linoleic acid (18:2n-6), which was opposed by increased n-3 PUFA mainly due to docosahexaenoic acid (22:6n-3) in phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). The content of arachidonic acid (20:4n-6) was unchanged in total phospholipids, but in PC it was increased in both ventricles (by 22%) and in PE decreased in LV only (by 20%). Chronic hypoxia increased the un-saturation index of PC and PE in both ventricles. The content of monounsaturated FA (MUFA) was increased and 18:2n-6 decreased in DPG. The proportion of saturated FA was increased in PC and PI of hypoxic RV but not LV. The FA composition of phosphatidylserine was not altered in hypoxic ventricles. It is concluded that chronic hypoxia led to only minor changes in individual phospholipid concentration in rat ventricular myocardium, but markedly altered their FA profile. These changes, in particular the greater incorporation of n-3 PUFA into phospholipids and increased un-saturation index, may lead to a better preservation of membrane integrity and thereby contribute to improved ischemic tolerance of chronically hypoxic hearts. [ABSTRACT FROM AUTHOR]

Because of its key role in proteosynthesis, the total content of elongation factor-2 (EF-2) and the distribution of six main EF-2 variants were investigated after Pseudomonas Exotoxin A catalyzed [32P]ADP-ribosylation using 1D-PAGE and isoelectric focusing (IEF) in a rat model of hemodynamic overload with variable degrees of cardiac hypertrophy: Chronic NO-synthase inhibition by L-NAME (N-omega-nitro-L-arginine-methyl-esther; 0.75 mg/ml drinking water) induced arterial hypertension without hypertrophy but myocardial apoptosis; additional treatment with IGF-1 (osmotic micropumps) did not modify hypertension but reduced apoptosis allowing moderate hypertrophy of the left ventricles. Total EF-2 did not significantly increase in rats with hemodynamic overload with or without IGF-1 supplementation. A positive correlation was found between an acidic EF-2 variant and apoptosis (p = 0.01). Hypertrophy under additional IGF-1 was combined with a shift of the EF-2 variants to basic subtypes (p < 0.01). This finding may be indicative of the trophic potency of IGF-1. [ABSTRACT FROM AUTHOR]

Myocardial ischaemia and reperfusion lead to myocardial cell death due, at least in part, to apoptotic mechanisms. Although cysteinyl aspartate-specific proteinase (caspase) activation is a major event and the most-cited culprit in the development of apoptosis, its potential contribution to ischaemic myocardial cell death is largely unknown. To study the role of caspase activation, isolated rat hearts (n=6 per group) were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion. A non-selective [0.1 or 0.5 µM acetyl-Tyr-Val-Ala-Asp chloromethylketone (YVAD-cmk)] or selective caspase inhibitors [0.07 or 0.2 µM acetyl-Asp-Glu-Val-Asp-cmk (Ac-DEVD-cmk, caspase-3 inhibitor); 0.07 or 0.2 µM benzoxycarbonyl-Leu-Glu-OMe-His-Asp(OMe)-fluoromethylketone (z-LEHD-fmk, caspase-9 inhibitor)] were added to the perfusate at the start of reperfusion. Non-selective caspase inhibition with 0.1 or 0.5 µM YVAD-cmk limited infarct size: (21±4%, P<0.05; 17±3%, P<0.05, respectively) compared with the ischaemic/reperfused control (32±5%). In hearts treated with 0.1 or 0.5 µM caspase II non-selective inhibitor, the fraction of terminal-deoxynucleotidyl-transferase deoxyuridine nick end labelling (TUNEL)-positive myocyte nuclei in the infarcted zone was reduced from the ischaemic/reperfused non-treated control of 11.2±2.1% to 6.2±1.6% (P<0.05) and 1.2±0.2% (P<0.05), respectively. The recovery of post-ischaemic cardiac function (coronary flow, aortic flow and left-ventricular developed pressure) improved significantly with the application of the non-selective caspase inhibitor as well. In hearts perfused with specific caspase inhibitors (caspase-3 and caspase-9) there was no significant reduction in the infarct size, no improvement in post-ischaemic cardiac function and no reduction of apoptotic cell death. We conclude that non-specific inhibition of caspases may be therapeutically beneficial in myocardial ischaemia/reperfusion-induced damage, while selective caspase inhibitors may fail to prevent such reperfusion-induced injury in our model system. [ABSTRACT FROM AUTHOR]

The regulatory neuropeptide calcitonin-gene related peptide (CGRP) has been shown to evoke a hypertrophic response in isolated cardiomyocytes in vitro, an effect which was attributed to PKC activation. Activation of PKC has previously been implicated in the development of cardiac hypertrophy. We therefore investigated the role of CGRP in pressure overload-induced hypertrophy in vivo, which has not previously been reported. Constriction of the ascending aorta of rats resulted in an increase in the heart weight to body weight ratio, increased myocyte diameter, re-expression of the fetal genes ANF, MHCβ and skeletal α-actin, and decreased expression of the adult genes GLUT4 and SERCA2a. Treatment of neonatal rat pups (1–2 days old) with capsaicin (50 mg/kg), resulted in the permanent de-afferentation of small-diameter unmyelinated CGRP-containing sensory C-fibres. Such treatment caused a 68% decrease in the CGRP-like immunoreactivity of hearts isolated from 10 week old rats (p < 0.001). Contrary to expectations, aortic constriction of capsaicin treated rats had no effect on the development of hypertrophy at the trophic, morphometric or gene expression levels. The results suggest that the development of pressure overload-induced hypertrophy in vivo does not require the regulatory neuropeptide CGRP. [ABSTRACT FROM AUTHOR]

Male Wistar rats aged 4 weeks, were subjected to hypobaric hypoxia (barometric pressure 505 hPa, PI,O2 106 hPa) or to diet restriction (reproducing the effect of hypoxia-induced anorexia) for 4 weeks. Each group (control, hypoxic, pair-fed, n=16), was divided into two sub-groups housed individually in either normal cages or cages with running wheels allowing evaluation of voluntary activity (n=8 each). The skinned-fibre technique was used to evaluate the functional properties of myofibrillar mitochondria from right and left ventricles in situ. The oxidative fibres from the soleus and diaphragm muscles were also investigated for comparison. Analysis of variance did not detect any significant effect of voluntary running activity. With calorie restriction, the maximal respiratory rate (Vmax) in the presence of 1 mM adenosine 5′-diphosphate (ADP) in myocardial fibres fell significantly (by about 25%) but was unchanged in skeletal myocytes. Following hypoxia, Vmax in myocardial fibres increased by 25% compared with the calorie restricted group and in soleus and diaphragm muscle fibres by about 30% compared with control. In myocardial fibres of control rats, creatine (20 mM) increased the sub-maximal respiratory rate by 80% in the presence of 0.1 mM ADP. Under calorie restriction or hypoxia the stimulatory effect was significantly reduced to 34–56%. This alteration was due to a decrease in the apparent Michaelis-Menten constant (Km) of mitochondrial respiration for ADP evaluated in the absence of creatine, while the Km in presence of creatine 20 mM was unchanged. In conclusion, reduced food intake decreased the oxidative capacity (Vmax) and the apparent Km for ADP of mitochondria in both left and right ventricles. Chronic hypoxia per se was responsible for an increase in the oxidative capacity of all oxidative muscles but did not exert significant effects on the control of respiration by ADP and creatine in myocardium. [ABSTRACT FROM AUTHOR]

Rhythm disorders are common complications in diabetic patients, due to their enhanced sensitivity to ischaemia. However, experimental studies are inconsistent, and both higher and lower vulnerability to injury has been reported. Our objectives were to compare susceptibility to ventricular arrhythmias in rats with prolonged duration of diabetes induced hy streptozotocin (45 mg/kg, i. v.), utilising two different models. Following 8 weeks, either anaestetised open-chest rats in vivo or isolated Langendorff-perfused hearts were subjected to 30 min regional zero-flow ischaemia induced by occlusion of LAD coronary artery. In addition, cardiac glycogenolysis and lactate production were measured. In open-chest rats, 90% of the controls exhibited ventricular tachycardia (VT) which represented 55.4% of total arrhythmias, whereby only 19.9% of arrhythmias occurred as VT in 44% of the diabetic rats (P < 0.05 vs controls). Duration of VT and ventricular fibrillation (VF) was reduced from 35.5 ± 11.1 and 224.8 ± 153.9 s in the controls to 4.8 ± 2.5 and 2.2 ± 0.2 s in the diabetics, respectively (P < 0.05). Accordingly, severity of arrhythmias (arrhythmia score, AS) was also lower in the diabetics (2.0 ± 0.38 vs 3.3 ± 0.3 in the controls; P < 0.05). In the isolated hearts, high incidence of VF was decreased in the diabetic hearts, and although VT occurred in almost all of the diabetic hearts, the duration of VT and VF was substantially shorter (61.5 ± 14.5 and 5.5 ± 0.5 s vs 221.5 ± 37 and 398.5 ± 55 s in the controls, respectively; P < 0.05). AS was reduced to 2.9 ± 0.12 from 4.1 ± 0.3 in the controls (P < 0.05). Postischaemic accumulation of lactate was lower in the diabetic than in the non-diabetic myocardium (20.4 ± 1.9 vs 29.5 ± 2.9 μmol/l/g w.wt.; P < 0.05). These results suggest that rat hearts with chronic diabetes, despite some differences in the arrhythmia profiles between the in vivo model and isolated heart preparation, are less sensitive to ischaemic injury and exhibit lower susceptibility to ventricular arrhythmias and reduced accumulation of glycolytic metabolites. [ABSTRACT FROM AUTHOR]

The in vitro effect of tedisamil on the specific activity and kinetic parameters of the sarcolemmal (Na,K)-ATPase as well as its ex vivo effect on the (Na,K)-ATPase in the isolated, perfused rat hearts was determined. Five μmol/l of tedisamil was added 5 min before the onset of 30 min global normothermic ischemia followed by 10 min reperfusion. At the conditions of its maximal cardioprotective effect (heart rate reduction, improved postischemic recovery of left ventricular developed pressure), the hearts were immediately used for isolation of sarcolemmal vesicles. In vitro, 1–100 μmol/l of tedisamil produced a concentration-dependent stimulatory effect on (Na,K)-ATPase activity, with a peak seen at 20 μmol/l (p < 0.01), while Mg-dependent ATPase was almost unchanged. Kinetic analysis revealed a significant increase in the affinity of the Na-binding sites on ATPase molecule at 20 μmol/l of tedisamil. These biochemical findings were confirmed by cytochemistry. Moreover, ex vivo experiments revealed that tedisamil rendered the sarcolemmal (Na,K)-ATPase activity to be a more resistant to detrimental effects of ischemia. In conclusion, the cardioprotective action of tedisamil was accompanied with a better preservation of the specific activity of (Na,K)-ATPase. [ABSTRACT FROM AUTHOR]

Diabetic hearts are suggested to exhibit either increased or lower sensitivity to ischemia. Detrimental effects of prolonged ischemia can be attenuated by preconditioning, however, relatively little is known about its effects in the diseased myocardium. This study was designed to test the susceptibility to ischemia-induced arrhythmias and the effect of preconditioning in the diabetic heart. Rats were made diabetic with streptozotocin (45 mg/kg, i.v.). After 1 week, isolated Langendorff-perfused hearts were subjected to 30 min occlusion of LAD coronary artery without or with preceding preconditioning induced by one cycle of 5 min ischemia and 10 min reperfusion. Glycogen and lactate contents were estimated in the preconditioned and non-preconditioned hearts before and after ischemia. Diabetic hearts were more resistant to ischemia-induced arrhythmias: incidence of ventricular tachycardia (VT) decreased to 42% and only transient ventricular fibrillation (VF) occurred in 17% of the hearts as compared to the non-diabetic controls (VT 100% and VF 70% including sustained VF 36%; p < 0.05). Preconditioning effectively suppressed the incidence and severity of arrhythmias (VT 33%, VF 0%) in the normal hearts. However, this intervention did not confer any additional protection in the diabetic hearts. Despite higher glycogen content in the diabetic myocardium and greater glycogenolysis during ischemia, production of lactate in these hearts was significantly lower than in the controls. Preconditioning caused a substantial decrease in the accumulation of lactate in the normal hearts, whereby in the diabetic hearts, this intervention did not cause any further reduction in the level of lactate. In conclusion, diabetic rat hearts exhibit lower susceptibility to ischemic injury and show no additional response to preconditioning. Reduced production of glycolytic metabolites during ischemia can account for the enhanced resistance of diabetic hearts to ischemia as well as for the lack of further protection by preconditioning. [ABSTRACT FROM AUTHOR]

Objectives. A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Methods. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 μM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Results. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. Conclusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function. [ABSTRACT FROM AUTHOR]

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg2+]i) and calcium ([Ca2+]i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg2+]i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li+, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca2+]i when either Na+ substitute was used. The possibility that Li+, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li+ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li+ displaces Mg2+ from intracellular binding sites. Having considered the binding constants for Mg2+ and Li+ to ATP, we conclude that Li+ can displace Mg2+ from Mg-ATP, thus causing a rise in [Mg2+]i. This work has implications for other studies where Li+ is used as a Na+ substitute. [ABSTRACT FROM AUTHOR]

Cardiac plasma membrane Ca2+/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+/Mg2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca2+-pump ATPase or sarcolemmal Ca2+-pump ATPase. On the other hand, the cardiac Ca2+/Mg2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca2+-pump ATPase or plasma membrane Ca2+-pump ATPase. Furthermore, the immune serum inhibited the Ca2+/Mg2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca2+/Mg2+ ecto-ATPase antibodies indicated the localization of Ca2+/Mg2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca2+/Mg2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca2+/Mg2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle. [ABSTRACT FROM AUTHOR]

The degree of a negative inotropic response of the isolated right ventricle to verapamil as well as the mortality rate were studied in rats during their postnatal development. Male Wistar rats aged 3, 15, 30, and 90 days were used. The isolated right ventricle was incubated in a glucose-free solution with a mixture of 95% O and 5% CO and electrically stimulated. The amplitude of isotonic contractions (AIC) was registered. In 90-day-old rats, AIC was 74.1±6.2% of initial amplitude 45 min after administration of verapamil; in 30-day-old, 41.1±6.4%; in 15-day-old, 38.2±4.1%; and in 3-day-old rats, only 2.6±1.5. The difference between the 3-day-old rats and all older groups was statistically highly significant. The mortality rate of verapamil-treated rats increased with decreasing age of animals. It is concluded that the sensitivity of the rat myocardium to verapamil is age dependent: the negative inotropic effect of this drug increases with decreasing age of the animal. This indicates a possible risk in the therapeutic use of verapamil when given to newborns and infants. [ABSTRACT FROM AUTHOR]

It was previously reported that the mitochondrial fraction of the rat heart contained a specific protein with a molecular weight of approximately ∼44kDa whose phosphorylation was inhibited by taurine (Lombardini,1994a). Isolation of the ∼44kDa phosphoprotein on a 1-dimensional polyacrylamide gel using traditional glycine buffers followed by re-electrophoresing the cut out proportion of the gel which corresponds to the ∼44kDa protein on a tricine-buffered gel resulted in sufficient pure protein for sequence analysis. The results indicate that the ∼44kDa phosphoprotein is pyruvate dehydrogenase. [ABSTRACT FROM AUTHOR]

The distribution of ionic lanthanum (La) was studied after perfusion of the isolated rat heart with a buffered salt solution containing lanthanum chloride. We have demonstrated that the localization of lanthanum differed from that shown in previous studies in which hearts were exposed to ionic or colloidal lanthanum during or after fixation. La passed rapidly through the intercellular cleft between endothelial cells. There was relatively little transport by micropinocytosis and no evidence of transendothelial passage through the cytoplasm. Perfusion with a solution containing 1 to 15 mM LaCl resulted in localization of La on the entire cytoplasmic membrane system of muscle cells, i.e.: the non-specialized parts of the muscle cell membranes were stained by La as well as the membranes of micropinocytotic vesicles, of the T-system and of the intercalated disc. The cytoplasmic leaflets of gap junctions were especially stained. However, the cell membranes of non-muscle cells were free of lanthanum precipitates. Freeze-fracturing of muscle cells showed concomitant alteration in the distribution of membrane-associated particles. The gap junction membranes also showed changes in particle aggregation. Intracellular binding of La was restricted to muscle cells. Lanthanum was found in the cell nucleus associated with heterochromatin, in the nucleolus, and along the nuclear membrane. The myofibrils were stained mainly over the I-band. [ABSTRACT FROM AUTHOR]

We have previously shown that hypercholesterolemia leads to the loss of pacing-induced preconditioning (PC), possibly due to the impairment of cardiac nitric oxide (NO) synthesis. It has been shown that excess exogenous cholesterol inhibits formation of several polyprenyl derivatives involved in signal transduction. In the present study, we examined whether PC and cardiac NO synthesis are restored by treatment with the key polyprenyl product, farnesol, in cholesterol-fed rats. Rats fed 2% cholesterol-enriched/control diet for 24 weeks were given i.p. 5 μM/kg farnesol/vehicle, respectively. An hour later, hearts were isolated and prepared for ‘working’ perfusion, then subjected to PC/non-PC protocols of 3 intermittent periods of pacing of 5 min duration at 10 Hz, followed by a 10 min coronary occlusion to test the effect of PC. PC increased ischemic aortic flow (AF) from its control value of 15.6 ± 1.5 to 27.3 ± 1.7 mL/min (p < 0.05). PC was not observed in hearts obtained from hypercholesterolemic rats (AF: 15.7 ± 1.2 mL/min), however, it reappeared in the farnesol-treated hypercholesterolemic group (AF: 31.8 ± 3.4 mL/min, p < 0.05). In tissue samples from the left ventricle, cholesterol-diet markedly decreased the intensity of the electron spin resonance spectra of NO obtained after in vivo spin trapping with Fe2+-diethyl-dithio-carbamate complex. Farnesol-treatment did not influence cardiac NO content in the cholesterol-fed or in the control group. These results show that the lost PC can be recaptured by farnesol-treatment in hypercholesterolemia, however, farnesol-treatment does not restore cardiac NO synthesis. [ABSTRACT FROM AUTHOR]

In the present report we describe an ATP diphosphohydrolase (apyrase EC 3.6.1.5) in rat cardiac sarcolemma. It is Ca2+ dependent and is insensitive to ouabain, orthovanadate, N-ethylmaleimide (NEM), lanthanum, and oligomycin that are classical ATPase inhibitors. Sodium azide that is a mitochondrial inhibitor at low concentrations, did not affect the enzyme activity at 5.0 mM or below. In contrast, at high concentrations (> 10 mM) sodium azide inhibited the enzyme. Levamisole, a specific inhibitor of alkaline phosphatase and P1, P5-di(adenosine 5′-)pentaphosphate (Ap5A), a specific inhibitor of adenylate kinase did not inhibit the enzyme. Mercury chloride showed a parallel inhibition of the hydrolysis of both substrates of apyrase. Similar inhibition profiles are powerful evidence for a common catalytic site for the hydrolysis of both substrates. The enzyme has an optimum pH range of 7.5–8.0 and catalyzes the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. The apparent Km (Michaelis constant) and Vmax (maximal velocity) are 62.1 ± 5.2 μM and 1255.7 ± 178 μmol inorganic phosphate liberated/min/mg with ATP and 59.4 ± 4.3 μM and 269.2 ± 39 μmol inorganic phosphate liberated/min/mg with ADP. Enzyme markers indicated that this apyrase is associated with the plasma membrane. A deposition of lead phosphate granules on the outer surface of the sarcolemmal vesicles was observed by electron microscopy in the presence of either ATP or ADP as substrate. It is suggested that the ATP diphosphohydrolase could regulate the concentration of extracellular adenosine, and thus is important in the control of vascular tone and coronary flow. [ABSTRACT FROM AUTHOR]

Lipid peroxidation (LPO) in rat testis and heart microsomes was compared using the ADP/Fe2+ as initiator with and without ascorbate at different concentrations. The extent of LPO was estimated by the levels of TBARS and PUFA. Without ascorbate, LPO was higher in heart than in testis despite elevated levels of catalase in heart. With increased ascorbate concentrations, a biphasic effect of LPO was observed. For a concentration ≤ 0.2 mM, ascorbate acted as pro-oxidant and increased TBARS correlated with decreased PUFA were observed both in testis and heart. Above 0.2 mM, ascorbate acts as antioxidant but differences in the rate of LPO were observed. In heart decreased TBARS correlated with increased PUFA whereas in testis TBARS only decreased, PUFA were not significantly modified. These results suggest different mechanisms in LPO initiation in the two organs. Increasing concentrations of H2O2 produced directly elevated TBARS levels in testis while a lag phase was observed in heart before the increase, suggesting that H2O2 was the essential ROS produced by ascorbate-ADP/Fe2+. The effects of scavengers such as catalase and ethanol showed an inhibitory effect on TBARS production only in testis, suggesting the role of H2O2/OH⋅ as an initiator of LPO. In heart, catalase produced a slight increase in TBARS levels whereas no modification was observed with ethanol, suggesting a possible direct activation by ADP/Fe2+ through a metal-oxo intermediate. [ABSTRACT FROM AUTHOR]

The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 × 10 trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K values, and a decreased total V. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100 - solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels. [ABSTRACT FROM AUTHOR]

Postnatal maturation of rat heart is characterized by increases in force production, velocity of shortening and heart rate. Simultaneously with the enhanced cardiac power production the size of ventricular myocytes markedly increases. Parallel increase in cardiac rate functions and cells size would be expected to require reorganization of cardiac Ca regulation so that adequate rate of Ca release and uptake can be maintained. In accordance with this the source of activator Ca shifts from extracellular space to intracellular stores within the first four or five weeks of postnatal life. Calcium handling of sarcoplasmic reticulum and sarcolemma change in complementary manner so that diminishing sarcolemmal Ca transport is compensated with enhanced Ca release and sequestration by the sarcoplasmic reticulum during the early postnatal development of rat heart. These functional changes are partly due to reciprocal alterations in surface area of sarcolemma and sarcoplasmic reticulum, partly due to age-dependent changes in the expression of different transport systems and their kinetic properties. [ABSTRACT FROM AUTHOR]

Inositol-1,4,5-trisphosphate (IP) has been proposed to be a second messenger in response to alpha-1-adrenoceptor stimulation also in myocardial cells. We studied the effect of alpha-1-adrenoceptor stimulation (5 × 10 mol/l phenylephrine or 5 × 10 mol/l noradrenaline both in the presence of 10 mol/l timolol) on IP mass content in isolated perfused rat hearts. IP content was determined by a specific receptor-binding assay-kit (TRK 1000, Amersham) after validating the method. For comparison also the effect of muscarinic stimulation (10 mol/l carbachol in the presence of 10 mol/l timolol) on IP content was measured in corresponding preparations. A basal IP level of about 75 pmol/mg protein was found. There were no prominent effects of alpha-1-adrenoceptor stimulation on total IP content in isolated perfused rat hearts. Phenylephrine gave a statistically significant increase of about 40% at 1/4 min and a statistically significant decrease of about 25% at 4 min after start of exposure. Noradrenaline, however, gave no statistically significant change of IP at the time-points studied. Muscarinic stimulation caused a slight, statistically insignificant, increase of IP at 1/4 min. The results are compatible with an assumption that agonist stimulation evokes a localized increase of IP3 which may be masked by a relatively high total IP mass content. The IP peak after phenylephrine coincided with the early positive inotropic phase of the response reported earlier in perfused rat hearts for alpha-1-adrenoceptor stimulation by phenylephrine. Although this might be compatible with a role for IP in this early and transient phase, a mediator function of IP in the inotropic response is not established. [ABSTRACT FROM AUTHOR]

Kinetic behaviour of rat heart pyruvate dehydrogenase kinase (PDHKα) was studied in the multi-enzyme complex (PDC) contained in two preparations: mitochondria (mPDC) and a high speed pellet of Triton-extracted tissue (hPDC). Two parameters were evaluated: Va, related to V, and Fractional Pyruvate Inhibition (FPI). Starvation of rats for 48 h led to a rise in V and a fall in FPI. Injection into starved rats of agents which reduce β-oxidation of fatty acids restored, in succession, FPI and then V, of hPDC, to levels found in hPDC from fed animals. In vitro incubation at 30°C of hPDC from starved animals restored FPI, but not V, to 'fed' values; both were restored during in vitro incubation of mPDC from starved animals within the same time frame as in the in vivo experiments. A sharp increase of FPI, but not V, of hPDC from both fed and starved rats was observed in later experiments. This could have been due to differential selection of the two genes for isoenzymes of PDHKa proposed by other workers. [ABSTRACT FROM AUTHOR]

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca-pump activity and thereby may cause the occurrence of intracellular Ca overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca uptake and Ca-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca-stimulated ATPase activity and ATP-dependent Ca accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3-20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase. [ABSTRACT FROM AUTHOR]

In order to examine the mechanisms of the beneficial effects of vanadate on cardiac dysfunction in chronic diabetes, rat hearts were perfused with xanthine plus xanthine oxidase, an oxyradical generating system in the absence or presence of vanadate. The heart failed to generate contractile force and increased the resting tension markedly within 5 min of perfusion with xanthine plus xanthine oxidase. These changes were prevented by the addition of 4 μM vanadate in the perfusion medium. The protective effects of vanadate on the loss of developed tension and increased resting tension due to xanthine plus xanthine oxidase were dose-dependent (0.1-5 μM). Perfusion of the hearts with glucose-free medium did not abolish the protective actions of vanadate. The sarcolemmal Ca-pump (ATP-dependent Ca uptake and Ca-stimulated ATPase) and Na-dependent Ca uptake activities were decreased upon perfusing the hearts with a medium containing xanthine plus xanthine oxidase for 5 min; these effects were prevented by the addition of 2-4 μM vanadate in the perfusion medium. The signals for superoxide radicals produced by xanthine plus xanthine oxidase, as detected by electron paramagnetic resonance spectroscopic technique, were inhibited by 5-100 μM vanadate. These results suggest that vanadate is an oxyradical scavenger and thus may prevent heart dysfunction under some pathological conditions by its antioxidant action. [ABSTRACT FROM AUTHOR]

Stimulation of α-adrenoceptors (AR) during ischaemia in the rat heart by exogenous phenylephrine exacerbates reperfusion arrhythmias, an effect apparently mediated by the α-AR subtype. We tested whether α-AR stimulation by endogenous catecholamines, released during ischaemia, could modulate reperfusion arrhythmias, using as pharmacological tools the selective α-AR antagonists abanoquil (UK52046) and WB4101. Isolated rat hearts (n=12/group) were subjected to dual coronary perfusion. After 15 min of aerobic perfusion of both coronary beds, abanoquil or WB4101 was infused selectively into the left coronary bed (LCB) for 5 min. The LCB was then subjected to 10 min of zero-flow ischaemia and 5 min of reperfusion. Effects on PR interval, width of the ventricular complex (QRST) and reperfusion arrhythmias were assessed. Abanoquil at concentrations of 0.03, 0.1 and 0.3 μM tended to reduce the incidence of reperfusion-induced ventricular fibrillation (VF) in a dose-dependent manner from 75% in controls to 58, 33 and 25%, but this effects did not achieve statistical significance. Similarly, WB4101 at 0.1, 0.3 and 1 μM also tended to reduce VF incidence from 67% in controls to 67, 42% and 33% (NS). The incidence of ventricular tachycardia (VT) was 100% in all groups and ECG parameters were not altered significantly by either drug. These results suggest that, in this denervated isolated heart preparation, α-AR stimulation during ischaemia by endogenous catecholamines does not significantly modulate reperfusion arrhythmias. [ABSTRACT FROM AUTHOR]

The rat model of myocardial infarction is characterized by progressive cardiac hypertrophy and failure. Rats with infarcts greater than 30% of the left ventricle exhibited early and moderate, stages of heart failure 4 and 8 weeks after the occlusion of the left coronary artery, respectively. As heart failure is usually associated with remodeling of the extracellular matrix, a histological and biochemical study of cardiac collagenous proteins was carried out using failing hearts. Total collagen content in the right ventricle increased at 2, 4, and 8 weeks following occlusion of the left coronary artery whereas such a change in viable left ventricle was seen after 4 and 8 weeks. Total cardiac hydroxyproline concentration was increased in both right and left ventricular samples from the infarcted animals when compared to those of control; this increase was due to elevation of pepsin-insoluble collagen fraction. The myocardial noncollagenous/collagenous protein ratio was decreased in experimental right and left ventricular samples when compared to control samples. These findings suggest that an increase in cross-linking of cardiac collagen as well as disparate synthesis of collagenous and noncollagenous proteins occurs in this model of congestive heart, failure. [ABSTRACT FROM AUTHOR]