Sodium-glucose cotransporter 2 inhibitor Dapagliflozin attenuates diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus type 2 (DM2) is a risk factor for developing heart failure but there is no specific therapy for diabetic heart disease. Sodium glucose transporter 2 inhibitors (SGLT2I) are recently developed diabetic drugs that primarily work on the kidney. Clinical data describing the cardiovascular benefits of SGLT2Is highlight the potential therapeutic benefit of these drugs in the prevention of cardiovascular events and heart failure. However, the underlying mechanism of protection remains unclear. We investigated the effect of Dapagliflozin-SGLT2I, on diabetic cardiomyopathy in a mouse model of DM2. METHODS: Cardiomyopathy was induced in diabetic mice (db/db) by subcutaneous infusion of angiotensin II (ATII) for 30 days using an osmotic pump. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water. Male homozygous, 12-14 weeks old WT or db/db mice (n = 4-8/group), were used for the experiments. Isolated cardiomyocytes were exposed to glucose (17.5-33 mM) and treated with Dapagliflozin in vitro. Intracellular calcium transients were measured using a fluorescent indicator indo-1. RESULTS: Angiotensin II infusion induced cardiomyopathy in db/db mice, manifested by cardiac hypertrophy, myocardial fibrosis and inflammation (TNFα, TLR4). Dapagliflozin decreased blood glucose (874 ± 111 to 556 ± 57 mg/dl, p < 0.05). In addition it attenuated fibrosis and inflammation and increased the left ventricular fractional shortening in ATII treated db/db mice. In isolated cardiomyocytes Dapagliflozin decreased intracellular calcium transients, inflammation and ROS production. Finally, voltage-dependent L-type calcium channel (CACNA1C), the sodium-calcium exchanger (NCX) and the sodium-hydrogen exchanger 1 (NHE) membrane transporters expression was reduced following Dapagliflozin treatment. CONCLUSION: Dapagliflozin was cardioprotective in ATII-stressed diabetic mice. It reduced oxygen radicals, as well the activity of membrane channels related to calcium transport. The cardioprotective effect manifested by decreased fibrosis, reduced inflammation and improved systolic function. The clinical implication of our results suggest a novel pharmacologic approach for the treatment of diabetic cardiomyopathy through modulation of ion homeostasis.

Induction of heme oxygenase-1 protects mouse liver from apoptotic ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is the main cause of primary graft dysfunction of liver allografts. Cobalt-protoporphyrin (CoPP)-dependent induction of heme oxygenase (HO)-1 has been shown to protect the liver from I/R injury. This study analyzes the apoptotic mechanisms of HO-1-mediated cytoprotection in mouse liver exposed to I/R injury. HO-1 induction was achieved by the administration of CoPP (1.5 mg/kg body weight i.p.). Mice were studied in in vivo model of hepatic segmental (70 %) ischemia for 60 min and reperfusion injury. Mice were randomly allocated to four main experimental groups (n = 10 each): (1) A control group undergoing sham operation. (2) Similar to group 1 but with the administration of CoPP 72 h before the operation. (3) Mice undergoing in vivo hepatic I/R. (4) Similar to group 3 but with the administration of CoPP 72 h before ischemia induction. When compared with the I/R mice group, in the I/R+CoPP mice group, the increased hepatic expression of HO-1 was associated with a significant reduction in liver enzyme levels, fewer apoptotic hepatocytes cells were identified by morphological criteria and by immunohistochemistry for caspase-3, there was a decreased mean number of proliferating cells (positively stained for Ki67), and a reduced hepatic expression of: C/EBP homologous protein (an index of endoplasmic reticulum stress), the NF-κB's regulated genes (CIAP2, MCP-1 and IL-6), and increased hepatic expression of IκBa (the inhibitory protein of NF-κB). HO-1 over-expression plays a pivotal role in reducing the hepatic apoptotic IR injury. HO-1 may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation.

Conditioned medium from hypoxic cells protects cardiomyocytes against ischemia.

The hypothesis of the present study is that cardiomyocytes subjected to prolonged ischemia, may release survival factors that will protect new cardiac cells from ischemic stress. We exposed neonatal rat cardiomyocyte primary cultures to hypoxia, collected the supernatant, treated intact cardiac cells by this posthypoxic supernatant, and exposed them to hypoxia. The results show cardioprotection of the treated cells compared with the untreated ones. We named the collected posthypoxic supernatant "conditioned medium" (CM), which acts in a dose-dependent manner to protect new cardiac cells from hypoxia: 100 or 75% of CM diluted in phosphate-buffered saline (PBS) protected cells as if they were not exposed to hypoxia (P < 0.001). When CM was removed from the cells before hypoxia, protection was not observed. CM also protected skeletal muscle cultures from hypoxia, but not cardiac cells against H(2)O(2)-induced cell damage. Finally, CM treatment protected the isolated heart in Langendorff set-up against ischemia. Smaller infarct size (9.9 ± 4.4% vs. 28.3 ± 8.5%, P < 0.05), better Rate Pressure Product (67 ± 11% vs. 48.6 ± 13.4%, P < 0.05) and better rate of contraction and relaxation were observed following ischemia and reperfusion (1341 ± 399 mmHg/s vs. 951 ± 349 mmHg/s, P < 0.05 and 1053 ± 347 mmHg/s vs. 736 ± 314 mmHg/s, P < 0.05). To conclude, there are factors that are released from the heart cells subjected to ischemia/hypoxia that protects cardiomyocytes from ischemic stress.

Adenosine A(1) and A (3) receptor agonists reduce hypoxic injury through the involvement of P38 MAPK.

Activation of either the A(1) adenosine receptor (A(1)R) or the A(3) adenosine receptor (A(3)R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K(ATP)) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K(ATP) channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K(ATP) channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A(1)R or A(3)R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K(ATP) channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.

CYP-450 Epoxygenase Derived Epoxyeicosatrienoic Acid Contribute To Reversal of Heart Failure in Obesity-Induced Diabetic Cardiomyopathy via PGC-1 α Activation.

We have previously shown that an Epoxyeicosatrienoic Acid (EET) -agonist has pleiotropic effects and reverses cardiomyopathy by decreasing inflammatory molecules and increasing antioxidant signaling. We hypothesized that administration of an EET agonist would increase Peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α), which controls mitochondrial function and induction of HO-1 and negatively regulates the expression of the proinflammatory adipokines CCN3/NOV in cardiac and pericardial tissues. This pathway would be expected to further improve left ventricular (LV) systolic function as well as increase insulin receptor phosphorylation. Measurement of the effect of an EET agonist on oxygen consumption, fractional shortening, blood glucose levels, thermogenic and mitochondrial signaling proteins was performed. Control obese mice developed signs of metabolic syndrome including insulin resistance, hypertension, inflammation, LV dysfunction, and increased NOV expression in pericardial adipose tissue. EET agonist intervention decreased pericardial adipose tissue expression of NOV, while normalized FS, increased PGC-1α, HO-1 levels, insulin receptor phosphorylation and improved mitochondrial function, theses beneficial effect were reversed by deletion of PGC-1α. These studies demonstrate that an EET agonist increases insulin receptor phosphorylation, mitochondrial and thermogenic gene expression, decreased cardiac and pericardial tissue NOV levels, and ameliorates cardiomyopathy in an obese mouse model of the metabolic syndrome.

Bax deficiency reduces infarct size and improves long-term function after myocardial infarction.

We have previously found that, following myocardial ischemia/reperfusion injury, isolated hearts from bax gene knockout mice [Bax(-/-)] exhibited higher cardioprotection than the wild-type. We here explore the effect of Bax(-/-), following myocardial infarction (MI) in vivo. Homozygotic Bax(-/-) and matched wild-type were studied. Mice underwent surgical ligation of the left anterior descending coronary artery (LAD). The progressive increase in left-ventricular end diastolic diameter, end systolic diameter, in Bax(-/-) was significantly smaller than in Bax(+/+) at 28 d following MI (p < 0.03) as seen by echocardiography. Concomitantly, fractional shortening was higher (35 +/- 4.1% and 27 +/- 2.5%, p < 0.001) and infarct size was smaller in Bax(-/-) compared to the wild-type at 28 days following MI (24 +/- 3.7 % and 37 +/- 3.3%, p < 0.001). Creatine kinase and lactate dehydrogenase release in serum were lower in Bax(-/-) than in Bax(+/+) 24 h following MI. Caspase 3 activity was elevated at 2 h after MI only in the wild-type, but reduced to baseline values at 1 and 28 d post-MI. Bax knockout mice hearts demonstrated reduced infarct size and improved myocardial function following permanent coronary artery occlusion. The Bax gene appears to play a significant role in the post-MI response that should be further investigated.

Aprotinin improves myocardial recovery after ischemia and reperfusion. Effects of the drug on isolated rat hearts.

The effects of aprotinin, a protease inhibitor, on the ischemic and nonischemic isolated rat heart was investigated with the use of the modified Langendorff model. During phase I of the study, hearts were perfused with either low-dose aprotinin (10(5) KIU/L), high-dose aprotinin (10(6) KIU/L), or normal saline solution added to modified Krebs-Henseleit solution. No statistically significant differences in contraction amplitude, contractility, coronary flow, and wet/dry heart weight ratio were observed among the three groups of hearts. In phase II, hearts were exposed to a 40-minute period of global ischemia at 31 degrees C. Ischemic arrest was induced by warm cardioplegia. Before ischemia and during cardioplegia, hearts were perfused with either aprotinin 10(6) KIU/L (n = 10) or normal saline solution (n = 10) for 30 minutes. On reperfusion, recovery of hearts treated with aprotinin was significantly better than that of control hearts, as reflected by better contractility (analysis of variance, p = 0.011), higher coronary flow (p < 0.025), and lower creatine kinase levels (p < 0.05). No statistically significant differences in contraction amplitude were observed between the two groups. When the effect of ischemia within each group of hearts was analyzed, the preserving effect of aprotinin was even more pronounced. In the control group, ischemia caused a decrease in contractility (p < 0.025) and a decrease in oxygen consumption (p = 0.006); by contrast, in the aprotinin group the preischemic values were maintained. Accordingly, we conclude that aprotinin at concentrations up to 10(6) KIU/L has no deleterious effect on normally perfused hearts and has a significant protective effect on the ischemic heart when used in high doses in the preischemic period.

Effect of experimental cardioplegia methods on normal and hypertrophied rat hearts.

The purpose of this study was to evaluate whether the addition of verapamil hydrochloride to oxygenated glucose-rich cardioplegic solution would improve myocardial preservation. The Langendorff preparation of the isolated rat heart was used. Groups of normal (WKY) and hypertrophied (SHR) hearts were treated by five different cardioplegic methods and subjected to 90 or 30 minutes of ischemia at 28 degrees to 29 degrees C and reperfusion at 37 degrees C. The following cardioplegic solutions were used: Group A, cold (16 degrees C) Krebs-Henseleit (KH) glucose free only; Group B, KH with KCL (30 mEq/L) (16 degrees C); Group C, same as B with verapamil (10 microM); Group D, perfusion with oxygenated KH solution containing KCL (30 mEq/L) for 15 minutes prior to ischemia; and Group E, same as D with verapamil (10 microM). Recovery of contraction amplitude, ischemic contracture, coronary perfusate volume, the amount of creatine kinase in the coronary perfusate, heart rate, time of revival, O2 consumption, and ischemic contracture were measured. After 30 minutes of ischemia, we did not find any significant difference among the combinations tested with respect to contraction amplitude recovery. The hearts recovered fully. After 90 minutes of ischemia, we found that the best-protected groups in the normal hearts were Groups D and E. In the hypertrophied hearts, the addition of verapamil to the enhancement solution was harmful. The use of enhancement solution without verapamil prior to ischemia provided the best myocardial protection in the hypertrophied hearts.

Ro 5-4864 has a negative inotropic effect on human atrial muscle strips that is not antagonized by PK 11195.

The effects of Ro 5-4864, a peripheral benzodiazepine receptor agonist (9 x 10(-6) M and 9 x 10(-5) M) and of PK 11195, a peripheral benzodiazepine receptor antagonist (3 x 10(-6) M and 3 x 10(-5) M), alone or together, on contraction parameters of human atrial muscle strips were studied. Atrial muscle strips were obtained from patients undergoing cardiac surgery. The strips were suspended in Krebs-Henseleit solution at 36.8 +/- 0.2 degrees C, connected to an isometric force transducer and then stimulated at 15 V above threshold and paced at 1.5 Hz. Ro 5-4864 at its higher concentration, alone or mixed with PK 11195 at both concentrations, depressed the contraction amplitude to 80% of the control value (P < 0.05). In conclusion, Ro 5-4864 had a small but significant depressant effect on the contraction amplitude of human atrial strips. Surprisingly, this effect was not reversed by the peripheral benzodiazepine receptor antagonist PK 11195.

Dose and dose-rate dependence of the frequency of HPRT deficient T lymphocytes in the spleen of the 137Cs gamma-irradiated mouse.

The frequency of hypoxanthine phosphoribosyl transferase (HPRT) deficient splenic T lymphocytes was measured in the 137Cs gamma-irradiated mouse by the T cell cloning method. Doses from 0.3 to 6 Gy were applied at the dose-rates 0.5 Gy/min, 1 Gy/day and 1 Gy/week. Mutants were determined 8-10 and 30-40 weeks after the end of exposure. Radiation-induced mutant frequency (MFi) was calculated by subtracting the age corrected spontaneous mutant frequency (MFsp) from total mutant frequency (MF) found in irradiated animals. Data were fitted to linear and linear-quadratic dose-response models. MFi depended markedly on dose, dose-rate and time after exposure. When mutants were determined 8-10 weeks after acute irradiation (0.5 Gy/min) the dose-effect curve fitted the linear-quadratic equation MFi = 6.9 x 10(-6) Gy + 1.2 x 10(-6) Gy2, whereas in low dose-rate experiments (1 Gy/day, 1 Gy/week) the dose-effect curves were linear. The slope of the linear regression was about 3 x 10(-6). When low dose-rate-irradiated animals were killed 30-40 weeks after irradiation, MFi was about one-third of that observed after 8 weeks. The dose dose-rate effectiveness factor (DDREF) for radiation mutagenicity was calculated in animals that had been exposed 8-10 weeks previously. For doses < 2 Gy the reduction in effectiveness was about 1.5 when the irradiation dose-rate was < or = 1 Gy/day. For higher doses DDREF was 3-5.

The effect of free-electron collection on the recombination correction to ionization measurements of pulsed radiation.

Three models of the charge collection process in small dosimetric ionization chambers exposed to pulsed radiation are discussed. All three models allow for the presence of a free-electron component in the charge transfer, incorporating this into the model in slightly different ways, and the resulting collection efficiency formulae are compared over the range of variables normally met within clinical dosimetry. Measurements of the free-electron fraction for plane-parallel ionization chambers and for a Baldwin-Farmer 0.6 cm3 chamber are presented. The proportion of free electrons at the normal operating voltage is often high in small chambers but it is obvious that this can only lead to an increase in collection efficiency if the f-value calculated for purely ionic conduction allows for some improvement. Thus, a 50% free-electron fraction in a chamber which collects ions with efficiency f = 0.9950 at low pulse doses will increase this efficiency to only 0.9982. The same chamber, at the same operating voltage, and therefore the same free-electron fraction, if exposed to larger pulse doses, yielding an efficiency of 0.9531 as calculated for ions alone, would have a true efficiency of 0.9830-a large change.

Time resolving ionization monitor for microsecond radiation impulses.

The current induced in the outer circuit of a fast response ionization chamber exposed to pulsed radiation consists of two components, a fast one induced by free electrons and a slow one induced by ions. The fast electron component may be used for the representation of the shape of the ionizing pulse. In order to avoid interference with the slow ion current, the latter has to be removed from the signal. This is achieved by deriving a voltage course from the chamber signal which fits the shape of the ion component and subtracting this from the entire signal. The function of the electronic circuit used for this purpose is described. Some considerations about the time resolution of the chamber gas are to be found in the appendix.

Role of protease inhibition in myocardial preservation following ischemia and reperfusion.

Aprotinin, a naturally occurring protease inhibitor, in concentrations of 10(6) KIU/L was found to have no effect on myocardial performance in normally perfused isolated rat hearts, before ischemia. Given during the preischemic period, the drug had a significant protective effect on the reperfused hearts, following cardioplegic ischemia: better contractility upon reperfusion (p < 0.011), faster decline of the ischemic contracture, higher coronary flow (p < 0.025), lower AV-difference (p < 0.05), and lower CPK levels (p < 0.01).

Mechanical, biochemical, and structural effects of vitamin D deficiency on the chick heart.

The effects of vitamin D deprivation on the chick heart were investigated from three aspects: cardiac contractility (+/- dP/dT), intracellular high-energy phosphorus compounds, and structural differences. Four-week-old vitamin D-deficient chicks were divided into four groups: Group A served as the normal group and received subcutaneous injections of cholecalciferol; Groups B and C were vitamin D-deficient hearts but perfused differently; Group D received daily subcutaneous injections of 5 micrograms of 1,25(OH)2D3. When the isolated spontaneously beating hearts (modified Langendorff preparation) were perfused with Krebs-Henseleit (KH) solution containing a calcium concentration of 2.5mM, the myocardial contractility of the vitamin D-deficient hearts was significantly increased when compared with group A. After the isolated heart had beaten for one hour, the myocardial contractility in the vitamin D-deficient hearts was found to decline to significantly lower values. Presacrifice administration of 1,25(OH)2D3 improved cardiac performance. Vitamin D deficiency resulted in an enhanced rate of decline of the intracellular high-energy phosphorus compounds. No differences were found in the microscopic study. These observations suggest that vitamin D has a role in cardiac function.

Combined effect of glucose and verapamil in experimental cardioplegia.

In this study, we determined whether pre-ischemic enhancement of the ATP level and the addition of verapamil (a calcium blocker) to the cardioplegic solution could improve myocardial protection during cardiac arrest. Using the Langendorff preparation of the isolated rat heart plus different cardioplegic solutions, five groups of normal rat hearts and five groups of hypertrophied rat hearts were subjected to 90-minute ischemic periods at 28 degrees +/- 1 and reperfusion of 30 minutes at 37 degrees C. Group A received no cardioplegic solution; Group B received KCl, 30 mEq/L; Group C received type B perfusion with verapamil; and Group D received 15 minutes of pre-ischemic oxygenated enhancement perfusion containing KCl, 30 mEq/L, and glucose as substrate at 37 degrees C. Group E received the same perfusion as Group D, with the addition of verapamil to the enhancement perfusion. Light and E/M microscopy was performed on representative samples of left ventricular muscle. We found that pre-ischemic enhancement with KCl, glucose, and verapamil was only protective in the normal hearts after 90 minutes of ischemia. In hypertrophied hearts, the addition of verapamil to the enhancement solution was harmful. The use of pre-ischemic enhancement solution without verapamil provided the best myocardial protection after 90 minutes of ischemia in the hypertrophied hearts.

The influence of aprotinin on myocardial function after liver ischemia-reperfusion.

BACKGROUND: The beneficial effect of aprotinin, a naturally occurring protease inhibitor, on preservation of organs such as the liver, kidney and lung has been documented. OBJECTIVE: To explore the effects of hepatic ischemia and reperfusion on both liver and myocardial function, using a dual isolated perfused organ model with and without aprotinin. METHODS: Isolated rat livers were stabilized for 30 minutes with oxygenated modified Krebs-Henseleit solution at 37 degrees C. Livers were then perfused continuously with KH or KH + aprotinin 10(6) KIU/L for an additional 135 min. Livers of two other groups were made globally ischemic for 120 min, then perfused for 15 min with KH or with KH + aprotinin. Isolated hearts (Langendorff preparation) were stabilized for 30 min and then reperfused with KH or KH + aprotinin exiting the liver for 15 min. The liver's circuit was disconnected, and hearts were re-circulated with the accumulated liver + heart effluent for an additional 50 min. RESULTS: In the ischemia and ischemia + aprotinin groups, portal vein pressure (1 and 15 min reperfusion) was 331 +/- 99% and 339 +/- 61% vs. 308 +/- 81% and 193 +/- 35% of baseline, respectively (P < 0.03 vs. ischemia). There were no other differences in the enzyme leakage between aprotinin-treated or untreated ischemic livers. Left ventricular pressure was stable in the controls. However, LV pressure in groups perfused with ischemic liver effluent declined within 65 min reperfusion, whether aprotinin treated or not (84 +/- 8% and 73 +/- 5% of baseline, respectively, P < 0.004 only for ischemia vs. control). CONCLUSION: When aprotinin was used, LV pressure was inclined to be higher while liver portal vein pressure was lower, thus providing protection against liver and heart reperfusion injury.

An ultra-low dose of tetrahydrocannabinol provides cardioprotection.

UNLABELLED: Tetrahydrocannabinol (THC), the major psychoactive component of marijuana, is a cannabinoid agonist that exerts its effects by activating at least two specific receptors (CB1 and CB2) that belong to the seven transmembrane G-protein coupled receptor (GPCR) family. Both CB1 and CB2 mRNA and proteins are present in the heart. THC treatment was beneficial against hypoxia in neonatal cardiomyocytes in vitro. We also observed a neuroprotective effect of an ultra low dose of THC when applied to mice before brain insults. The present study was aimed to test and characterize the cardioprotective effects of a very low dose (0.002mg/kg) of THC which is 3-4 orders of magnitude lower than the conventional doses, administered before myocardial infarction in mice in vivo. Three regimens of THC administration were tested: single THC application 2h or 48h before the induction of infarct, or 3 weeks continuous treatment before MI. All protocols of THC administration were found to be beneficial. In the case of THC treatment 2h before MI, fractional shortening was elevated (37±4% vs. 42±1%, p<0.04), troponin T leakage to the blood was reduced (14±3ng/ml vs. 10±4ng/ml, p<0.008), infarct size decreased (29±4% vs. 23±4%, p<0.02), and the accumulation of neutrophils to the infarct area declined (36±10cells/field vs. 19±4cells/field, p<0.007) in THC- compared to vehicle-pretreated mice, 24h after MI. ERK1/2 phosphorylation following infarct was also inhibited by pre-treatment with THC (p<0.01). CONCLUSION: A single ultra low dose of THC before ischemia is a safe and effective treatment that reduces myocardial ischemic damage.

A histone deacetylase inhibitory prodrug - butyroyloxymethyl diethyl phosphate - protects the heart and cardiomyocytes against ischemia injury.

Butyroyloxymethyl diethylphosphate (AN-7) is a prodrug of butyric acid effective in reducing cardiotoxicity caused by chemotherapy. In this study, we tested whether AN-7 protects the heart and cardiomyocytes against ischemia injury. A single oral dose of AN-7 was given to mice or rats. Animals were sacrificed 1.5 or 24 h later and the hearts were subjected to ischemia and reperfusion ex-vivo (Langendorff). The mechanical performance was recorded throughout and the infarct size was measured at the end of reperfusion. Neonatal rat cardiomyocytes were subjected to 24-48 h hypoxia (1% O(2)) in the absence or presence of AN-7 and mitochondria damage and cell death were assessed. Proteins were analyzed by Western immunoblotting. In the two rodents, a single dose of AN-7 given in vivo preconditioned the hearts for improved functional recovery from ischemia and reperfusion performed ex-vivo. Both 1.5 h and 24 h treatments improved the pressure-related parameters whereas the coronary flow was ameliorated in the 24 h treatment only. Infarct size was smaller in the AN-7 treated hearts. In cardiomyocytes, AN-7 diminished the hypoxia induced dissipation of mitochondria membrane potential and cell death. Compared with untreated controls, AN-7-treated hearts recovering from global ischemia and cardiomyocytes undergoing hypoxia, displayed significantly higher levels of the cytoprotective heme oxygenase-1. Our findings indicate that AN-7 imparts cardioprotection against ischemia both in vivo and in vitro and emerges as a potential treatment modality for cardiac injury.

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor.

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7±4.08% vs. 33.5±2.7% respectively, p<0.001). However, the FS of P2Y(2)(-/-) receptor KO mice were not affected by UTP treatment (34.7±5.3% vs. 35.9±2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8±4.6 vs. 12±3.1 p<0.05). In contrast, P2Y(2)(-/-) receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y(2) receptor mediates UTP cardioprotection, in vivo.