[AMP-activated kinase activation inhibits transforming growth factor-ß1 production in cardiac fibroblasts via targeting C/EBPß].

AMP-activated protein kinase (AMPK) activation has been shown to protect against fibrosis. However, the underlying mechanism remains unclear. Here we explored the effect of AMPK activation on transforming growth factor-ß1 (TGFß1) production induced by angiotensin II (AngII) in cardiac fibroblasts and the underlying mechanisms. Adult mouse cardiac fibroblasts were isolated. TGFß1 and AMPK activity were determined by ELISA and Western blots, respectively. Pretreatment of AMPK activator AICAR inhibited TGFß1 production induced by AngII in cardiac fibroblasts, which was reversed by AMPK inhibitor compound C. Furthermore, bioinformatics predicted a potential CCAAT/enhancer-binding protein ß (C/EBPß) binding site in the promoter region of the mouse Tgfb1 gene. Luciferase reporter with wild type, but not deleted, C/EBPß binding sites transfection in mouse embryonic fibroblasts showed increased TGFß1 transcriptional activity induced by AngII, indicating that C/EBPß mediates AngII-induced TGFß1 transcript expression. Pretreatment of AICAR inhibited C/EBPß expression induced by AngII. In conclusion, AMPK activation inhibited TGFß1 production induced by AngII in cardiac fibroblasts through targeting C/EBPß. This finding provides a new mechanism underlying the anti-fibrogenic effects of AMPK activation.

The protective effect of rutin against ischemia/reperfusion-associated hemodynamic alteration through antioxidant activity.

Reactive oxygen species exert toxic effects during ischemia-reperfusion (I/R) injury of various organs. This study was designed to evaluate the preventive effects of various isoflavonoids such as biochanin A, daidzein, genistein, rutin and quercetin. These compounds are wellknown naturally occurring compounds with beneficial health effects and antioxidant activity. Free radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and superoxide dismutase (SOD) assay. Among the isoflavonoids tested, biochanine A, quercetin and rutin showed significant DPPH free radical scavenging activity. Similarly, treatment of biochanine A, genistein and rutin significantly increased SOD activity in neonant rat heart myocyte primary cells as well as in H9C2 cells. For ex vivo study, hearts from Sprague-Dawley rats were perfused in Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O(2) and 5% CO(2). Hearts were subjected to 20 min of pre-ischemia followed by 20 min of global ischemia, and then 50 min of reperfusion at 37°C. The test compounds were perfused 10 min before ischemia and during the entire reperfusion period. Among the isoflavonoids tested, only rutin significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/- dP/dtmax). In left ventricular end diastolic pressure (LVEDP) analysis, rutin, daidzein and biochanin A were effective. Among the isoflavonoids, rutin had consistent protective effects in I/R injury by affecting cardiac dynamic factors as well as by enhancing SOD and DPPH activity.

The Protective Effect of Epigallocatechin-3 Gallate on Ischemia/Reperfusion Injury in Isolated Rat Hearts: An ex vivo Approach.

The aim of this study was to evaluate the preventive role of epigallocatechin-3 gallate (EGCG, a derivative of green tea) in ischemia/reperfusion (I/R) injury of isolated rat hearts. It has been suggested that EGCG has beneficial health effects, including prevention of cancer and heart disease, and it is also a potent antioxidant. Rat hearts were subjected to 20 min of normoxia, 20 min of zero-flow ischemia and then 50 min of reperfusion. EGCG was perfused 10 min before ischemia and during the whole reperfusion period. EGCG significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/-dP/dtmax). EGCG also significantly increased the coronary flow (CF) at baseline before ischemia and at the onset of the reperfusion period. Moreover, EGCG decreased left ventricular end diastolic pressure (LVEDP). This study showed that lipid peroxydation was inhibited and Mn-SOD and catalase expressions were increased in the presence of EGCG. In addition, EGCG increased levels of Bcl-2, Mn-superoxide dismutase (SOD), and catalase expression and decreased levels of Bax and increased the ratio of Bcl-2/Bax in isolated rat hearts. Cleaved caspase-3 was decreased after EGCG treatment. EGCG markedly decreased the infarct size while attenuating the increase in lactate dehydrogenase (LDH) levels in the effluent. In summary, we suggest that EGCG has a protective effect on I/R-associated hemodynamic alteration and injury by acting as an antioxidant and anti-apoptotic agent in one.

Measuring adriamycin-induced cardiac hemodynamic dysfunction with a proteomics approach.

Adriamycin is a potent antitumor drug that causes severe cardiotoxicity. However, the toxic mechanisms are not clear. We used a proteomics approach to analyze changes in protein profiles after adriamycin-induced changes in hemodynamic factors. Although adriamycin itself did not affect left ventricular developed pressure (LVDP) or left ventricular end diastolic pressure (LVEDP), the drug did enhance susceptibility to ischemia-reperfusion-induced changes in LVDP, LVEDP and heart rate. Adriamycin altered the expression of 52 proteins, primarily energy metabolism and cytoskeleton proteins. Adriamycin decreased the expression of the metabolism-related proteins, ATP synthase, Sdha protein, Triose phosphate isomerase 1 (TPI-1), pyruvate dehydrogenase E1 alpha1, 6-phosphofructokinase, and fructose-1,6-bisphosphatase, as did cytoskeletal proteins, such as actin. Alterations in energy metabolism and subsequent free radical production may affect cytoskeletal protein expression, producing adriamycin-induced changes in cardiac hemodynamics.

Sulforaphane protects ischemic injury of hearts through antioxidant pathway and mitochondrial K(ATP) channels.

Reactive oxygen species are important mediators that exert a toxic effect during ischemia-reperfusion (I/R) injury of various organs. Sulforaphane is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. In this study, we investigated whether sulforaphane protects heart against I/R injury. Sprague-Dawley rats received sulforaphane (500microg/kg/day) or vehicle intraperitoneally for 3 days and global ischemia was performed using isolated perfused Langendorff hearts. Hearts were perfused with Krebs-bicarbonate buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Treatment with sulforaphane inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), +/-dP/dt, and coronary flow as compared with the untreated control hearts. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial K(ATP) channel blocker, for 10min before ischemia attenuated the improvement of LVEDP, LVDP, +/-dP/dt, and coronary flow induced by sulforaphane. Sulforaphane markedly decreased the infarcted size and attenuated the increased lactate dehydrogenase level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of sulforaphane. Post-ischemia increased the concentration of atrial natriuretic peptide in coronary effluent, which attenuated by sulforaphane treatment. Decreases on Mn-superoxide dismutase (SOD), catalase, and heme oxygenase-1 levels by I/R were increased by sulforaphane treatment and pretreatment of 5-HD blocked the sulforaphane effects. Increases in Bax and caspase-3 levels, and decrease in Bcl-2 level by I/R were attenuated by sulforaphane treatment. These results suggest that the protective effects of sulforaphane against I/R injury may be partly mediated through mitochondrial K(ATP) channels and antioxidant pathway.

Estrogen attenuates cardiac ischemia-reperfusion injury via inhibition of calpain-mediated bid cleavage.

Although several studies have shown that the administration of 17beta-estradiol (estrogen) is cardioprotective to ischemia-reperfusion (I/R), the molecular mechanisms are largely unknown. Therefore, we investigated the effects of estrogen on myocardial I/R injury in rat that were sham operated (Sham), ovariectomized (OVX), or ovariectomized and then given estrogen supplementation (OE). Langendorff-perfused rat hearts were subjected to I/R stimuli and the effects of estrogen were examined on cardiac performance. Additionally, we examined the mechanism of estrogen-mediated inhibition of apoptosis. Depression in cardiac contractile function and an increment of calpain activity were observed during I/R in the OVX rats. Estrogen replacement recovered cardiac contractile function and attenuated calpain activity, Bid cleavage, and caspases activities. Through in vitro assay using cardiomyocytes, we demonstrated that addition of H2O2 (100 microM) significantly increased calpain activity, which was attenuated by estrogen. Moreover, calpain activity was inhibited by calpain inhibitors such as ALLN or leupeptin, but not by caspase-8 inhibitor peptide. These results suggest that estrogen protects the heart against I/R injury through the decrease of calpain activity, Bid cleavage and caspase-8 activity. These apoptotic mechanisms may play a critical role on I/R-associated cardiac damage.

Dendroaspis natriuretic peptide protects the post-ischemic myocardial injury.

The present study used isolated rat hearts to investigate whether (1) Dendroaspis natriuretic peptide (DNP) is protective against post-ischemic myocardial dysfunction, and (2) whether the cardioprotective effects of DNP is related to alteration of Bcl-2 family protein levels. The excised hearts of Sprague-Dawley rats were perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O2 and 5% CO2. Left ventricular end-diastolic pressure (LVEDP, mmHg), left ventricular developed pressure (LVDP, mmHg) and coronary flow (CF, ml/min) were continuously monitored. In the presence of 50 nM DNP, all hearts were perfused for a total of 100 min consisting of a 20 min pre-ischemic period followed by a 30 min global ischemia and 50 min reperfusion. Lactate dehydrogenase (LDH) activity in the effluent was measured during reperfusion. Treatment with DNP alone improved the pre-ischemic LVEDP and post-ischemic LVEDP significantly comparing with the untreated control hearts during reperfusion. However, DNP did not affect the LVDP, heart rate (HR, beats/min), and CF. Bcl-2, an anti-apoptotic protein expressed in ischemic myocardium of DNP+ischemia/reperfusion (I/R) group, was higher than that in I/R alone group. Bax, a pro-apoptotic protein expressed in ischemic myocardium of DNP+I/R group, has no significant difference compared with I/R alone group. These results suggest that the protective effects of DNP against I/R injury would be mediated, at least in part, through the increased ratio of Bcl-2 to Bax protein after ischemia-reperfusion.

Dendroaspis natriuretic peptide induces the apoptosis of cardiac muscle cells.

Early heart failure is characterized by elevated plasma Dendroaspis natriuretic peptide-like immunoreactivity (DNP-LI). However, the direct effects of DNP on heart or the heart-associated cell system are not well known. Therefore, we investigated whether DNP induces the apoptosis of H9c2 cardiac muscle cells. H9c2 cardiac muscle cells and rat neonatal cardiomyocytes were treated with various concentrations of DNP. Cell viability and nuclear morphology change were determined by trypan blue staining and Hoechst 33258 staining, respectively. Caspase-3-like activity was measured using specific fluorogenic substrates. Pro-and antiapoptotic proteins were assayed by Western blotting. DNP induced the apoptosis of H9c2 cardiac muscle cells in a dose-dependent manner. Maximum effects occurred at 100 nM concentration of DNP, with a 7-8-fold increase in apoptotic cells, to reach a maximum apoptotic index of 17%. We also identified that H9c2 cardiac muscle cells expressed Natriuretic peptide reactor -A and -B, which respond to DNP to generate cGMP. The treatment with DNP also markedly reduced levels of Bcl-2, inhibitor of apoptosis protein-1, and inhibitor of apoptosis protein-2 and increased the level of Bax and cytochrome c release into cytoplasm and subsequent caspase-3 activation, which co-occurred with increased apoptosis. DNP-induced apoptosis was mediated by cyclic GMP, and this effect was mimicked by dibutylyl-cGMP (30 microM), a membrane permeable analog of cGMP. Furthermore, DNP-induced apoptosis was observed in rat neonatal cardiomyocytes. These results suggest that DNP induces the apoptosis of H9c2 cardiac muscle cells and of cardiomyocytes via cGMP and demonstrate that the operative mechanism includes the regulation of Bcl-2 family proteins.