AST-120, an Adsorbent of Uremic Toxins, Improves the Pathophysiology of Heart Failure in Conscious Dogs.

PURPOSE: Several lines of evidence suggest that renal dysfunction is associated with cardiovascular toxicity through the action of uremic toxins. The levels of those uremic toxins can be reportedly reduced by the spherical carbon adsorbent AST-120. Because heart failure (HF) causes renal dysfunction by low cardiac output and renal edema, the removal of uremic toxins could be cardioprotective. METHOD: To determine whether blood levels of the uremic toxin indoxyl sulfate (IS) increase in HF and whether AST-120 can reduce those levels and improve HF. We induced HF in 12 beagle dogs by 6 weeks of rapid right ventricular pacing at 230 beats per min. We treated six dogs with a 1-g/kg/day oral dosage of AST-120 for 14 days from week 4 after the start of rapid ventricular pacing. The other six dogs did not receive any treatment (control group). RESULTS: In the untreated dogs, IS levels increased as cardiac function deteriorated. In contrast, plasma IS levels in the treated dogs decreased to baseline levels, with both left ventricular fractional shortening and pulmonary capillary wedge pressure also improving when compared with untreated dogs. Finally, AST-120 treatment was shown to reduce both myocardial apoptosis and fibrosis along with decreases in extracellular signal-regulated kinase phosphorylation, the Bax/Bcl-2 ratio, and TGF-ß1 expression and increases in AKT phosphorylation. CONCLUSIONS: IS levels are increased in HF. AST-120 treatment reduces the levels of IS and improves the pathophysiology of HF in a canine model. AST-120 could be a novel candidate for the treatment of HF.

Dynamic changes in plasma total and high molecular weight adiponectin levels in acute heart failure.

BACKGROUND: Elevated levels of total plasma adiponectin (APN) and high molecular weight (HMW)-APN have been observed in chronic heart failure (HF) and are associated with poor prognosis, however, the response of APN levels in acute HF is not known. The purpose of this study was to clarify the dynamic changes of the plasma total APN, HMW-APN levels, and the ratio of HMW-APN to total APN (HMWR) in acute HF. METHODS: From February 2006 to January 2007, 20 patients with acute HF (non-ischemic and non-valvular origin, 17 men, aged 63±11 years) were enrolled, and blood was sampled before the onset of the treatment and at discharge. Ten patients admitted for the treatment of supraventricular arrhythmia (8 men, aged 45±13 years) were included as controls. RESULTS: The medians and interquartile ranges of the plasma total APN, HMW-APN levels, and HMWR at admission were 20.8 (14.5-38.9) µg/mL, 12.4 (7.7-23.3) µg/mL, and 0.60 (0.50-0.69), respectively. The total APN and HMW-APN values were significantly higher than the values of the control. The plasma total APN, HMW-APN, and HMWR values at discharge decreased to 19.4 (7.2-27.3)µg/mL, 10.5 (3.2-12.8) µg/mL, and 0.52 (0.46-0.57), respectively. An exploratory survival analysis showed that the higher HMWR values at admission and the larger decrease in HMWR were associated with a better prognosis after discharge. CONCLUSION: Plasma total APN and HMW-APN values are elevated at the admission for acute HF. Plasma total APN, HMW-APN, and HMWR values decrease following treatment. Higher HMWR at admission and its larger decrease may be the signs of favorable treatment responsiveness in acute HF.

Natriuretic peptides enhance the production of adiponectin in human adipocytes and in patients with chronic heart failure.

OBJECTIVES: We investigated the functional relationship between natriuretic peptides and adiponectin by performing both experimental and clinical studies. BACKGROUND: Natriuretic peptides are promising candidates for the treatment of congestive heart failure (CHF) because of their wide range of beneficial effects on the cardiovascular system. Adiponectin is a cytokine derived from adipose tissue with various cardiovascular-protective effects that has been reported to show a positive association with plasma brain natriuretic peptide (BNP) levels in patients with heart failure. METHODS: The expression of adiponectin messenger ribonucleic acid (mRNA) and its secretion were examined after atrial natriuretic peptide (ANP) or BNP was added to primary cultures of human adipocytes in the presence or absence of HS142-1 (a functional type A guanylyl cyclase receptor antagonist). Changes of the plasma adiponectin level were determined in 30 patients with CHF who were randomized to receive intravenous ANP (0.025 microg/kg/min human ANP for 3 days, n = 15) or saline (n = 15). RESULTS: Both ANP and BNP dose-dependently enhanced the expression of adiponectin mRNA and its secretion, whereas such enhancement was inhibited by pre-treatment with HS142-1. The plasma adiponectin level was increased at 4 days after administration of human ANP compared with the baseline value (from 6.56 +/- 0.40 microg/ml to 7.34 +/- 0.47 microg/ml, p < 0.05), whereas there was no change of adiponectin in the saline group (from 6.53 +/- 0.57 microg/ml to 6.55 +/- 0.56 microg/ml). CONCLUSIONS: Natriuretic peptides enhance adiponectin production by human adipocytes in vitro and even in patients with CHF, which might have a beneficial effect on cardiomyocytes in patients receiving recombinant natriuretic peptide therapy for heart failure.

Metformin prevents progression of heart failure in dogs: role of AMP-activated protein kinase.

BACKGROUND: Some studies have shown that metformin activates AMP-activated protein kinase (AMPK) and has a potent cardioprotective effect against ischemia/reperfusion injury. Because AMPK also is activated in animal models of heart failure, we investigated whether metformin decreases cardiomyocyte apoptosis and attenuates the progression of heart failure in dogs. METHODS AND RESULTS: Treatment with metformin (10 micromol/L) protected cultured cardiomyocytes from cell death during exposure to H2O2 (50 micromol/L) via AMPK activation, as shown by the MTT assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and flow cytometry. Continuous rapid ventricular pacing (230 bpm for 4 weeks) caused typical heart failure in dogs. Both left ventricular fractional shortening and left ventricular end-diastolic pressure were significantly improved in dogs treated with oral metformin at 100 mg x kg(-1) x d(-1) (n=8) (18.6+/-1.8% and 11.8+/-1.1 mm Hg, respectively) compared with dogs receiving vehicle (n=8) (9.6+/-0.7% and 22+/-0.9 mm Hg, respectively). Metformin also promoted phosphorylation of both AMPK and endothelial nitric oxide synthase, increased plasma nitric oxide levels, and improved insulin resistance. As a result of these effects, metformin decreased apoptosis and improved cardiac function in failing canine hearts. Interestingly, another AMPK activator (AICAR) had effects equivalent to those of metformin, suggesting the primary role of AMPK activation in reducing apoptosis and preventing heart failure. CONCLUSIONS: Metformin attenuated oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with activation of AMPK. Therefore, metformin may be a potential new therapy for heart failure.

Crossveinless-2 controls bone morphogenetic protein signaling during early cardiomyocyte differentiation in P19 cells.

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of down-stream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.

Atorvastatin slows the progression of cardiac remodeling in mice with pressure overload and inhibits epidermal growth factor receptor activation.

The aim of this study was to investigate whether atorvastatin inhibits epidermal growth factor receptor (EGFR) activation in cardiomyocytes in vitro and slows the progression of cardiac remodeling induced by pressure overload in mice. Either atorvastatin (5 mg/kg/day) or vehicle was orally administered to male C57BL/6J mice with transverse aortic constriction (TAC). Physiological parameters were obtained by echocardiography or left ventricular (LV) catheterization, and morphological and molecular parameters of the heart were also examined. Furthermore, cultured neonatal rat cardiomyocytes were studied to clarify the underlying mechanisms. Four weeks after TAC, atorvastatin reduced the heart/body weight and lung/body weight ratios (8.69+/-0.38 to 6.45+/-0.31 mg/g (p<0.001) and 10.89+/-0.68 to 6.61+/-0.39 mg/g (p<0.01) in TAC mice with and without atorvastatin, respectively). Decrease of LV end-diastolic pressure and the time constant of relaxation, increased fractional shortening, downregulation of a disintegrin and metalloproteinase (ADAM)12, ADAM17 and heparin-binding epidermal growth factor genes, and reduction of the activity of EGFR and extracellular signal-regulated kinase (ERK) were observed in the atorvastatin group. Phenylephrine-induced protein synthesis, phosphorylation of EGFR, and activation of ERK in neonatal rat cardiomyocytes were all inhibited by atorvastatin. These findings indicated that atorvastatin ameliorates cardiac remodeling in mice with pressure overload, and its actions are associated with inhibition of the EGFR signaling pathway.

Identification of p32 as a novel substrate for ATM in heart.

Chemotherapeutic agents to induce DNA damage have been limited to use due to severe side effects of cardiotoxicity. ATM (Ataxia-telangiectasia mutated) is an essential protein kinase in triggering DNA damage responses. However, it is unclear how the ATM-mediated DNA damage responses are involved in the cardiac cell damage. To elucidate these functions in heart, we searched for specific substrates of ATM from mouse heart homogenate. Combining an in vitro phosphorylation following anion-exchange chromatography with purification by reverse-phase high-performance liquid chromatography (HPLC), we successfully identified p32, an ASF/SF2-associated protein, as a novel substrate for ATM. An in vitro kinase assay using recombinant p32 revealed that ATM directly phosphorylated p32. Furthermore, we determined Ser 148 of p32 as an ATM phosphorylation site. Since p32 is known to regulate mRNA splicing and transcription, p32 phosphorylation by ATM might be a new transcriptional regulatory pathway for specific DNA damage responses in heart.

Impact of blockade of histamine H2 receptors on chronic heart failure revealed by retrospective and prospective randomized studies.

OBJECTIVES: The goal of this work was to determine whether the blockade of histamine H2 receptors is beneficial for the pathophysiology of chronic heart failure (CHF). BACKGROUND: Because CHF is one of the major life-threatening diseases, we need to find a novel effective therapy. Intriguingly, our previous study, which predicts the involvement of histamine in CHF, suggests that we should test this hypothesis in patients with CHF. METHODS: We selected 159 patients who received famotidine among symptomatic CHF patients for the retrospective study. We blindly selected age- and gender-matched CHF patients receiving drugs for gastritis other than histamine H2 receptor blockers as a control group. For the prospective study, 50 symptomatic CHF patients were randomly divided into 2 groups. One group received famotidine of 30 mg/day for 6 months, and the other group received teprenone. RESULTS: In the retrospective study, famotidine of 20 to 40 mg decreased both left ventricular end-diastolic and end-systolic lengths (LVDd and LVDs, respectively) and the plasma B-type natriuretic peptide (BNP) levels (182 +/- 21 vs. 259 +/- 25 pg/ml, p < 0.05) with unaltered fractional shortening (FS). In a randomized, open-label study, compared with teprenone, famotidine of 30 mg prospectively decreased both New York Heart Association functional class (p < 0.05) and plasma BNP levels (183 +/- 26 pg/ml vs. 285 +/- 41 pg/ml, p < 0.05); this corresponded to decreasing both LVDd (57 +/- 2 mm vs. 64 +/- 2 mm, p < 0.05) and LVDs (47 +/- 2 mm vs. 55 +/- 2 mm, p < 0.05) with unaltered FS (15 +/- 1% vs. 17 +/- 1%). The frequency of readmission because of worsening of CHF was lower in the famotidine group (4% and 24%, p < 0.05). On the other hand, teprenone had no effects on CHF. CONCLUSIONS: Famotidine improved both cardiac symptoms and ventricular remodeling associated with CHF. Histamine H2 receptor blockers may have therapeutic benefits for CHF.

Granulocyte colony-stimulating factor mediates cardioprotection against ischemia/reperfusion injury via phosphatidylinositol-3-kinase/Akt pathway in canine hearts.

PURPOSE: Recent studies suggest that G-CSF prevents cardiac remodeling following myocardial infarction (MI) likely through regeneration of the myocardium and coronary vessels. However, it remains unclear whether G-CSF administered at the onset of reperfusion prevents ischemia/reperfusion injury in the acute phase. We investigated acute effects of G-CSF on myocardial infarct size and the incidence of lethal arrhythmia and evaluated the involvement of the phosphatidylinositol-3 kinase (PI3K) in the in vivo canine models. METHODS: In open-chest dogs, left anterior descending coronary artery (LAD) was occluded for 90 minutes followed by 6 hours of reperfusion. We intravenously administered G-CSF (0.33 micro/kg/min) for 30 minutes from the onset of reperfusion. Wortmannin, a PI3K inhibitor, was selectively administered into the LAD after the onset of reperfusion. RESULTS: G-CSF significantly (p<0.05) reduced myocardial infarct size (38.7+/-4.3% to 15.7+/-5.3%) and the incidence of ventricular fibrillation during reperfusion periods (50% to 0%) compared with the control. G-CSF enhanced Akt phospholylation in ischemic canine myocardium. Wortmannin blunted both the infarct size-limiting and anti-arrhythmic effects of G-CSF. G-CSF did not change myeloperoxidase activity, a marker of neutrophil accumulation, in the infarcted myocardium. CONCLUSION: An intravenous administration of G-CSF at the onset of reperfusion attenuates ischemia/reperfusion injury through PI3K/Akt pathway in the in vivo model. G-CSF administration can be a promising candidate for the adjunctive therapy for patients with acute myocardial infarction.

Blockade of histamine H2 receptors protects the heart against ischemia and reperfusion injury in dogs.

We have previously reported that histamine H(2) blockers may be cardioprotective in patients with chronic heart failure. Since both endogenous histamine and histamine H(2) receptors are present in heart tissue, we tested the hypothesis that the blockade of histamine H(2) receptors mediates protection against reversible or irreversible ischemia and reperfusion injury. In open-chest dogs, the left anterior descending coronary artery was occluded for 90 minutes, followed by reperfusion for 6 hours. Administration of famotidine and cimetidine from 10 minutes before occlusion until after 1 hour of reperfusion reduced infarct size (17.0 +/- 4.1% and 17.8 +/- 2.9% vs. 36.9 +/- 5.9% of the solvent group, respectively) Famotidine administration only during the reperfusion period for 1 hour also attenuated infarct size (22.5 +/- 3.5%). There were no differences in either area at risk or collateral flow among the groups. In another set of experiments, we decreased coronary perfusion pressure in dogs so that the coronary blood flow decreased to 50% of the non-ischemic level. In such conditions, we observed the increases in histamine release compared with non-ischemic conditions (0.04 +/- 0.03 to 0.28 +/- 0.13 ng/ml, p < 0.05). Famotidine improved anaerobic myocardial metabolism gauged by both lactate extraction ratio and myocardial oxygen consumption. We conclude that the blockade of histamine H(2) receptors mediates improvements in the anaerobic myocardial metabolism, and thus protects against ischemia and reperfusion injury.

A role of opening of mitochondrial ATP-sensitive potassium channels in the infarct size-limiting effect of ischemic preconditioning via activation of protein kinase C in the canine heart.

The opening of mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels triggers or mediates the infarct size (IS)-limiting effect of ischemic preconditioning (IP). Because ecto-5'-nucleotidase related to IP is activated by PKC, we tested whether the opening of mitoK(ATP) channels activates PKC and contributes to either activation of ecto-5'-nucleotidase or IS-limiting effect. In dogs, IP procedure decreased IS and activated ecto-5'-nucleotidase, both of which were mimicked by transient exposure to either cromakalim or diazoxide, and these effects were blunted by either GF109203X (a PKC inhibitor) or 5-hydroxydecanoate (a mitoK(ATP) channel blocker), but not by HMR-1098 (a surface sarcolenmal K(ATP) channel blocker). Either cromakalim or diazoxide activated both PKC and ecto-5'-nucleotidase, which was blunted by either GF109203X or 5-hydroxydecanoate, but not by HMR-1098. We concluded that the opening of mitoK(ATP) channels contributes to either activation of ecto-5'-nucleotidase or the infarct size-limiting effect via activation of PKC in canine hearts.

A calcium channel blocker amlodipine increases coronary blood flow via both adenosine- and NO-dependent mechanisms in ischemic hearts.

Amlodipine reduces oxidative stress that decreases NO and adenosine release. This study was undertaken to examine whether amlodipine mediates coronary vasodilation and improves myocardial metabolism and contractility in ischemic hearts via either adenosine- or NO-dependent mechanisms. In open-chest dogs, amlodipine (2 mug kg per min) was infused at the minimum dose that caused maximal coronary vasodilation. The perfusion pressure was reduced in the left anterior descending coronary artery so that coronary blood flow (CBF) decreased by 50%. Amlodipine increased the difference of the adenosine level (VAD (Ado): 119+/-14 to 281+/-46 nM) and the nitrate+nitrite level (VAD (NOx): 7.8+/-1.3 to 16.1+/-1.1 muM) between coronary venous and coronary arterial blood, and also increased CBF (50+/-3 to 69+/-6 ml/100 g/min). These changes were partially reversed by either 8-sulfophenyeltheophylline (8SPT) or l(omega)-nitro arginine methyl ester (l-NAME), and were completely blocked by both 8SPT and l-NAME. The reduction of CBF increased VAD (8-iso-prostaglandin F(2alpha)), and this increase was reduced by amlodipine (10.8+/-1.1 to 5.0+/-0.5 pg/ml). In addition, pretreatment with superoxide dismutase mimicked the coronary effects of amlodipine and blunted the response to amlodipine administration. Amlodipine-induced coronary vasodilation via both adenosine- and NO-dependent mechanisms. Adenosine and NO may interact in ischemic hearts to mediate coronary vasodilation by amlodipine.

Aldosterone nongenomically worsens ischemia via protein kinase C-dependent pathways in hypoperfused canine hearts.

Rapid nongenomic actions of aldosterone independent of mineralocorticoid receptors (MRs) on vascular tone are divergent. Until now, the rapid nongenomic actions of aldosterone on vascular tone of coronary artery and cardiac function in the in vivo ischemic hearts were not still fully estimated. Furthermore, although aldosterone can modulate protein kinase C (PKC) activity, there is no clear consensus whether PKC is involved in the nongenomic actions of aldosterone on the ischemic hearts. In open chest dogs, the selective infusion of aldosterone into the left anterior descending coronary artery (LAD) reduced coronary blood flow (CBF) in the nonischemic hearts in a dose-dependent manner. Also, in the ischemic state that CBF was decreased to 33% of the baseline, the intracoronary administration of aldosterone (0.1 nmol/L) rapidly decreased CBF (37.4+/-3.8 to 19.3+/-5.2 mL/100 g/min; P<0.05), along with decreases in fractional shortening (FS) (8.4+/-0.7 to 5.4+/-0.4%; P<0.05) and lactate extraction rate (LER) (-31.7+/-2.9 to -41.4+/-3.7%; P<0.05). The decrease in CBF was reproduced by the infusion of bovine serum albumin-conjugated aldosterone. Notably, these aldosterone-induced deteriorations of myocardial contractile and metabolic functions were blunted by the co-administration of GF109203X, an inhibitor of PKC, but not spironolactone. In addition, aldosterone activated vascular PKC. These results indicate that aldosterone nongenomically induces vasoconstriction via PKC-dependent pathways possibly through membrane receptors, which leads to the worsening of the cardiac contractile and metabolic functions in the ischemic hearts. Elevation of plasma or cardiac aldosterone levels may be deleterious to ischemic heart disease through its nongenomic effects.

Benidipine, a long-acting calcium channel blocker, inhibits cardiac remodeling in pressure-overloaded mice.

OBJECTIVE: The effects of long-acting calcium channel blockers (CCBs) on pressure overload-induced cardiac remodeling are seldom studied in animals. We evaluated the effects of benidipine, a long-acting CCB, on cardiac remodeling. METHODS: Rat neonatal cardiac myocytes were used to examine the influence of benidipine on protein synthesis. Cardiac remodeling was induced in C57 B6/J mice by transverse aortic constriction (TAC). Then the effects of benidipine (10 mg/kg/d) were assessed on myocardial hypertrophy and heart failure, cardiac histology, and gene expression. RESULTS: Benidipine significantly inhibited protein synthesis by cardiac myocytes stimulated with phenylephrine (PE), and this effect was partially abolished by cotreatment with a nitric oxide synthase (NOS) inhibitor [N(G)-nitro-l-arginine methylester (l-NAME)]. Four weeks after the onset of pressure overload, benidipine therapy potently inhibited cardiac hypertrophy and prevented heart failure. The heart to body weight ratio was 6.89+/-0.48 mg/g in treated mice vs. 8.76+/-0.33 mg/g in untreated mice (P<0.01), and the lung to body weight ratio was 7.39+/-0.93 mg/g vs. 10.53+/-0.99 mg/g, respectively (P<0.05). Left ventricular fractional shortening (LVFS) was improved on echocardiography. Plasma NO levels were increased, while B type natriuretic peptide, protein inhibitor of neuronal NOS, and procollagen IV alpha were down-regulated in benidipine-treated mice. CONCLUSION: These results indicate that benidipine inhibits cardiac remodeling due to pressure overload at least partly by acting on the nitric oxide signaling pathway.

Optimal windows of statin use for immediate infarct limitation: 5'-nucleotidase as another downstream molecule of phosphatidylinositol 3-kinase.

BACKGROUND: Although statins are reported to have a cardioprotective effect, their immediate direct influence on ischemia-reperfusion injury and the underlying mechanisms remain obscure. We investigated these issues an in vivo canine model. METHODS AND RESULTS: Dogs were subjected to coronary occlusion (90 minutes) and reperfusion (6 hours) immediately after injection of pravastatin (0.2, 2, or 10 mg/kg), pitavastatin (0.01, 0.1, or 0.5 mg/kg), or cerivastatin (0.5, 5, or 50 microg/kg). Then myocardial phosphatidylinositol 3-kinase (PI3-K) and 5'-nucleotidase activities were measured, as well as infarct size. After 15 minutes of reperfusion, pravastatin caused dose-dependent activation of Akt and ecto-5'-nucleotidase in the ischemic zone, and the effect was significant at higher doses. Pitavastatin also significantly increased these activities, and its optimal dose was within the clinical range, whereas cerivastatin caused activation at the lowest dose tested. In all cases, both Akt and ecto-5'-nucleotidase showed activation in parallel, and this activation was completely abolished by wortmannin, a PI3-K inhibitor. The magnitude of the infarct-limiting effect paralleled the increase in Akt and ecto-5'-nucleotidase activity and was blunted by administration of wortmannin, alpha,beta-methyleneadenosine-5'-diphosphate, or 8-sulfophenyltheophylline during reperfusion. Both collateral flow and the area at risk were comparable for all groups. CONCLUSIONS: Activation of ecto-5'-nucleotidase after ischemia by PI3-K activation may be crucial for immediate infarct-size limitation by statins. There seems to be an optimal dose for each statin that is independent of its clinical cholesterol-lowering effect.

Celiprolol, a vasodilatory beta-blocker, inhibits pressure overload-induced cardiac hypertrophy and prevents the transition to heart failure via nitric oxide-dependent mechanisms in mice.

BACKGROUND: The blockade of beta-adrenergic receptors reduces both mortality and morbidity in patients with chronic heart failure, but the cellular mechanism remains unclear. Celiprolol, a selective beta(1)-blocker, was reported to stimulate the expression of endothelial NO synthase (eNOS) in the heart, and NO levels have been demonstrated to be related to myocardial hypertrophy and heart failure. Thus, we aimed to clarify whether celiprolol attenuates both myocardial hypertrophy and heart failure via the NO-signal pathway. METHODS AND RESULTS: In rat neonatal cardiac myocytes, celiprolol inhibited protein synthesis stimulated by either isoproterenol or phenylephrine, which was partially suppressed by N(G)-nitro-L-arginine methyl ester (L-NAME). Four weeks after transverse aortic constriction (TAC) in C57BL/6 male mice, the ratio of heart weight to body weight (mg/g) (8.70+/-0.42 in TAC, 6.61+/-0.44 with celiprolol 100 mg x kg(-1) x d(-1) PO, P<0.01) and the ratio of lung weight to body weight (mg/g) (10.27+/-1.08 in TAC, 7.11+/-0.70 with celiprolol 100 mg x kg(-1) x d(-1) PO, P<0.05) were lower and LV fractional shortening was higher in the celiprolol-treated groups than in the TAC group. All of these improvements were blunted by L-NAME. Celiprolol treatment significantly increased myocardial eNOS and activated phosphorylation of eNOS. Myocardial mRNA levels of natriuretic peptide precursor type B and protein inhibitor of NO synthase, which were increased in the TAC mice, were decreased in the celiprolol-treated mice. CONCLUSIONS: These findings indicated that celiprolol attenuates cardiac myocyte hypertrophy both in vitro and in vivo and halts the process leading from hypertrophy to heart failure. These effects are mediated by a selective beta1-adrenergic receptor blockade and NO-dependent pathway.

Protein kinase A as another mediator of ischemic preconditioning independent of protein kinase C.

BACKGROUND: We and others have reported that transient accumulation of cyclic AMP (cAMP) in the myocardium during ischemic preconditioning (IP) limits infarct size independent of protein kinase C (PKC). Accumulation of cAMP activates protein kinase A (PKA), which has been demonstrated to cause reversible inhibition of RhoA and Rho-kinase. We investigated the involvement of PKA and Rho-kinase in the infarct limitation by IP. METHODS AND RESULTS: Dogs were subjected to 90-minute ischemia and 6-hour reperfusion. We examined the effect on Rho-kinase activity during sustained ischemia and infarct size of (1) preischemic transient coronary occlusion (IP), (2) preischemic activation of PKA/PKC, (3) inhibition of PKA/PKC during IP, and (4) inhibition of Rho-kinase or actin cytoskeletal deactivation during myocardial ischemia. Either IP or dibutyryl-cAMP treatment activated PKA, which was dose-dependently inhibited by 2 PKA inhibitors (H89 and Rp-cAMP). IP and preischemic PKA activation substantially reduced infarct size, which was blunted by preischemic PKA inhibition. IP and preischemic PKA activation, but not PKC activation, caused a substantial decrease of Rho-kinase activation during sustained ischemia. These changes were cancelled by preischemic inhibition of PKA but not PKC. Furthermore, either Rho-kinase inhibition (hydroxyfasudil or Y27632) or actin cytoskeletal deactivation (cytochalasin-D) during sustained ischemia achieved the same infarct limitation as preischemic PKA activation without affecting systemic hemodynamic parameters, the area at risk, or collateral blood flow. CONCLUSIONS: Transient preischemic activation of PKA reduces infarct size through Rho-kinase inhibition and actin cytoskeletal deactivation during sustained ischemia, implicating a novel mechanism for cardioprotection by ischemic preconditioning independent of PKC and a potential new therapeutic target.

Beta-adrenoceptor blocker carvedilol provides cardioprotection via an adenosine-dependent mechanism in ischemic canine hearts.

BACKGROUND: Carvedilol is a beta-adrenoceptor blocker with a vasodilatory action that is more effective for the treatment of congestive heart failure than other beta-blockers. Recently, carvedilol has been reported to reduce oxidative stress, which may consequently reduce the deactivation of adenosine-producing enzymes and increase cardiac adenosine levels. Therefore, carvedilol may also have a protective effect on ischemia and reperfusion injury, because adenosine mediates cardioprotection in ischemic hearts. METHODS AND RESULTS: In anesthetized dogs, the left anterior descending coronary artery was occluded for 90 minutes, followed by reperfusion for 6 hours. Carvedilol reduced the infarct size (15.0+/-2.8% versus 40.9+/-4.2% in controls), and this effect was completely reversed by the nonselective adenosine receptor antagonist 8-sulfophenyltheophylline (45.2+/-5.4%) or by an inhibitor of ecto-5'-nucleotidase (44.4+/-3.6%). There were no differences of either area at risk or collateral flow among the various groups. When the coronary perfusion pressure was reduced in other dogs so that coronary blood flow was decreased to 50% of the nonischemic level, carvedilol increased coronary blood flow (49.4+/-5.6 to 73.5+/-7.5 mL x 100 g(-1) x min(-1); P<0.05) and adenosine release (112.3+/-22.2 to 240.6+/-57.1 nmol/L; P<0.05) during coronary hypoperfusion. This increase of coronary blood flow was attenuated by either 8-sulfophenyltheophylline or superoxide dismutase. In human umbilical vein endothelial cells cultured with or without xanthine and xanthine oxidase, carvedilol caused an increase of ecto-5'-nucleotidase activity. CONCLUSIONS: Carvedilol shows a cardioprotective effect against ischemia and/or reperfusion injury via adenosine-dependent mechanisms.

Methotrexate and MX-68, a new derivative of methotrexate, limit infarct size via adenosine-dependent mechanisms in canine hearts.

Methotrexate, an anti-rheumatic agent, has recently been reported to show an anti-inflammatory action via ecto-5'-nucleotidase- and adenosine-dependent mechanisms. Because ecto-5'-nucleotidase contributes to the production of adenosine and adenosine has a potent cardioprotective effect against ischemia/reperfusion injury, we investigated whether methotrexate or MX-68 [N-1-((2,4-diamino-6-pteridinyl) methyl)-3,4-dihydro-2H-1,4-benzothiazine-7- carbonyl]-N-2- aminoadipic acid] could reduce infarct size via adenosine-dependent mechanisms. In beagle dogs, the left anterior descending coronary artery was perfused through a bypass tube, which was occluded for 90 minutes followed by 6 hours of reperfusion. The size of infarcts was assessed by TTC staining. MX-68 reduced infarct size compared with that in untreated dogs (13.7 +/- 1.9 versus 38.6 +/- 5.3%, P < 0.01). This effect was completely blunted by either the adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) (45.0 +/- 4.6% and 46.8 +/- 5.8% in the 8-SPT and MX-68 + 8-SPT groups, respectively) or by the ecto-5'-nucleotidase inhibitoralpha,beta-methylenadenosine 5'-diphosphate (AMP-CP) (44.0 +/- 4.5% and 46.7 +/- 5.8% in the AMP-CP and MX-68 + AMP-CP groups, respectively). Methotrexate also reduced infarct size to a level comparable with that in the MX-68 group, and its effect was also blunted by 8-SPT. There were no significant differences of collateral blood flow or risk area between the groups. We conclude that methotrexate and its derivative (MX-68) both limit infarct size via adenosine-dependent mechanisms.

Lamr1 functional retroposon causes right ventricular dysplasia in mice.

Arrhythmogenic right ventricular dysplasia (ARVD) is a hereditary cardiomyopathy that causes sudden death in the young. We found a line of mice with inherited right ventricular dysplasia (RVD) caused by a mutation of the gene laminin receptor 1 (Lamr1). This locus contained an intron-processed retroposon that was transcribed in the mice with RVD. Introduction of a mutated Lamr1 gene into normal mice by breeding or by direct injection caused susceptibility to RVD, which was similar to that seen in the RVD mice. An in vitro study of cardiomyocytes expressing the product of mutated Lamr1 showed early cell death accompanied by alteration of the chromatin architecture. We found that heterochromatin protein 1 (HP1) bound specifically to mutant LAMR1. HP1 is a dynamic regulator of heterochromatin sites, suggesting that mutant LAMR1 impairs a crucial process of transcriptional regulation. Indeed, mutant LAMR1 caused specific changes to gene expression in cardiomyocytes, as detected by gene chip analysis. Thus, we concluded that products of the Lamr1 retroposon interact with HP1 to cause degeneration of cardiomyocytes. This mechanism may also contribute to the etiology of human ARVD.