Nicorandil attenuates monocrotaline-induced vascular endothelial damage and pulmonary arterial hypertension

Background

An antianginal K_ATP channel opener nicorandil has various beneficial effects on cardiovascular systems; however, its effects on pulmonary vasculature under pulmonary arterial hypertension (PAH) have not yet been elucidated. Therefore, we attempted to determine whether nicorandil can attenuate monocrotaline (MCT)-induced PAH in rats.

Materials and Methods

Sprague-Dawley rats injected intraperitoneally with 60 mg/kg MCT were randomized to receive either vehicle; nicorandil (5.0 mg·kg⁻¹·day⁻¹) alone; or nicorandil as well as either a K_ATP channel blocker glibenclamide or a nitric oxide synthase (NOS) inhibitor N ^ω-nitro-l-arginine methyl ester (l-NAME), from immediately or 21 days after MCT injection. Four or five weeks later, right ventricular systolic pressure (RVSP) was measured, and lung tissue was harvested. Also, we evaluated the nicorandil-induced anti-apoptotic effects and activation status of several molecules in cell survival signaling pathway in vitro using human umbilical vein endothelial cells (HUVECs).

Results

Four weeks after MCT injection, RVSP was significantly increased in the vehicle-treated group (51.0±4.7 mm Hg), whereas it was attenuated by nicorandil treatment (33.2±3.9 mm Hg; P<0.01). Nicorandil protected pulmonary endothelium from the MCT-induced thromboemboli formation and induction of apoptosis, accompanied with both upregulation of endothelial NOS (eNOS) expression and downregulation of cleaved caspase-3 expression. Late treatment with nicorandil for the established PAH was also effective in suppressing the additional progression of PAH. These beneficial effects of nicorandil were blocked similarly by glibenclamide and l-NAME. Next, HUVECs were incubated in serum-free medium and then exhibited apoptotic morphology, while these changes were significantly attenuated by nicorandil administration. Nicorandil activated the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in HUVECs, accompanied with the upregulation of both eNOS and Bcl-2 expression.

Conclusions

Nicorandil attenuated MCT-induced vascular endothelial damage and PAH through production of eNOS and anti-apoptotic factors, suggesting that nicorandil might have a promising therapeutic potential for PAH.     

VIP and endothelin receptor antagonist: An effective combination against experimental pulmonary arterial hypertension

Background

Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy?

Methods

Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival.

Results

Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days.

Conclusions

1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.     

HMGB1 Promotes the Development of Pulmonary Arterial Hypertension in Rats

Rationale

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. However, the molecular and cellular mechanisms driving inflammation have not been fully elucidated.

Objectives

To elucidate the roles of high mobility group box 1 protein (HMGB1), a ubiquitous DNA-binding protein with extracellular pro-inflammatory activity, in a rat model of PAH.

Methods

Male Sprague-Dawley rats were administered monocrotaline (MCT). Concentrations of HMGB1 in bronchoalveolar lavage fluid (BALF) and serum, and localization of HMGB1 in the lung were examined over time. The protective effects of anti-HMGB1 neutralizing antibody against MCT-induced PAH were tested.

Results

HMGB1 levels in BALF were elevated 1 week after MCT injection, and this elevation preceded increases of other pro-inflammatory cytokines, such as TNF-α, and the development of PAH. In contrast, serum HMGB1 levels were elevated 4 weeks after MCT injection, at which time the rats began to die. Immunohistochemical analyses indicated that HMGB1 was translocated to the extranuclear space in periarterial infiltrating cells, alveolar macrophages, and bronchial epithelial cells of MCT-injected rats. Anti-HMGB1 neutralizing antibody protected rats against MCT-induced lung inflammation, thickening of the pulmonary artery wall, and elevation of right ventricular systolic pressure, and significantly improved the survival of the MCT-induced PAH rats.

Conclusions

Our results identify extracellular HMGB1 as a promoting factor for MCT-induced PAH. The blockade of HMGB1 activity improved survival of MCT-induced PAH rats, and thus might be a promising therapy for the treatment of PAH.     

Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?

Background

The leading cause of mortality due to pulmonary arterial hypertension (PAH) is failure of the cardiac right ventricle. It has long been hypothesized that during the development of chronic cardiac failure the heart becomes energy deprived, possibly due to shortage of oxygen at the level of cardiomyocyte mitochondria. However, direct evaluation of oxygen tension levels within the in vivo right ventricle during PAH is currently lacking. Here we directly evaluated this hypothesis by using a recently reported technique of oxygen-dependent quenching of delayed fluorescence of mitochondrial protoprophyrin IX, to determine the distribution of mitochondrial oxygen tension (mitoPO₂) within the right ventricle (RV) subjected to progressive PAH.

Methods

PAH was induced through a single injection of monocrotaline (MCT). Control (saline-injected), compensated RV hypertrophy (30 mg/kg MCT; MCT30), and RV failure (60 mg/kg MCT; MCT60) rats were compared 4 wk after treatment. The distribution of mitoPO₂ within the RV was determined in mechanically-ventilated, anaesthetized animals, applying different inspired oxygen (FiO₂) levels and two increment dosages of dobutamine.

Results

MCT60 resulted in RV failure (increased mortality, weight loss, increased lung weight), MCT30 resulted in compensated RV hypertrophy. At 30% or 40% FiO₂, necessary to obtain physiological arterial PO₂ in the diseased animals, RV failure rats had significantly less mitochondria (15% of total mitochondria) in the 0-20 mmHg mitoPO₂ range than hypertrophied RV rats (48%) or control rats (54%). Only when oxygen supply was reduced to 21% FiO_2, resulting in low arterial PO₂ for the MCT60 animals, or when oxygen demand increased with high dose dobutamine, the number of failing RV mitochondria with low oxygen became similar to control RV. In addition, metabolic enzyme analysis revealed similar mitochondrial mass, increased glycolytic hexokinase activity following MCT, with increased lactate dehydrogenase activity only in compensated hypertrophied RV.

Conclusions

Our novel observation of increased mitochondrial oxygenation suggests down-regulation of in vivo mitochondrial oxygen consumption, in the absence of hypoxia, with transition towards right ventricular failure induced by pulmonary arterial hypertension.     

Divergent Cardiopulmonary Actions of Heme Oxygenase Enzymatic Products in Chronic Hypoxia

Background

Hypoxia and pressure-overload induce heme oxygenase-1 (HO-1) in cardiomyocytes and vascular smooth muscle cells (VSMCs). HO-1^−/− mice exposed to chronic hypoxia develop pulmonary arterial hypertension (PAH) with exaggerated right ventricular (RV) injury consisting of dilation, fibrosis, and mural thrombi. Our objective was to indentify the HO-1 product(s) mediating RV protection from hypoxic injury in HO-1^−/− mice.

Methodology/Principal Findings

HO-1^−/− mice were exposed to seven weeks of hypoxia and treated with inhaled CO or biliverdin injections. CO reduced right ventricular systolic pressure (RVSP) and prevented hypoxic pulmonary arteriolar remodeling in both HO-1^−/− and control mice. Biliverdin had no significant effect on arteriolar remodeling or RVSP in either genotype. Despite this, biliverdin prevented RV failure in the hypoxic HO-1^−/− mice (0/14 manifested RV wall fibrosis or thrombus), while CO-treated HO-1^−/− mice developed RV insults similar to untreated controls. In vitro, CO inhibited hypoxic VSMC proliferation and migration but did not prevent cardiomyocyte death from anoxia-reoxygenation (A-R). In contrast, bilirubin limited A-R-induced cardiomyocyte death but did not inhibit VSMC proliferation and migration.

Conclusions/Significance

CO and bilirubin have distinct protective actions in the heart and pulmonary vasculature during chronic hypoxia. Moreover, reducing pulmonary vascular resistance may not prevent RV injury in hypoxia-induced PAH; supporting RV adaptation to hypoxia and preventing RV failure must be a therapeutic goal.     

Nicorandil alleviates myocardial injury and post-infarction cardiac remodeling by inhibiting Mst1

Background

Cardiomyocyte autophagy and apoptosis are crucial events underlying the development of cardiac abnormalities and dysfunction after myocardial infarction (MI). A better understanding of the cell signaling pathways involved in cardiac remodeling may support the development of new therapeutic strategies for the treatment of heart failure (HF) after MI.

The Soluble Guanylate Cyclase Stimulator Riociguat Ameliorates Pulmonary Hypertension Induced by Hypoxia and SU5416 in Rats

Background

The nitric oxide (NO)–soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63–2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO–sGC–cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH.

Methods and Results

Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55±0.02, p<0.05), increased cardiac output (60.8±.8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03±0.3 mmHg min⁻¹ ml⁻¹ 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05).

Conclusion

Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.     

Reversal of MicroRNA Dysregulation in an Animal Model of Pulmonary Hypertension

Background

Animals models have played an important role in enhancing our understanding of the pathogenesis of pulmonary arterial hypertension (PAH). Dysregulation of the profile of microRNAs (miRNAs) has been demonstrated in human tissues from PAH patients and in animal models. In this study, we measured miRNA levels in the monocrotaline (MCT) rat model of PAH and examined whether blocking a specific dysregulated miRNA not previously reported in this model, attenuated PAH. We also evaluated changes in miRNA expression in lung specimens from MCT PAH rats overexpressing human prostacyclin synthase, which has been shown to attenuate MCT PAH.

Methods

Expression levels of a panel of miRNAs were measured in MCT-PAH rats as compared to naïve (saline) control rats. Subsequently, MCT PAH rats were injected with a specific inhibitor (antagomiR) for miR-223 (A223) or a nonspecific control oligonucleotide (A-control) 4 days after MCT administration, then weekly. Three weeks later, RV systolic pressure and RV mass were measured. Total RNA, isolated from the lungs, microdissected pulmonary arteries, and right ventricle, was reverse transcribed and real-time quantitative PCR was performed. MiRNA levels were also measured in RNA isolated from paraffin sections of MCT-PAH rats overexpressing prostacyclin synthase.

Results

MiRs 17, 21, and 223 were consistently upregulated, whereas miRs 126, 145, 150, 204, 424, and 503 were downregulated in MCT PAH as compared to vehicle control. A223 significantly reduced levels of miR-223 in PA and lungs of MCT PAH rats as compared to levels measured in A-control or control MCT PAH rats, but A223 did not attenuate MCT PAH. Right ventricular mass and right ventricular systolic pressure in rats treated with A223 were not different from values in A-control or MCT PAH rats. In contrast, analysis of total RNA from lung specimens of MCT PAH rats overexpressing human prostacyclin synthase (hPGIS) demonstrated reversal of MCT-induced upregulation of miRs 17, 21, and 223 and an increase in levels of miR-424 and miR-503. Reduction in bone morphogenetic receptor 2 (BMPR2) messenger (m)RNA expression was not altered by A223, whereas human prostacyclin synthase overexpression restored BMPR2 mRNA to levels in MCT PAH to levels measured in naive controls.

Conclusions

Inhibition of miR-223 did not attenuate MCT PAH, whereas human prostacyclin synthase overexpression restored miRNA levels in MCT PAH to levels detected in naïve rats. These data may establish a paradigm linking attenuation of PAH to restoration of BMPR2 signaling.     

Human immunodeficiency virus transgenic rats exhibit pulmonary hypertension

Human immunodeficiency virus (HIV)-associated pulmonary arterial hypertension (PAH) is a serious noninfectious disease involving an aberrant increase in pressure in the blood vessels of the lung, which leads to right ventricular (RV) heart failure and can eventually result in death. A lack of viable animal models of HIV-PAH has limited the identification of signaling pathways involved in HIV-mediated onset and progression of PAH. To determine whether the HIV-1 transgenic (HIV Tg) rat displays pathophysiological end points associated with PAH, we evaluated peak RV systolic pressure (RVSP), RV hypertrophy, pulmonary vessel remodeling, and alterations in gene expression by real-time PCR and microarray. RVSP was measured by RV catheterization via the right jugular vein in 3- and 9-mo-old HIV Tg and age-matched Fischer 344 (control) male rats while under 2% isoflurane anesthesia. RVSP was elevated in the HIV Tg rats (34.2 ± 2.5 mmHg) compared with the F344 controls (21.2 ± 2.5 mmHg), with more significant elevations in the 9-mo-old HIV Tg rats (42.5 ± 3.7 mmHg). We observed significant increases in RV wall thickness in HIV Tg rats compared with controls, both histologically and by echocardiograph measurement. HIV Tg rats also show increased thickening of the pulmonary artery and remodeling of small pulmonary arteries, as well as altered expression of gene pathways associated with PAH. These data represent the first analysis of PAH in HIV Tg rats and suggest that this model will be useful for investigating pathways and identifying potential therapies for HIV-PAH.     

N-acetylcysteine improves established monocrotaline-induced pulmonary hypertension in rats

Background

The outcome of patients suffering from pulmonary arterial hypertension (PAH) are predominantly determined by the response of the right ventricle to the increase afterload secondary to high vascular pulmonary resistance. However, little is known about the effects of the current available or experimental PAH treatments on the heart. Recently, inflammation has been implicated in the pathophysiology of PAH. N-acetylcysteine (NAC), a well-known safe anti-oxidant drug, has immuno-modulatory and cardioprotective properties. We therefore hypothesized that NAC could reduce the severity of pulmonary hypertension (PH) in rats exposed to monocrotaline (MCT), lowering inflammation and preserving pulmonary vascular system and right heart function.

Methods

Saline-treated control, MCT-exposed, MCT-exposed and NAC treated rats (day 14–28) were evaluated at day 28 following MCT for hemodynamic parameters (right ventricular systolic pressure, mean pulmonary arterial pressure and cardiac output), right ventricular hypertrophy, pulmonary vascular morphometry, lung inflammatory cells immunohistochemistry (monocyte/macrophages and dendritic cells), IL-6 expression, cardiomyocyte hypertrophy and cardiac fibrosis.

Results

The treatment with NAC significantly decreased pulmonary vascular remodeling, lung inflammation, and improved total pulmonary resistance (from 0.71 ± 0.05 for MCT group to 0.50 ± 0.06 for MCT + NAC group, p < 0.05). Right ventricular function was also improved with NAC treatment associated with a significant decrease in cardiomyocyte hypertrophy (625 ± 69 vs. 439 ± 21 μm² for MCT and MCT + NAC group respectively, p < 0.001) and heart fibrosis (14.1 ± 0.8 vs. 8.8 ± 0.1% for MCT and MCT + NAC group respectively, p < 0.001).

Conclusions

Through its immuno-modulatory and cardioprotective properties, NAC has beneficial effect on pulmonary vascular and right heart function in experimental PH.     

Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats

Hill, Nicholas S., Rod R. Warburton, Linda Pietras, andJames R. Klinger. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats.J. Appl. Physiol. 83(4):1209-1215, 1997.Endothelin-1 (ET-1), a potent vasoactive andmitogenic peptide, has been implicated in the pathogenesis ofseveral forms of pulmonary hypertension. We hypothesized thatnonspecific blockade of ET receptors would blunt the development ofmonocrotaline (MCT)-induced pulmonary hypertension in rats. Asingle dose of the nonspecific ET blocker bosentan (100 mg/kg) given tointact rats by gavage completely blocked the pulmonary vasoconstrictoractions of Big ET-1 and partially blunted hypoxic pulmonaryvasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavagedonce daily with bosentan (200 mg/kg) had lower right ventricular (RV)systolic pressure (RVSP), RV-to-body weight (RV/BW) andRV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratiosand less percent medial thickness of small pulmonary arteries thancontrol MCT-injected rats. Lower dose bosentan (100 mg/kg) had noeffect on these parameters after MCT or saline injection. Bosentanraised plasma ET-1 levels but had no effect on lung ET-1 levels.Bosentan (200 mg/kg) also had no effect on wet-to-dry lung weightratios 6 days after MCT injection. When given during the last 10 days,but not the first 11 days of a 3-wk period after MCT injection,bosentan reduced RV/(LV+S) compared with MCT-injected controls. Weconclude that ET-1 contributes to the pathogenesis of MCT-inducedpulmonary hypertension and acts mainly during the later inflammatoryrather than the acute injury phase after injection.

     

Cardioprotective effect of nicorandil, a mitochondrial ATP-sensitive potassium channel opener, prolongs survival in HSPB5 R120G transgenic mice

Background

Transgenic (TG) mice with overexpression of an arg120gly (R120G) missense mutation in HSPB5 display desmin-related cardiomyopathy, which is characterized by formation of aggresomes. It is also known that progressive mitochondrial abnormalities and apoptotic cell death occur in the hearts of R120G TG mice. The role of mitochondrial dysfunction and apoptosis in disease progression, however, remains uncertain.

Methods and Results

Mitochondrial abnormalities and apoptotic cell death induced by overexpression of HSPB5 R120G were analyzed in neonatal rat cardiomyocytes. Overexpression of mutant HSPB5 led to development of aggresomes with a concomitant reduction in cell viability in the myocytes. Overexpression of mutant HSPB5 induced a reduction in the cytochrome c level in the mitochondrial fraction and a corresponding increase in the cytoplasmic fraction in the myocytes. Down-regulation of BCL2 and up-regulation of BAX were detected in the myocytes expressing the mutant HSPB5. Concomitant with mitochondrial abnormality, the activation of caspase-3 and increased apoptotic cell death was observed. Cell viability was dose-dependently recovered in myocytes overexpressing HSPB5 R120G by treatment with nicorandil a mitochondrial ATP-sensitive potassium channel opener. Nicorandil treatment also inhibited the increase in BAX, the decrease in BCL2, activation of caspase-3 and apoptotic cell death by mutant HSPB5. To confirm the results of the in-vitro study, we analyzed the effect of nicorandil in HSPB5 R120G TG mice. Nicorandil treatment appeared to reduce mitochondrial impairment and apoptotic cell death and prolonged survival in HSPB5 R120G TG mice.

Conclusions

Nicorandil may prolong survival in HSPB5 R120G TG mice by protecting against mitochondrial impairments.     

Nicorandil improves post-fatigue tension in slow skeletal muscle fibers by modulating glutathione redox state

Fatigue is a phenomenon in which force reduction has been linked to impairment of several biochemical processes. In skeletal muscle, the ATP-sensitive potassium channels (K_ATP) are actively involved in myoprotection against metabolic stress. They are present in sarcolemma and mitochondria (mitoK_ATP channels). K⁺ channel openers like nicorandil has been recognized for their ability to protect skeletal muscle from ischemia-reperfusion injury, however, the effects of nicorandil on fatigue in slow skeletal muscle fibers has not been explored, being the aim of this study. Nicorandil (10 μM), improved the muscle function reversing fatigue as increased post-fatigue tension in the peak and total tension significantly with respect to the fatigued condition. However, this beneficial effect was prevented by the mitoK_ATP channel blocker 5-hydroxydecanoate (5-HD, 500 μM) and by the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 1 mM), but not by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 μM). Nicorandil also decreased lipid peroxidation and maintained both reduced glutathione (GSH) levels and an elevated GSH/GSSG ratio, whereas total glutathione (TGSH) remained unaltered during post-fatigue tension. In addition, NO production, measured through nitrite concentrations was significantly increased with nicorandil during post-fatigue tension; this increase remained unaltered in the presence of nicorandil plus L-NAME, nonetheless, this effect was reversed with nicorandil plus MPG. Hence, these results suggest that nicorandil improves the muscle function reversing fatigue in slow skeletal muscle fibers of chicken through its effects not only as a mitoK_ATP channel opener but also as NO donor and as an antioxidant.     

Up-regulation of hexokinase1 in the right ventricle of monocrotaline induced pulmonary hypertension

Background

Pulmonary arterial hypertension (PAH) is a proliferative arteriopathy associated with a glycolytic shift during heart metabolism. An increase in glycolytic metabolism can be detected in the right ventricle during PAH. Expression levels of glycolysis genes in the right ventricle during glycolysis that occur in monocrotaline (MCT)-induced pulmonary hypertension (PH) remain unknown.

Methods

PH was induced by a single subcutaneous injection of MCT (50 mg/kg) into rats, eventually causing right heart failure. Concurrently, a control group was injected with normal saline. The MCT-PH rats were randomly divided into three groups according to MCT treatment: MCT-2 week, 3 week, and 4 week groups (MCT-2w, 3w, 4w). At the end of the study, hemodynamics and right ventricular hypertrophy were compared among experimental groups. Expression of key glycolytic candidate genes was screened in the right ventricle.

Results

We observed an increase in mean pulmonary arterial pressure, right ventricular systolic pressure and right ventricular hypertrophy index three weeks following MCT injection. Alterations in the morphology and structure of right ventricular myocardial cells, as well as the pulmonary vasculature were observed. Expression of hexokinase 1 (HK1) mRNA began to increase in the right ventricle of the MCT-3w group and MCT-4w group, while the expression of lactate dehydrogenase A (LDHA) was elevated in the right ventricle of the MCT-4w group. Hexokinase 2(HK2), pyruvate dehydrogenase complex α1 (PDHα1), and LDHA mRNA expression showed no changes in the right ventricle. HK1 mRNA expression was further confirmed by HK1 protein expression and immunohistochemical analyses. All findings underlie the glycolytic phenotype in the right ventricle.

Conclusions

There was an increase in the protein and mRNA expression of hexokinase-1 (HK1) three and four weeks after the injection of monocrotaline in the right ventricle, intervention of HK1 may be amenable to therapeutic intervention.     

Chronic treatment with probucol effectively inhibits progression of pulmonary hypertension in rats

It has been reported that probucol is a lipid-lowering agent having a strong antioxidative effect and inhibitory action on vascular smooth muscle cell proliferation. In this work, we studied the effect of treatment with a 1% probucol diet on pulmonary hypertension induced by monocrotaline (MCT) in rats. Rats were fed a control or 1% probucol-supplemented diet for 7 days, then given a single subcutaneous injection of 60 mg/kg MCT or saline, and continuously fed the same diet for 20 days, respectively. MCT caused an increase in right ventricular systolic pressure (RVSP), an indicator of pulmonary hypertension, and central venous pressure (CVP) on day 20. In rats receiving a diet containing 1% probucol, RVSP was significantly lower than that in rats treated with control diet, and CVP remained essentially at the basal level. On day 20, MCT also caused an increase in the ratio of right ventricular (RV) to body weight (BW), compared to the control value, indicating the development of RV hypertrophy in MCT rats. RV hypertrophy was significantly inhibited in 1% probucol-treated rats. These findings suggest that chronic treatment with probucol effectively inhibits the progression of pulmonary hypertension in rats.     

Biventricular Remodeling in Murine Models of Right Ventricular Pressure Overload

Right ventricular (RV) failure is a major cause of mortality in acute or chronic lung disease and left heart failure. The objective of this study was to demonstrate a percutaneous approach to study biventricular hemodynamics in murine models of primary and secondary RV pressure overload (RVPO) and further explore biventricular expression of two key proteins that regulate cardiac remodeling: calcineurin and transforming growth factor beta 1 (TGFβ1).

Methods

Adult, male mice underwent constriction of the pulmonary artery or thoracic aorta as models of primary and secondary RVPO, respectively. Conductance catheterization was performed followed by tissue analysis for changes in myocyte hypertrophy and fibrosis.

Results

Both primary and secondary RVPO decreased biventricular stroke work however RV instantaneous peak pressure (dP/dt_max) and end-systolic elastance (Ees) were preserved in both groups compared to controls. In contrast, left ventricular (LV) dP/dt_max and LV-Ees were unchanged by primary, but reduced in the secondary RVPO group. The ratio of RV:LV ventriculo-arterial coupling was increased in primary and reduced in secondary RVPO. Primary and secondary RVPO increased RV mass, while LV mass decreased in primary and increased in the secondary RVPO groups. RV fibrosis and hypertrophy were increased in both groups, while LV fibrosis and hypertrophy were increased in secondary RVPO only. RV calcineurin expression was increased in both groups, while LV expression increased in secondary RVPO only. Biventricular TGFβ1 expression was increased in both groups.

Conclusion

These data identify distinct effects of primary and secondary RVPO on biventricular structure, function, and expression of key remodeling pathways.     

Modulation of myocardial mitochondrial mechanisms during severe polymicrobial sepsis in the rat

Background

We tested the hypothesis that 5-Hydroxydecanoic acid (5HD), a putative mitoK_ATP channel blocker, will reverse sepsis-induced cardiodynamic and adult rat ventricular myocyte (ARVM) contractile dysfunction, restore mitochondrial membrane permeability alterations and improve survival.

Methodology/Principal Findings

Male Sprague-Dawley rats (350–400 g) were made septic using 400 mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. The Voltage Dependent Anion Channels (VDAC1), Bax and cytochrome C levels were determined in isolated single ARVMs obtained from sham and septic rat heart. Mitochondria and cytosolic fractions were isolated from ARVMs treated with norepinephrine (NE, 10 µmoles) in the presence/absence of 5HD (100 µmoles). A continuous infusion of 5HD using an Alzet pump reversed sepsis-induced mortality when administered at the time of induction of sepsis (−40%) and at 6 hr post-sepsis (−20%). Electrocardiography revealed that 5HD reversed sepsis-induced decrease in the average ejection fraction, Simpsons+m Mode (53.5±2.5 in sepsis and 69.2±1.2 at 24 hr in sepsis+5HD vs. 79.9±1.5 basal group) and cardiac output (63.3±1.2 mL/min sepsis and 79.3±3.9 mL/min at 24 hr in sepsis+5HD vs. 85.8±1.5 mL/min basal group). The treatment of ARVMs with 5HD also reversed sepsis-induced depressed contractility in both the vehicle and NE-treated groups. Sepsis produced a significant downregulation of VDAC1, and upregulation of Bax levels, along with mitochondrial membrane potential collapse in ARVMs. Pretreatment of septic ARVMs with 5HD blocked a NE-induced decrease in the VDAC1 and release of cytochrome C.

Conclusion

The data suggest that Bax activation is an upstream event that may precede the opening of the mitoK_ATP channels in sepsis. We concluded that mitoK_ATP channel inhibition via decreased mitochondrial membrane potential and reduced release of cytochrome C provided protection against sepsis-induced ARVM and myocardial contractile dysfunction.     

ACE2 improves right ventricular function in a pressure overload model

Background

Right ventricular (RV) dysfunction is a complication of pulmonary hypertension and portends a poor prognosis. Pharmacological therapies targeting RV function in pulmonary hypertension may reduce symptoms, improve hemodynamics, and potentially increase survival. We hypothesize that recombinant human angiotensin-converting enzyme 2 (rhACE2) will improve RV function in a pressure overload model.

Results

rhACE2 administered at 1.8 mg/kg/day improved RV systolic and diastolic function in pulmonary artery banded mice as measured by in vivo hemodynamics. Specifically, rhACE2 increased RV ejection fraction and decreased RV end diastolic pressure and diastolic time constant (p<0.05). In addition, rhACE2 decreased RV hypertrophy as measured by RV/LV+S ratio (p<0.05). There were no significant negative effects of rhACE2 administration on LV function. rhACE2 had no significant effect on fibrosis as measured by trichrome staining and collagen1α1 expression. In pulmonary artery banded mice, rhACE2 increased Mas receptor expression and normalized connexin 37 expression.

Conclusion

In a mouse RV load-stress model of early heart failure, rhACE2 diminished RV hypertrophy and improved RV systolic and diastolic function in association with a marker of intercellular communication. rhACE2 may be a novel treatment for RV failure.     

Activation of PPAR-γ ameliorates pulmonary arterial hypertension via inducing heme oxygenase-1 and p21: An in vivo study in rats

Aims

Our previous study has indicated that activation of PPAR-γ inhibits the proliferation of rat pulmonary artery smooth muscle cells (PASMCs) in vitro through inducing the expression of heme oxygenase-1 (HO-1), which in turn up-regulates the p21^WAF1 expression. In the present study, we intended to determine whether similar mechanisms have been involved in activation of PPAR-γ inhibition of development of rat PAH model.

Material and methods

Rat pulmonary arterial hypertension (PAH) model was established by subcutaneous injection of monocrotaline (MCT). Rosiglitazone was administered to activate PPAR-γ. Zinc protoporphyria IX (ZnPP-IX), was used to confirm the role of HO-1 in mediating PPAR-γ function. Parameters including the right ventricle systolic pressure (RVSP), the right ventricular hypertrophy (RVH) and the percentage of medial wall thickness were used to evaluate the development of PAH. Immunoblotting was used to determine the expression of HO-1 and p21^WAF1.

Key findings

Rosiglitazone significantly decreased the RVSP and inhibited the RVH in MCT-induced rat PAH model, and partially inhibited the pulmonary vascular remodeling. These effects were coupled with the sequential increase of HO-1 and p21^WAF1 expressions by rosiglitazone.

Significance

Activation of PPAR-γ benefits PAH by inhibiting proliferation of PASMCs and reducing pulmonary vascular remodeling. The present study suggests that enhancing PPAR-γ activity might have potential value in clinical treatment of PAH.