Renalase Deficiency In Heart Failure——A Novel Mechanism Underlying Circulating Catecholamine Accumulation AND Increased Expression Of Integrin-Linked Kinase Improves Cardiac Function Of Heart Failure

Background and Objective-Chronic heart failure is a clinic syndrome occuring as a result of a variety of cardiovascular diseases, the most common being is ischemic heart disease and in particular myocardial infarction. One of the most important pathophysiologic characteristics of heart failure is sympathetic overactivity and circulating catecholamine accumulation, which will contribute to the progression of cardiac dysfunction. The mechanisms underlying circulating catecholamine accumulation in heart failure is poorly understood while the finding of "renalase pathway" enlightened us. As a novel secreted monoamine oxidase, renalase can degrade circulating catecholamine directly. On the other hand, catecholamine can also regulate the activity, synthesis and secretion of renalase. Here we evaluate the influence of renal blood flow or perfusion pressure on renalase synthesis using unilateral renal artery stenosis model and isolated perfused rat kidney model. And also we investigate the relationship between the expression and activity of renalase in the kidney and the concentration of plasma norepinephrine in heart failure.Methods-Male Sprague-Dawley (SD) rats received angiotensin-converting enzyme inhibitor (ACEI) for one week before surgery to block the activation of renin-angiotensin-aldosterone system (RAAS) whilst Rats given a corresponding volume of normal saline (NS) served as the control group to exclude the potential influence of RAAS on renalase expression. At the end of this time, unilateral renal artery stenosis model was constructed with 0.2mm artery clip, while the sham-operation group was also set up. The renalase expression in the kidney at mRNA and protein level were evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting respectively. To investigate whether norepinephrine can be degraded directly by the kidney, and whether kidney perfusion flow or pressure can influence its clearance rate, we constructed isolated perfused rat kidney model while 20ng/mL norepinephrine was added to the Krebs-Henseleit bicarbonate (KHS) perfusate previously. Norepinephrine concentration in the perfusion medium and urine at different time points was determined by a commercially available norepinephrine Enzyme linked immunosorbent assay (ELISA) kit. To investigate whether renalase deficiency existed and its relationship with circulating norepinephrine concentration in heart failure, acute myocardial infarction (AMI) model was constructed by ligation of left descending coronary artery of rats on a ventilator, sham-operation group and normal control group were also set up. The construction of AMI was confirmed by weakening of beating of the apex cordis and anterior wall of the left ventricle, as well as a change in color from rubicund to pale and 2,3,4-triphenyltetrazolium chloride dye (TTC) staining 3 days after the surgery. Heart function was evaluated by echocardiography and hemodynamic assessment four weeks later. Circulating renalase and norepinephrine concentration were determined using ELISA. The mRNA and protein expression of renalase in the kidney were quantified by RT-PCR and Western blotting respectively. The activity of renalase were measured by Amplex(?) Red Monoamine Oxidase Assay Kit.Results-No difference was found in renalase expression, both at the mRNA and protein level, between rats treated with ACEI and those treated with corresponding vehicle, thus effectively excluding the possibility that RAAS activity could potentially influence renalase expression. On the other hand, both mRNA and protein expression levels were lower in the ischemic than the non-ischemic kidney. In the isolated perfused kidney model, the concentration of norepinephrine in the perfusion medium decreased gradually during perfusion. Moreover, the clearance rate of norepinephrine decreased with reduction of either perfusion flow or pressure. In heart failure model, the concentration of plasma norepinephrine and renalase were both higher in heart failure rats than the other two groups while renalase expression decreased at the protein level in the kidneys of heart failure rats compared with those of control rats. Renalase activity showed no difference among groups.Conclusions-Renalase expression is influenced by renal blood flow. Norepinephrine can be degraded directly by the kidney and as blood flow or pressure declined, the rate of clearance of norepinephrine decreased in parallel. The impaired synthesis of renalase by the kidney may represent a novel mechanism underlying circulating norepinephrine accumulation in heart failure.Background and Objective-Chronic heart failure is the end-stage of many kinds of cardiovascular disease. Being a disease characterized by a loss of cardiomyocytes, deteriorated function of remained cells and imbalanced ratio of blood flow to cardiomyocytes, stimulating cardiomyocytes proliferation and angiogenesis, inhibiting apoptosis as well as ameliorating micro-circumstances of cardiomyocytes have become promising strategies. Integrin-linked kinase (ILK), a functional serine/threonine protein kinase, as an critical component of the cardiac stretch sensor has been reported to play an important part in regulating cell apoptosis, proliferation, hypertrophy and angiogenesis. In the present study, we investigated whether integrin-linked kinase (ILK) could improve cardiac function of heart failure and the underlying mechanisms of the beneficial effects.Methods-Recombinant adenoviral vector containing both human wild-type ILK and humanized recombinant green fluorescent protein (hrGFP), as well as null-content adenoviral vector, was prepared. Rat model of dilated cardiomyopathy were induced by six equal doses of intraperitoneal administration of doxorubicin (DOX, total dose of 15 mg/kg) within 2 weeks.5 weeks after the first injection, echocardiographic analysis demonstrated exacerbated heart function and ILK or vehicle was injected into the myocardium. ILK expression and activity were confirmed using western blotting. Echocardiographic and hemodynamic analyses were performed 4 weeks after transfection. Then myocardial tissues were harvested and fixed for subsequent histological, immunohistochemical and TUNEL examination. H&E (hematoxylin & eosin) staining was performed for histological (morphological) analysis as well as Masson’s trichrome for fibrosis. Apoptosis was measured by TUNEL analysis. Cardiomyocyte proliferation was estimated by phosphohistone-H3 staining. We also detected the concentration of malonaldehyde (MDA) and the superoxide dismutase (SOD) activity in the plasma which may represent the degree of oxidative stress of different groups and plasma TNF-a concentration for inflammation state.Results-Western blotting analysis revealed higher expression of ILK and hrGFP of ad-ILK heart as compared with ad-null controls. Elevated expression of pAkt/Akt which representing the activity of ILK was also seen in ad-ILK heart. Four weeks after transfection, echocardiographic and hemodynamic analysis demonstrated relatively preserved cardiac function in the ILK group. Compared with vehicle injection, ILK treatment significantly ameliorates inflammation infiltration and cardiomyocyte degeneration which were induced by doxorubicin. ILK treatment was also associated with preserved left ventricular (LV) geometry (including LV diameter, LV wall thickness, cardiomyocyte size and myofilament density). Reduced fibrosis, oxidase stress and apoptosis as well as increased cardiomyocyte proliferation were observed in ad-ILK animals.Conclusions-ILK gene therapy improves cardiac remodeling and function of heart failure following dilated cardiomyopathy. The beneficial effect is associated with reduced apoptosis, increased cardiomyocyte proliferation, decreased oxidase stress and inflammatory level.