Youyou Zhao

Nox2 NADPH oxidase promotes pathologic cardiac remodeling associated with Doxorubicin chemotherapy.

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2-/-) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2-/- versus WT mice (fractional shortening: 29.5±1.4 versus 25.7±1.0%; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2-/- versus WT mice (ejection fraction: 52.6±2.5 versus 28.5±2.3%, LVdP/dtmin: -8,379±416 versus -5,198±527 mmHg s(-1); end-diastolic pressure-volume relation: 0.051±0.009 versus 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2-/- mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-β3, and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

Differential Expression of Components of the Cardiomyocyte Adrenomedullin/Intermedin Receptor System following Blood Pressure Reduction in Nitric Oxide-Deficient Hypertension

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nω-nitro-l-arginine methyl ester (l-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from l-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-α-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency.

AbstractBackground: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-α-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

Background: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD’s actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). Aim/method: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. Results: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. Conclusion: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

SRIF Receptor Subtype Expression and Involvement in Positive and Negative Contractile Effects of Somatostatin-14 (SRIF-14) in Ventricular Cardiomyocytes

Background/Aims: Somatostatin-14 (SRIF-14), a neuropeptide co-stored with acetylcholine in the cardiac parasympathetic innervation, exerts both positive and negative influences directly on contraction of ventricular cardiomyocytes, indicative of involvement of more than one of five known SRIF (SSTR) receptor subtypes. The aim was to characterize receptor subtype expression in adult rat ventricular cardiomyocytes and to investigate the influence of a series of SRIF (SSTR) subtype-selective agonists on contractile parameters. Methods: mRNA and protein expression of each receptor subtype were quantified by RT-PCR and immunoblotting respectively; for contraction studies, cells were stimulated at 0.5 Hz under basal conditions and in the presence of isoprenaline (ISO, 10-8M). Results: all five SRIF (SSTR) receptor subtypes were expressed in cardiomyocytes although SRIF1A (SSTR2) and SRIF2A (SSTR1) were less abundant than the other subtypes. L803087 (10-8M), a SRIF2B (SSTR4) agonist, attenuated ISO-stimulated peak contractile amplitude and prolonged relaxation time (T50). L796778 (10-7M), a SRIF1C (SSTR3) agonist, augmented basal and ISO-stimulated peak contractile amplitude; L779976 (10-8M) and L817818 (10-9M), agonists at SRIF1A (SSTR2) and SRIF1B (SSTR5) receptors, respectively, also augmented ISO-stimulated peak amplitude. Conclusion: these data support involvement of SRIF2B (SSTR4) receptors in the negative contractile effects of SRIF-14, while one or more of the three SRIF1 receptor subtypes (SSTR2, 3 or 5) may contribute to the positive contractile effects of SRIF-14.

Modulation of contractile function through neuropeptide Y receptors during development of cardiomyocyte hypertrophy

Severity of left ventricular hypertrophy (LVH) correlates with elevated plasma levels of neuropeptide Y (NPY) in hypertension. NPY elicits positive and negative contractile effects in cardiomyocytes through Y1 and Y2 receptors, respectively. This study tested the hypothesis that NPY receptor-mediated contraction is altered during progression of LVH. Ventricular cardiomyocytes were isolated from spontaneously hypertensive rats (SHRs) pre-LVH (12 weeks), during development (16 weeks), and at established LVH (20 weeks) and age-matched normotensive Wistar Kyoto (WKY) rats. Electrically stimulated (60 V, 0.5 Hz) cell shortening was measured using edge detection and receptor expression determined at mRNA and protein level. The NPY and Y1 receptor-selective agonist, Leu31Pro34NPY, stimulated increases in contractile amplitude, which were abolished by the Y1 receptor-selective antagonist, BIBP3226 [R-N2-(diphenyl-acetyl)-N-(4-hydroxyphenyl)methyl-argininamide)], confirming Y1 receptor involvement. Potencies of both agonists were enhanced in SHR cardiomyocytes at 20 weeks (2300- and 380-fold versus controls). Maximal responses were not attenuated. BIBP3226 unmasked a negative contraction effect of NPY, elicited over the concentration range (10–12 to 3 × 10–9 M) in which NPY and PYY3–36 attenuated the positive contraction effects of isoproterenol, the potencies of which were increased in cardiomyocytes from SHRs at 20 weeks (175- and 145-fold versus controls); maximal responses were not altered. Expression of NPY-Y1 and NPY-Y2 receptor mRNAs was decreased (55 and 69%) in left ventricular cardiomyocytes from 20-week-old SHRs versus age-matched WKY rats; parallel decreases (32 and 80%) were observed at protein level. Enhancement of NPY potency, producing (opposing) contractile effects on cardiomyocytes together with unchanged maximal response despite reduced receptor number, enables NPY to contribute to regulating cardiac performance during compensatory LVH.

Signalling mechanisms underlying doxorubicin and Nox2 NADPH oxidase-induced cardiomyopathy: involvement of mitofusin-2

The anthracycline doxorubicin (DOX), although successful as a first‐line cancer treatment, induces cardiotoxicity linked with increased production of myocardial ROS, with Nox2 NADPH oxidase‐derived superoxide reported to play a key role. The aim of this study was to identify novel mechanisms underlying development of cardiac remodelling/dysfunction further to DOX‐stimulated Nox2 activation.

MODULATION OF OXIDATIVE STRESS INDUCED APOPTOSIS AND AUTOPHAGY IN CARDIOMYOCYTES BY INTERMEDIN

The heart is subjected to oxidative stress in conditions of increased reactive oxygen species (ROS) production, such as doxorubicin (DOX) chemotherapy. A major cause of associated ventricular dysfunction is cardiomyocyte loss through apoptosis, while autophagic processes can also be detrimental. Intermedin (IMD) has emerged as a major counter-regulatory peptide with cytoprotective properties. Here we examined its potential to be upregulated and attenuate indices of cardiomyocyte death by a mechanism involving NADPH oxidase-derived superoxide. In isolated adult C57BL/6J wild type (WT) mouse ventricular cardiomyocytes, DOX (5×10−7 M) increased preproIMD mRNA expression 3.5-fold, which was significantly decreased in NOX2-deficient cells. Similarly, IMD mRNA was upregulated by the direct pro-oxidant, H2O2 (10−7 M) in both WT and HL-1 cardiomyocytes, as was NOX2; DOX increased IMD protein (immuno-cytochemistry). Superoxide production stimulated by DOX or H2O2 (by ∼35%, lucigenin-enhanced chemiluminescence), was abolished by IMD (10−10 M–10−8 M) in both WT and HL-1 cardiomyocytes. Apoptosis in HL-1 cells, shown by DOX-induced increases in caspase 3/7 activity (3.5-fold), was decreased significantly by IMD (10−9 M), which also increased cell viability. Increased autophagosome formation after serum starvation or DOX treatment (by ∼50%, Cyto-ID fusion with LC3 protein), was decreased to control values by IMD (10−9 M). Confocal imaging showed numerous cytoplasmic punctate structures with DOX, whereas addition of IMD showed a diffuse LC-3 staining pattern similar to control. These findings indicate that a NOX2-mediated increase in IMD in cardiomyocytes could have a potential autocrine effect, acting at nM concentration to reduce levels of superoxide and so limit cell death by apoptotic and autophagic mechanisms.