c - type natriuretic peptide ( cnp ) belongs to the natriuretic peptides ( nps ) family , which was originally isolated from porcine brain tissues and later established as a positive regulator of endochondral ossification . in the cardiovascular system , cnp is secreted from endothelial cells and cardiac fibroblasts , and acts as an autocrine / paracrine regulator [ 2 , 3 ] . myocardial production and circulating cnp levels are increased in patients with chronic heart failure [ 4 , 5 ] , suggesting that cnp has an important role in the pathogenesis of heart failure . anp and bnp bind to natriuretic peptide receptor ( npr)-a , inducing natriuresis and diuresis , whereas cnp preferentially binds to its specific receptor , npr - b . because of the relative abundance of npr - b over npr - a in cardiac fibroblasts and in cardiomyocytes , cnp reportedly has more potent antihypertrophic and antifibrotic properties , despite less hypotensive and less natriuretic effects compared with anp and bnp [ 6 , 7 ] . the recently designed novel compound cd - np , which is a chimera of cnp and dnp , is a promising candidate for new - generation therapeutics using nps . it is therefore of interest to elucidate the precise role of cnp in cardiovascular pathophysiology from both a basic and clinical medicine perspective . previous experimental studies showed that long - term administration of cnp attenuates cardiac remodeling and maintains systolic function in murine models of myocardial infarction and myocarditis , independent of its hypotensive effect [ 9 , 10 ] . cardiomyocyte - specific overexpression of cnp attenuated cardiac hypertrophy in a murine model of myocardial infarction . conversely , transgenic rats with overexpression of a dominant - negative mutant of npr - b showed exacerbated cardiac hypertrophy . in vitro , it has been shown that cnp prevents agonist - induced cardiomyocyte hypertrophy via a cyclic gmp - dependent protein kinase - mediated mechanism [ 7 , 13 ] . cnp also inhibits fibroblast proliferation and extracellular matrix production of cardiac fibroblasts more potently than anp and bnp . these data indicate that cnp acts directly on myocardial cells to protect against pathological stimuli . experimental and clinical evidence implicates a central role for angiotensin ii ( ang ii ) in all stages of the cardiovascular continuum [ 14 , 15 ] , and the detrimental effects of ang ii are mediated , at least in part , by reactive oxygen species ( ros ) [ 1618 ] . to this end however , little is known about the effects of cnp on ang ii - induced cardiac ros generation . here , we investigated whether chronic cnp infusion could attenuates myocardial superoxide production in a mouse model of cardiac hypertrophy and remodeling induced by ang ii . ang ii ( peptide institute ) or saline alone was subcutaneously infused via an osmotic minipump ( alzet model 1002 ) at the rate of 3.2  mg / kg / day for 2 weeks . simultaneously with the initiation of ang ii treatment , mice were infused with cnp ( peptide institute , 0.05  g / kg / min ) or vehicle by continuous subcutaneous infusion for 2 weeks . systolic blood pressure ( sbp ) and heart rate ( hr ) of the conscious mice were monitored by the tail - cuff method ( mk-2000st ; muromachi ) . the serum level of cnp was measured using the cnp-22 eia kit ( phoenix pharmaceuticals , karlsruhe , germany ) according to the manufacturer 's protocol . all procedures were performed in accordance with the kumamoto university animal care guidelines , which conformed to the guide for the care and use of laboratory animals published by the us national institutes of health ( nih publication no . transthoracic ultrasound cardiogram ( ucg ) was performed using a xario system ( toshiba , tokyo , japan ) equipped with a 12-mhz linear array transducer . m - mode images were recorded from the short - axis view at the high papillary muscle level . left ventricular end - diastolic dimension ( lvdd ) , end - systolic dimension ( lvds ) , intraventricular septum ( ivs ) , and posterior wall thickness ( pw ) were measured . fractional shortening ( % fs ) was calculated using the following equation : % fs = ( lvdd  lvds)/lvdd  100 . all echocardiography was performed by investigators who were blinded to the identity of the treatment group . total rna was prepared using a qiagen rneasy fibrous minikit , using the protocol supplied by the manufacturer , and cdna was produced using the thermoscript rt - pcr systems ( invitrogen , carlsbad , ca ) . transcript expression levels were determined as the number of transcripts relative to those for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , and normalized to the mean value from control hearts . heart sections were prepared and stained with hematoxylin and eosin ( h&e ) for overall morphology or masson 's trichrome ( mt ) to assess myocardial interstitial fibrosis . at least 10 fields were selected randomly to determine myofiber size and myocardial interstitial fibrosis using image j image analysis software , as described previously . dihydroethidium ( dhe ) was used to evaluate superoxide levels of cardiac tissue as described in detail previously . comparisons among groups were made by one - way anova with fisher 's plsd test . to examine the effect of exogenous cnp supplementation on ang ii - induced cardiac remodeling , ang ii- and saline - infused mice were treated with cnp or vehicle for 2 weeks ( table 2 ) . ang ii infusion for 2 weeks significantly increased sbp compared with the saline - infused control group ( 136  6 versus 97  4  mmhg , resp . ) . circulating cnp levels were significantly increased in cnp - treated mice compared with vehicle - treated controls . the dose of cnp used ( 0.05  g / kg / min ) had no additional effect on sbp in either ang ii- or saline - infused mice . hr was unaffected by ang ii infusion in both cnp- and vehicle - treated mice compared to saline - infused mice . figure 1(a ) shows representative lv m - mode echocardiographic recordings . as shown in figure 1(b ) , ivs and pwt were significantly increased by ang ii infusion in vehicle - treated mice , and these changes were suppressed by cnp treatment ( without affecting sbp , as stated ) . echocardiographic analysis also revealed significantly increased lvedd and % fs in vehicle - treated mice 2 weeks after ang ii infusion ( figure 1(c ) ) ; however , these effects were significantly attenuated by cnp treatment . in contrast , long - term infusion of cnp had no effect on echocardiographic parameters in saline - infused mice . heart weight to tibial length ( hw / tl ) ratios were significantly increased by ang ii infusion in vehicle - treated mice ( 9.6    0.5 versus 6.5    0.2  mg / mm ; figure 2(b ) ) , while treatment with cnp decreased this ang ii - induced cardiac hypertrophy by 16% . analysis of myocyte cross - sectional area in histological sections corroborated these findings ( figure 2(a ) ) ; the calculated area of myocytes was increased in vehicle - treated mice that underwent ang ii infusion , but this increase was largely blocked in mice given cnp ( 276  16 versus 375  11  m ; figure 2(c ) ) . cnp treatment of vehicle - treated mice had no effect on myocyte cross - sectional area . consistent with this observation , ang ii infusion increased expression of the fetal - type cardiac genes anp , bnp , and -myosin heavy chain , and these upregulations were blocked by cnp treatment ( figure 2(e ) ) . two weeks after ang ii infusion , the lungs wet weight to tl ratios ( lw / tl ) , a parameter used to assess pulmonary congestion , were significantly decreased in cnp - treated mice compared with vehicle - treated controls ( 8.1    0.1 versus 10.6    0.8  mg / g ; figure 2(d ) ) . to investigate the consequence of exogenous cnp administration on cardiac remodeling interstitial fibrosis was significantly increased in vehicle - treated mice 2 weeks after ang ii infusion ( figure 3(b ) ) . however , treatment with cnp caused a significant decrease in this measure ( 4.0    0.8 versus 9.4    0.4% ) . in agreement with this observation , ang ii - induced gene upregulations of collagen i and iii were significantly decreased by exogenous cnp treatment ( figure 3(c ) ) . cnp infusion did not detectably affect fibrosis or collagen expression in saline - infused mice . myocardial superoxide generation is a key mechanism underlying the ang ii - induced cardiac remodeling . we therefore investigated whether cnp administration could attenuates ang ii - induced superoxide production in mice heart . myocardial tissue sections were stained with dhe to determine superoxide production in vehicle- and cnp - treated mice 2 weeks after ang ii infusion ( figure 4(a ) ) . ang ii infusion increased superoxide generation in vehicle - treated mice by a factor of 1.5 , but treatment with cnp markedly diminished ang ii - induced cardiac superoxide production ( figure 4(b ) ) . furthermore , ang ii induced the gene upregulation of nox4 , an nadph oxidase subunit , and this was clearly suppressed by treatment with cnp ( figure 4(c ) ) . in contrast , cnp treatment of vehicle - treated mice had no effect on myocardial superoxide production or nox4 gene expression . transcript expression levels of other nadph oxidase subunits such as p22 and p47 were not changed by cnp treatment . this study provides the first in vivo evidence that continuous infusion of exogenous cnp attenuated ang ii - induced myocardial superoxide production and cardiac dysfunction . administration of cnp significantly prevented ang ii - induced cardiac hypertrophy and lv dilatation , thereby maintaining myocardial contractile function without affecting sbp . cnp reduced ang ii - induced increases in cardiomyocyte size and interstitial fibrosis , and markedly suppressed hypertrophic and fibrotic gene expression . these beneficial effects were accompanied by reduced superoxide production and nox4 gene expression in the ang ii - infused myocardium . together , these findings suggested that cnp exerts antioxidant activity under conditions that promote cardiac hypertrophy and fibrosis , as a result , maintaining left ventricular systolic function . cardiac hypertrophy is initially an adaptive response designed to maintain cardiac function in response to hemodynamic or neurohumoral stress . however , a prolonged external load leads to decompensated cardiac hypertrophy characterized by ventricular dilation and loss of contractile function . although the role of anp and bnp in cardiac hypertrophy and remodeling has been investigated in several in vitro and in vivo studies , limited data are available on the role of cnp in this process . we used an ang ii infusion model in this study because multiple lines of evidence established that ang ii induces not only hypertension , but also directly contributes to cardiac remodeling . for example , essick et al . reported that contractile function was not impaired by same dose ( 3.2  mg / kg / day ) and same duration ( 2 weeks ) of ang ii infusion as our present study . on the other hands , fujita et al . reported that lower dose of ang ii infusion ( 1.2  mg / kg / day ) for 2 weeks reduced contractile function . the reason for the discrepancy was probably due to different reagents , equipments , or mice strain . ang ii - induced ros generation is recognized as a key mechanism regulating cardiovascular remodeling . with regard to the cellular source of ros , ang ii - induced upregulation of nadph oxidase subunits has been documented in various cardiovascular cell types including cardiomyocytes , fibroblasts , and vascular smooth muscle cells ( vsmc ) [ 2729 ] . one of these subunits , nox4 , was implicated in ang ii infusion and/or pressure overload - induced myocardial hypertrophy [ 3032 ] . it has been demonstrated that cnp prevents agonist - induced cardiomyocyte hypertrophy via a cyclic gmp - dependent protein kinase - mediated mechanism in vitro [ 7 , 13 ] . thus , we speculate that the antihypertrophic effect of chronic cnp infusion observed in this study was mediated by cyclic gmp - dependent manner and inhibition of nox4 production .   another natriuretic peptide , anp , can suppress ros production in the cardiovascular system . for example , the antihypertrophic actions of anp in vitro were accompanied by reduced levels of superoxide , mediated via the npr - a / cgmp pathway . we previously demonstrated that intravenous infusion of carperitide , the human form of anp , significantly decreased serum ros levels in patients with chronic heart failure and exogenous anp exerted direct antioxidant properties in cardiomyocytes in vitro . in contrast to anp and bnp , cnp binds to and activates its specific receptor , npr - b . both npr - a and npr - b activate cgmp - dependent signaling upon binding of their specific ligands , suggesting that the antioxidative properties of cnp were mediated via the npr - b / cgmp pathway . recent reports indicated that stimulation of npr - c decreases nadph oxidase activity and production of superoxide by attenuating the expressions of nox4 and p47 subunits of nadph oxidase in vsmc from spontaneous hypertensive rats . we speculate that cnp inhibit the nox4 expression by same mechanism in the present experiments . the synthetic reagents for anp and bnp are currently used in a clinical setting with reported antiremodeling effects . however , due to their prompt vasodilatation and natriuretic actions , these agents can not be used in patients with severe hypotension as sometimes seen in acute heart failure . compared to anp and bnp , cnp exerts lower hypotensive and natriuretic effects , and more potent antihypertrophic and antifibrotic properties . these functional differences accounted for the relative abundance of npr - b over npr - a in cardiovascular tissue [ 6 , 7 ] . furthermore , these hemodynamic and antiremodeling aspects are ideal for long - term administration , because cardiac remodeling is generally developed as a result of long - term external stimulus . in this study , chronic cnp infusion had little effect on sbp ; however , ang ii - induced cardiac remodeling was markedly prevented . our results thus support the notion that long - term cnp administration is potentially useful in anti - remodeling strategies for chronic heart diseases .   in conclusion , our data indicated that cnp protects against the development of ang ii - induced cardiac remodeling which was accompanied by reduced cardiac superoxide production .
myocardial oxidative stress and inflammation are key mechanisms in cardiovascular remodeling . 
 c - type natriuretic peptide ( cnp ) is an endothelium - derived cardioprotective factor , although its effect on cardiac superoxide generation has not been investigated in vivo . 
 this study tested the hypothesis that suppression of superoxide production contributes to the cardioprotective action of cnp . 
 angiotensin ii 
 ( ang ii ) or saline was continuously infused subcutaneously into mice using an osmotic minipump . simultaneously with the initiation of ang ii treatment , mice were infused with cnp ( 0.05  g / kg / min ) or vehicle for 2 weeks . 
 the heart weight to tibial length ratio was significantly increased by ang ii in vehicle - treated mice . 
 treatment with cnp decreased ang ii - induced cardiac hypertrophy without affecting systolic blood pressure . 
 echocardiography showed that cnp attenuated ang ii - induced increase in wall thickness , left ventricular dilatation , and decrease in fractional shortening . 
 cnp reduced ang ii - induced increases in cardiomyocyte size and interstitial fibrosis and suppressed hypertrophic- and fibrosis - related gene expression . finally , cnp decreased ang ii - induced cardiac superoxide production . 
 these changes were accompanied by suppression of nox4 gene expression . 
 our data indicate that treatment with cnp attenuated ang ii - induced cardiac hypertrophy , fibrosis , and contractile dysfunction which were accompanied by reduced cardiac superoxide production .