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European Journal of Heart Failure 2007 9(6-7):548-557; doi:10.1016/j.ejheart.2007.02.006
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© 2007 European Society of Cardiology

Cardiomyocyte-restricted over-expression of C-type natriuretic peptide prevents cardiac hypertrophy induced by myocardial infarction in mice

Yong Wanga,b,1, Monique C. de Waardc,1, Anja Sterner-Kockd, Holger Stepane, Heinz-Peter Schultheissa, Dirk J. Dunckerc and Thomas Walthera,b,*

a Department Cardiology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin (CBF), Germany
b Department of Pharmacology, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands
c Experimental Cardiology, Erasmus Medical Center, Rotterdam, The Netherlands
d Institute for Veterinary Pathology, Freie Universität, Berlin, Germany
e Department of Obstetrics and Gynecology, University of Leipzig, Germany

* Corresponding author. Department of Pharmacology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands. Tel.: +31 10 4087530; fax: +31 10 4089458. E-mail address: thomas.walther{at}charite.de


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Infused C-type natriuretic peptide (CNP) was recently found to play a cardioprotective role in preventing myocardial ischaemia/reperfusion (I/R) injury and improving cardiac remodelling after myocardial infarction (MI) in rats. Our study aimed to investigate the effect of cardiomyocyte-specific CNP over-expression on I/R injury and MI in transgenic mice.

Methods and results: We generated transgenic (TG) mice over-expressing CNP in cardiomyocytes. Elevated CNP expression on RNA and protein levels was demonstrated by RNase-protection assay and radioimmunoassay. Male TG mice and age-matched wild-type (WT) littermates were subjected to 1-hour global myocardial ischaemia and 23 h of reperfusion or permanent ligation of the coronary artery for 3 weeks.

Infarct size did not differ between the WT and TG groups in mice subjected to I/R. In mice that underwent permanent ligation of coronary arteries, both left and right ventricular hypertrophy were prevented by CNP over-expression 3 weeks post-MI. Histological analysis revealed less necrosis, muscular degeneration and inflammation in infarcted TG mice. Impairment of cardiac function was less pronounced in transgenic animals than in the wild-type controls.

Conclusions: Over-expression of CNP in cardiomyocytes does not affect I/R-induced infarct size but prevents cardiac hypertrophy induced by MI. Therefore, CNP may represent a potent therapeutic target for the treatment of patients with cardiac hypertrophy induced by myocardial infarction or other aetiology.

Key Words: Natriuretic peptide • Infarction • Hypertrophy • Ischaemia • Reperfusion

Received September 15, 2006; Revised January 16, 2007; Accepted February 19, 2007


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Ischaemic heart disease is the most common cause of death in many countries around the world. Temporary or permanently stopped blood supply to the cardiomyocytes results in ischaemia/reperfusion (I/R) injury or myocardial infarction (MI). After MI, left ventricular (LV) remodelling occurs, in which the myocardium changes shape, size, and function in response to increased mechanical and neurohumoral stress. These adaptations include scar maturation and cardiac hypertrophy of remote myocardium to compensate for myocardial loss and increased wall stress [1,2]. Despite the apparent appropriateness of this remodelling, it constitutes an independent risk factor for the progression from LV dysfunction to overt congestive heart failure.

C-type natriuretic peptide (CNP) belongs to the natriuretic peptide family, which consists of three structurally related peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and CNP [3]. In contrast to ANP and BNP, which are produced mainly in the cardiac atria and ventricles, CNP is mainly released in an autocrine/paracrine fashion from endothelial cells as an endothelium-derived vasodilator [4,5]. The biological actions of natriuretic peptides are modulated by three different membrane-bound receptor subtypes, natriuretic peptide receptors A, B, and C (NPRA, NPRB and NPRC) [6]. NPRA and NPRB are guanylyl cyclase (GC)-coupled receptors, which can convert guanosine triphosphate (GTP) to guanosine 3',5'-cyclic monophosphate (cGMP). NPRC is a single transmembrane receptor with a short 37-amino-acid intracellular tail that lacks GC activity but contains a pertussin toxin-sensitive Gi binding domain [7]. ANP and BNP are ligands of NPRA, while CNP preferentially binds to NPRB. NPRC has similar affinity to all three natriuretic peptides and is commonly considered a clearance receptor [8,9].

Since Wei and colleagues first confirmed the presence of CNP within the myocardium by immunohistochemistry and radioimmunoassay [10], accumulating evidence suggests that CNP exhibits important autocrine and paracrine functions within the heart and coronary circulation [11]. Hobbs et al. demonstrated that, in addition to interacting with its receptor NPRB, endothelium-derived CNP is involved in the regulation of rat coronary circulation via the activation of NPRC [12]. They further showed that this newly defined CNP/NPRC pathway represents a protective mechanism against I/R injury in isolated perfused hearts, since infusion of CNP, either prior to or following ischaemic insult, resulted in a 30-50% reduction in the infarct size [12]. Furthermore, in vivo administration of CNP has been shown to improve cardiac function and attenuate cardiac remodelling after myocardial infarction in rats [13]. However, it is still not certain whether the cardiac CNP effects are dependent on the endothelium or cardiomyocytes. Thus, we generated transgenic mice selectively over-expressing CNP in cardiomyocytes. This in-vivo model was used to investigate the effect of cardiomyocyte-specific CNP over-expression on I/R injury and MI.


   2. Materials and methods
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Generation of transgenic mice
All animal experiments were done according to the guidelines of the Federal Law on the Use of Experimental Animals in Germany or guidelines provided by the animal committee of The Netherlands and were approved by the local authorities. cDNA encoding rat CNP (RNCNP) was cloned by RT-PCR, then ligated into an expression vector containing the {alpha}-myosin heavy chain ({alpha}MHC) gene promoter that directs transgene over-expression to cardiomyocytes [14] and the bovine growth hormone (BGH) polyadenylation signal. After linearizing the generated recombinant plasmid (pMHCRNCNP) by Bgl II/Bln I, a 2750-bp fragment was microinjected into the pronuclei of fertilized oocytes isolated from FVB/N mice according to the standard protocol. Chromosomal integration of the CNP transgene was proven by Southern blot analysis as described previously [15]. Ten µg of genomic DNA digested by EcoR I were used to screen for potential founder lines.

2.2. Genotyping of transgenic mice
Primers in the {alpha}MHC promoter region (MHC51: 5'-CAT CTG TCT CTA CTC TCT CTG CC-3') and the CNP gene (RNCNP3: 5'-CCG CCT GGA GTC TTG TCA CC-3') were used to perform PCR to detect the presence of the CNP transgene (35 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 50 s with a hot-start at 94 °C for 3 min, and a final extension at 72 °C for 10 min). The PCR reactions were carried out in a total volume of 45 µl containing 1.25 U of BioTherm– DNA polymerase, 2 mmol/L of MgCl2, 0.26 mmol/L of dNTP (Rapidozym GmbH, Berlin, Germany), and 2 nmol/L of each primer (BioTeZ GmbH, Berlin, Germany).

2.3. RNase protection assay (RPA)
Total RNA was isolated from heart, lung, kidney, testis and brain using the TRIzol reagent (Invitrogen GmbH, Karlsruhe, Germany) with subsequent chloroform-isopropanol extraction according to the manufacturer's instructions. RNCNP, mouse BNP and collagen type III mRNA expression were identified by RPA using the Ambion RPA II kit (Ambion (Europe) Ltd., Huntingdon, U.K.) as described elsewhere [16]. In brief, T7- or SP6-RNA polymerase transcribed radioactive antisense probes complementary to target mRNA (RNCNP: 405 bp; BNP 290 bp; collagen type III: 476 bp). RNA complementary to 127 nucleotides of rL32 mRNA was used as a positive control. Ten to 15 µg of each RNA sample was hybridized with approximately 40,000 cpm of RNCNP-, BNP- or collagen type III- and 40,000 cpm of rL32-radiolabeled antisense probe in the same reaction. The hybridized fragments protected from RNase A+T1 digestion were separated by electrophoresis on a denaturing gel (5% [w/v] polyacrylamide, 8M urea) and analyzed using a FUJIX BAS 2000 Phospho-Imager system (Raytest GmbH, Straubenhardt, Germany). Quantitative analyses were performed by measuring the intensity of the target bands normalized by the intensity of rL32.

2.4. Bioassay for CNP
After weighing the hearts, lungs and kidneys from both genotypes seven volumes of ice-cold 0.5 N acetic acid containing 0.01% Triton X-100 were added and homogenized by a T8 homogenizer (IKA GmbH, Staufen, Germany). The homogenates were boiled for 10 min, and then centrifuged at 15,000 rpm for 40 min at 4 °C. The protein concentration of the supernatants was determined with the BCA protein assay kit (Perbio Science GmbH, Bonn, Germany). The supernatants or plasma were extracted using Sep-Pak C18 cartridges, and the eluates were lyophilized and dissolved in an assay buffer of a commercial radioimmunoassay kit for CNP (Immundiagnostik AG, Bensheim, Germany) as previously described [17]. CNP concentration was normalized by total protein concentration and expressed as pg/mg for organs and pg/ml for plasma.

2.5. cGMP measurement
Levels of cGMP in the ventricles and plasma were measured using a commercially available low-pH cGMP immunoassay kit (R&D Systems, Minneapolis, MN, USA). The frozen tissue was weighed and homogenized in 10 volumes of 0.1 N HCl. One hundred µl of supernatant were used to measure the cGMP concentration following the non-acetylation procedure. cGMP concentration was expressed as pmol/mg for organs and pmol/ml for plasma. The sensitivity of the assay was approximately 0.6 pmol/ml.

2.6. Animal experimental protocols
2.6.1. Blood pressure measurement
Mice were sedated with 4% isoflurane, intubated and artificially ventilated with a mixture of 70% room air and 30% oxygen including 2% isoflurane for anesthesia. A PE10 cannula was inserted into the left carotid artery to monitor systolic blood pressure (SBP), mean arterial pressure (MAP), diastolic blood pressure (DBP) and heart rate (HR) for 15 min using BMON software (TSE GmbH, Bad Homburg, Germany).

2.6.2. Cardiac surgery
Before starting instrumentation, the mice received 0.05 mg/kg analgesic (buprenorphine hydrochloride) subcutaneously, which was repeated once 12 h after surgery. Surgical techniques were employed according to Tarnavski et al. [18] with some minor revisions. Briefly, mice were anaesthetized and artificially ventilated with a mixture of oxygen and N2O [1:2 (vol/vol)] using a rodent ventilator, to which 2-2.5% isoflurane was added for anaesthesia [19]. The ventilation rate was set at 80 strokes/min, a peak inspiration pressure of 18 cm H2O and a positive end expiration pressure of 4 cm H2O. Mice were placed on a heating pad to maintain body temperature at 37 °C. Regional I/R was produced by 1-hour occlusion of the left anterior descending coronary artery (LAD) with a sterile 7.0 silk suture followed by 23 h of reperfusion. Mice were then sacrificed and myocardial infarct size was measured.

In another group of mice, MI was produced by permanent ligation of the LAD. Echocardiographic and haemodynamic measurements were performed 3 weeks after MI, after which RV, LV and wet lung weights were obtained. One half of the LV was snap frozen in liquid nitrogen, and the other half was fixed in 4% paraformaldehyde for histological analysis.

2.6.3. Echocardiographic and haemodynamic analysis
Three weeks after MI, echocardiographic and haemodynamic measurements were performed as previously described [19]. M-mode echocardiograms of the LV with simultaneous electrocardiograph (ECG) (ProSound SSD-4000, Aloka, Tokyo, Japan) were obtained using a 13-MHz probe. LV diameters were measured at end diastole (LVEDD) and end systole (LVESD), and fractional shortening was calculated as FS=(LVEDD–LVESD)/LVEDDx100%. After echocardiography, a polyethylene catheter (PE-10) was inserted into the left carotid artery and advanced into the aortic arch to measure mean aortic blood pressure (MAP). A 1.4-F-microtipped pressure transducer catheter (Millar Instruments, Houston, TX, USA) was inserted into the LV lumen via the right carotid artery to measure LV pressure. Subsequently, baseline recordings were obtained for MAP, HR and LV systolic pressure. In addition, we measured the contractility parameter LV dP/dtmax, the afterload independent LV dP/dtP30 (positive LV dP/dt at LV pressure of 30 mm Hg) and the relaxation parameters Tau ({tau}) and LV dP/dtmin [19].

2.7. Measurement of infarct size
After reperfusion, the ligature around the LAD was retied and 1 ml of 1% Evan's blue dye was injected into the jugular vein to delineate the area at risk (AAR). The heart was quickly excised, frozen for a few minutes at –20 °C, and then immediately sliced with a scalpel into 1-mm-thick sections perpendicular to the long axis of the heart. Slices were incubated individually in 2% triphenyltetrazolium chloride (TTC) (Fluka, Buchs, Switzerland) in Sörensen buffer (pH 7.4) at 37 °C for 5 min. Thereafter, the RV was removed, and each slice of LV was weighed and photographed on both sides. Evan's blue stained area, Evan's blue negative area (AAR), TTC stained area, and TTC stain negative area (infarct area) were digitally measured using SigmaScan (SPSS, Chicago, IL, USA). The infarct size was calculated according to the method of Kurrelmeyer et al. [20], and expressed as a percentage of infarct area (IA) over total AAR.

2.8. Histological analysis
Paraffin-embedded LVs were sectioned along the short axis into 5-µm-thick slices. After staining with hematoxylin-eosin (H.-E.), muscular degeneration and inflammation (mononuclear inflammatory infiltrate) in the infarct area were evaluated by one pathologist who was blinded to the genotypes. Ladewig staining was performed to assess the degree of fibrosis in the infarcted area and the stains graded with 0x + to 4x +.

2.9. Data and statistical analysis
All data were expressed as mean±SEM. Differences between groups were determined using two-way ANOVA followed by post-hoc testing using Student's t test. Delta values were calculated by subtracting the mean of LV/tibia or RV/tibia in sham-operated mice from each sample in MI group and the difference in change between wild-type (WT) and transgenic (TG) lines was compared by an unpaired t-test. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Generation and basic characterization of CNP-transgenic mice
We generated transgenic mice over-expressing rat CNP. The transgene was driven by the rat {alpha}MHC to ensure specific transgene over-expression in cardiomyocytes. The transgene integration in the chromosomal DNA was proven by Southern blot. Five independent founders 2, 3, 8, 9, and 12 were detected in the first litter (Fig. 1A). Transgenic line 1 was identified from a second litter (data not shown). PCR confirmed the transgenic offspring of founders crossed with wild-type FVB/N mice. All lines were fertile and did not differ in body weight compared to the wild-type controls. To prove CNP transgene mRNA expression, RPA was used to investigate the ventricles of 3- to 5-month-old males of the transgenic lines 1, 2, 9, and 12 (data not shown). Since quantitative analysis showed that CNP mRNA expression was the highest in line 1 among the four lines, this line was selected for the experiments.


Figure 01
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  		Fig. 1 Generation and identification of transgenic mice over-expressing CNP mRNA. (A) Chromosomal integration of the transgene was detected by Southern blot using EcoR I-digested genomic DNA isolated from tail biopsies of possible founders. Transgenic lines 2, 3, 8, 9, 12 were identified from this litter. Different length and number of bands in founder mice result from random integration of the transgenic cDNA in to the chromosomal DNA. IF: injected fragment, which was released through digestion by Bgl II and Bln I. Plasmid: recombinant pMHCRNCNP plasmid linearized by EcoR I. (B) Representative RNase-protection assay showing CNP mRNA expression in different investigated organs of 3-month-old CNP1-transgenic (+) and wild-type (–) mice. Y+: yeast RNA with RNase treatment; Y–: yeast RNA without RNase treatment. (C) Quantification of CNP transgene expression in different organs of CNP1 transgenic (+) and control (–) mice after autoradiographic signal analysis. Data are shown as multiples after normalization to rL32 mRNA levels, n=3.

 
To investigate the ontogenetic regulation of CNP transgene expression, cardiac CNP mRNA of transgenic mice was determined at different time points. CNP transgene over-expression was detected at all investigated time points with no significant variation in aging (data not shown). To exclude ectopic expression of the transgene, CNP mRNA in the atrium, ventricle, lung, kidney, liver, testis, forebrain, and hindbrain was detected in 3-month-old male TG mice and their WT littermates using RPA (Fig. 1B). As expected, high transgene CNP expression was detected in the atrium and ventricle, whereas none of the other investigated organs showed detectable transgene expression (Fig. 1C).

Peptide concentrations were measured in cardiac tissue, lung, kidney and plasma to confirm that CNP mRNA over-expression also led to more CNP generation. CNP levels were significantly increased in atria (WT: 0.15±0.02 vs. TG: 0.30±0.07 pg/mg; P<0.05) and ventricles (LV: WT: 0.01±0.01 vs. TG: 0.74±0.12 pg/mg; RV: WT: 0.01±0.01 vs. TG: 0.92±0.14 pg/mg; P<0.001) of transgenic mice. No change was found in the other organs or plasma (Fig. 2A).


Figure 02
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  		Fig. 2 Characterization of CNP peptide expression and cGMP accumulation in transgenic mice. (A) Quantification of CNP peptide concentration from atrium, ventricle, lung, kidney, and plasma of CNP1-transgenic (TG) and wild-type (WT) mice, analyzed by radioimmunoassay. *P<0.05, ***P<0.001 vs. WT, n=6-8. (B) cGMP concentrations in the ventricles and plasma of CNP-transgenic (TG) and wild-type (WT) mice. *P<0.05 vs. WT, n=7.

 
Due to its interaction with the NPRB receptor, which couples to guanylyl cyclase, CNP stimulates the generation of second messenger cGMP, an important mediator of effects initiated by natriuretic peptides. cGMP levels in ventricles and plasma were measured to test the hypothesis that CNP over-expression results in higher cGMP concentration. While the cGMP level was significantly increased in ventricles of transgenic mice (WT: 2.5±0.2 vs. TG: 3.5±0.2 pmol/mg; P<0.05), it did not differ in plasma in either of the two lines (Fig. 2B).

CNP-transgenic mice showed no change in cardiac weight as shown by the ratios of LV and RV to body weight (data not shown) or to tibia length (Table 1). Furthermore, CNP over-expression in cardiomyocytes did not modify blood pressure and heart rate compared to their age-matched wild-type littermates (data not shown).


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  		Table 1 Characteristics of wild-type (WT) and transgenic (TG) mice 3 weeks after induction of myocardial infarction or sham operation

 
3.2. Effect of CNP on I/R injury
To assess impact of CNP over-expression on I/R injury, infarct size was measured after 1 h of regional myocardial ischaemia and 23 h of reperfusion. AAR/LV was similar in both groups (WT: 45±2% vs. TG: 43±3%, n=9, per group). No significant difference was observed in myocardial infarct size as defined as the percentage of IA/AAR (WT: 68±8% vs. TG: 69±7%, n=9) compared to wild-type controls. Consequently, the infarct size in relation to the left ventricle was also unaltered (WT: 31±4% vs. TG: 30±3%, n=9).

3.3. Analysis of cardiac remodelling and function after myocardial infarction
Echocardiographic and haemodynamic measurements were performed to evaluate cardiac remodelling and function 3 weeks after MI induction. The LV weight/tibia length ratio did not differ for sham-operated WT and TG mice (WT: 46±2 vs. TG: 47±2 mg/cm) (Table 1). All other measured parameters were comparable in sham-operated wild-type and transgenic mice (Table 1). However, 3 weeks after MI, infarct-induced cardiac hypertrophy was prevented by CNP over-expression in cardiomyocytes. While LV mass was significantly increased in wild-type mice, no significant increase in LV weight was observed in mice with elevated CNP expression ({Delta}LV/tibia: WT: 8.6±1.2 vs. TG: 1.8±1.3 mg/cm; P<0.01) (Fig. 3A). The RV/tibia length ratio was also significantly increased in wild-type mice (Table 1), but CNP over-expression in cardiomyocytes prevented MI-induced RV hypertrophy ({Delta}RV/tibia: WT: 2.4±0.8 vs. TG: – 1.4±0.5 mg/cm; P<0.01) (Fig. 3A). It should be noted that lung wet weight was significantly higher in wild-type mice after MI than in sham-operated mice (sham: 158±7 vs. MI: 198±11 mg/g; P<0.05), while the increase was not statistically different in the TG line (sham: 169±10 vs. MI: 189±10 mg/g) (Table 1).


Figure 03
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  		Fig. 3 CNP over-expression in cardiomyocytes prevents cardiac hypertrophy. (A) Left-ventricle (LV) and right-ventricle (RV) hypertrophy as indicated by {Delta}LV/tibia length and {Delta}RV/tibia length (mg/cm) were prevented in transgenic mice (TG). **P<0.01 vs. wild-type (WT) controls, n=7 (TG) or n=8 (WT). (B) Upper panel: RNase-protection assay showing collagen type III (col III) expression in non-infarcted left ventricles of TG and WT control mice 3weeks after myocardial infarction (MI) or sham operation. Lower panel: Quantification of col III expression in non-infarcted left ventricular myocardium normalized by rL32 mRNA levels.

 
Cardiac function was significantly impaired in both MI groups except for HR and Tau. Whereas MAP was significantly decreased after MI in the wild-type group as described for this experimental model [21,22], it did not differ in transgenic mice (Table 1). Moreover, indices of LV systolic and diastolic function, as dP/dtmax and dP/dtmin, fractional shortening, and LVEDD indicated better performance in transgenic mice, although they did not reach statistical significance (Table 1).

3.4. Quantification of parameters of cardiac failure
To investigate whether CNP had other beneficial effects besides preventing cardiac hypertrophy, we examined mRNA expression levels of collagen type III and BNP, which are associated with fibrosis and cardiac failure. In the non-infarcted LV, mRNA levels of collagen type III were significantly elevated after MI in both groups without a significant inter-group difference. However, the increase in the transgenic group was less pronounced than in the wild-type mice (WT: 3.7±0.3 vs. TG: 1.8±0.2-fold) (Fig. 3B). BNP mRNA, which is known to be upregulated in the ventricles under pathophysiological conditions like cardiac failure, was significantly elevated after MI in wild-type group, while the increase in the transgenic group did not reach statistical significance (data not shown).

3.5. Histological analysis
H.-E. staining was carried out to prove whether the prevented cardiac hypertrophy also had an impact on histological structure and inflammation after myocardial infarction. Normal cardiomyocytes were evident in both sham-operated lines (Fig. 4A and B). However, there was prominent muscular degeneration and a moderate mononuclear inflammatory infiltration in the infracted area of wild-type mice 3 weeks after MI compared to only mild degeneration of myofibrils and no mononuclear inflammatory infiltrate in the transgenic group (Fig. 4C and D).


Figure 04
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  		Fig. 4 Cardiac morphological changes after myocardial infarction. Normal-appearing cardiomyocytes were found in sham-operated wild-type (WT) and transgenic (TG) mice (A and B). There was prominent muscular degeneration and a moderate mononuclear inflammatory infiltrate in the infarcted area of wild-type animals (C), while only mild degeneration of myofibrils was observed in the infarcted transgenic group, and there is no mononuclear inflammatory infiltrate (D). Representative photographs stained with hematoxylin and eosin (H.-E.) are given. Bar=100 µm.

 
Ladewig staining was used to assess the extent of cardiac fibrosis. No fibrosis was observed in sham-operated mice of either group (Fig. 5A and B). Marked fibrosis (blue stain) was observed in the infarcted area of both groups that underwent myocardial infarction surgery. However, confirming the findings of H.-E. staining, there were more abundant muscle fibers (muscle = red stain) preserved in the TG group than in the wild-type controls (Fig. 5C and D).


Figure 05
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  		Fig. 5 Histological analysis to determine the effect of CNP over-expression on cardiac fibrosis. Normal myofibrils (red staining) were found in sham-operated wild-type (WT) and transgenic (TG) mice with no detectable fibrosis (blue staining) (A and B). Marked fibrosis and few muscle fibers were observed in the infarcted area of wild-type mice (C). Infarcted transgenic mice showed still marked fibrosis but more abundant myofibrils (D). Representative Ladewig staining photographs are given. Bar=100 µm.

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In this study, we generated transgenic mice selectively over-expressing rat CNP in cardiomyocytes, we confirmed the over-expression on mRNA and peptide levels, and demonstrated that cardiomyocyte-restricted CNP over-expression does not reduce infarct size produced by 1-hour global myocardial ischaemia and 23-hour reperfusion, but prevents cardiac hypertrophy 3 weeks post-myocardial infarction.

CNP mediates most of its physiological actions (e.g. vasorelaxation) via interaction with its receptor NPRB. However, it was also recently identified as an endothelium-derived hyperpolarizing factor (EDHF) in rat mesenteric arteries involving activation of the clearance receptor NPRC [23]. This CNP/NPRC signalling pathway has been implicated in the regulation of coronary blood flow and the reduction of I/R-induced infarct size in isolated perfused Langendorff rat hearts [12]. In contrast to this described cardioprotective role of CNP against I/R, we did not observe a significant difference between transgenic mice over-expressing CNP in cardiomyocytes and their controls in our studies. Species-specific differences, independent experimental protocols, and unequal origin of CNP elevation may account for this discrepancy. Firstly, Langendorff preparations from rat hearts were used in previous studies, whereas we investigated transgenic mice in vivo. However, we do not consider this to be the main reason for the different findings, since the rat and mouse share similar signalling pathways in cardiac remodelling. Secondly, mice were subjected to 1 h of ischaemia and 23 h of reperfusion in our in vivo studies, but a protocol of 25 min of global ischaemia and 120 min of reperfusion was used in the previous ex vivo experiments. Thirdly, CNP originated from the cardiomyocytes in our transgenic animals and thus acted in an autocrine way; however, CNP was supplied intravascularly in retrogradely perfused hearts in the Hobbs study, thereby exposing the endothelium to the highest concentrations. Thus, the discrepancy between Hobbs' et al. and our findings could result from the need to stimulate NPRC on endothelial cells in order to mediate the beneficial effects of CNP in an I/R model, which did not occur in our transgenic mice.

Pre-ischaemic infusion of ANP or BNP has been shown to elicit protective effects against I/R in isolated rat hearts [24,25]. However, the possible mechanisms for the cardioprotective effects of the three natriuretic peptides on I/R seem to be controversial. NPRC activation is thought to be responsible for the cardioprotective role of CNP as mentioned above, and the selective agonist of NPRC (cANF4-23) also reduces infarct size and maintains coronary perfusion pressure [12]. Studies of ANP and BNP effects show the involvement of cGMP generated via the NPRA receptor in the reduction of infarct size [24,25]. Interestingly, increased cGMP supply in transgenic mice selectively over-expressing endothelial nitric oxide synthase (eNOS) in cardiomyocytes was also implicated in attenuating myocardial I/R injury in an ex vivo model [26]. Thus, although we can exclude the beneficial effect of CNP over-expression on I/R in our in vivo model based on infarct size measurement, the demonstrated cGMP increase in CNP-transgenic hearts may still indicate cardioprotective effects against I/R in an ex vivo model. Consequently, further studies need to evaluate the possible effects of CNP in different I/R models and their relevance under in vivo conditions.

Although we did not see beneficial effects of CNP on I/R injury in our in vivo model, cardiomyocyte-restricted over-expression of this peptide identified significantly improved cardiac remodelling 3 weeks after myocardial infarction. Both MI-induced LV and RV hypertrophy were prevented in our transgenic mice. The results provide clear in vivo evidence that CNP is a potent antihypertrophic agent after MI. This agrees in part with a previous report by Soeki and colleagues in rats [13]. In vivo administration of CNP via osmotic mini-pumps significantly improved cardiac function and led to less pronounced cardiac hypertrophy and fibrosis in rats 2 weeks post-MI [13]. In our present study, however, the improved cardiac remodelling in TG mice did not result in significantly improved cardiac function compared to their infarcted wild-type controls, although impairment of cardiac function compared to the sham group was less pronounced in transgenic mice than in the infarcted wild-type mice, probably due to inhibited cardiac remodelling. However, while Soeki et al. applied CNP by mini-pump, we exclusively elevated cardiac CNP concentrations, showing for the first time that the local cardiac CNP mediates the antihypertrophic effects.

The prevention of hypertrophy by CNP over-expression in our transgenic mice that underwent myocardial infarction, agrees with data on the antihypertrophic effects of its receptor NPRB, as recently reported by Langenickel et al. [27]. Transgenic rats expressing a dominant-negative mutant of NPRB (NPR-B {Delta}KC) developed progressive, blood pressure-independent cardiac hypertrophy [27]. Histological assessment and echocardiography revealed cardiac hypertrophy in these NPR-B {Delta}KC transgenic rats, which was aggravated with age accompanied by increasing cardiac markers of heart failure. Interestingly, there was no evidence for increased interstitial or perivascular fibrosis in these rats, supporting our finding of non-significant differences in the grade of stimulated fibrosis post-MI in CNP-transgenic mice compared to wild-type controls. Furthermore, chronic volume overload by an infrarenal aortocaval shunt in 8-week-old rats resulted in exaggerated cardiac hypertrophy in NPR-B {Delta}KC transgenic rats 6 weeks after surgery [27]. Although the affinity of CNP to NPRA is much less than to the NPRB receptor [28], and CNP does not increase cGMP accumulation in cells expressing human NPRA [11,29], we cannot finally exclude that transgenic CNP mediates part of its antihypertrophic effect via NPRA. Nevertheless, the combination of data from Langenickel et al. and our findings provides clear evidence that the CNP/NPRB axis is implicated in the regulation of cardiomyocyte growth but not in cardiac fibrosis.

Furthermore, a limitation of Langenickel et al.'s findings was the increased heart rate in the NPR-B {Delta}KC transgenic rats compared to controls, which was possibly caused by reduced CNP actions on the central nervous system facilitating baroreflexes by downregulation of functional NPRB. Therefore, they could not discriminate whether central or peripheral alterations were responsible for the cardiac hypertrophy observed in this animal model. Since our transgenic mice did not show altered systemic blood pressure and heart rate and specifically over-express CNP in the heart, we can finally define the cardiac CNP/NPRB axis to be crucial in preventing cardiac hypertrophy.

Although we cannot conclusively prove exocytosis of transgene CNP to the extracellular space of cardiomyocytes, new research has shown that intracellular compartmentation of a peptide ligand allows interaction with its receptor and thus stimulation of receptor signalling [30]. Furthermore, that the CNP/NPRB axis is responsible for the cardiac CNP effects is also supported by cell-based studies. CNP modulates the growth, proliferation, and hypertrophy of smooth muscle cells, cardiomyocytes and fibroblasts [31-33], and cGMP is implicated in all of these studies when CNP binds to its receptor NPRB. Additionally, our data on increased cGMP in the hearts of CNP-transgenic mice is congruent with findings in isolated rat and rabbit cardiomyocytes generating cGMP after CNP stimulation [34,35].

The importance of the CNP/NPRB axis is furthermore illustrated by a series of studies about nitric oxide synthase, supporting the concept that cGMP, a second messenger not stimulated by interaction of CNP with NPRC, is a key player in the local regulation of cardiac remodelling and function after myocardial infarction [36-39].

In conclusion, the present study indicates that the CNP/NPRB/cGMP signalling pathway plays an important role in the local regulation of cardiac hypertrophy under pathophysiological conditions. Thus, modulation of this pathway represents a potent new therapeutic target for the treatment of patients with myocardial infarction or hypertension-induced cardiac hypertrophy.


   Acknowledgement
 
This study was supported by the grant WA1441/15-2 from the ‘Deutsche Forschungsgemeinschaft’ (German Research Foundation).


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
1 Both authors contributed equally to this work. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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