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Circulation Research. 2007;101:e32-e42
Published online before print August 2, 2007, doi: 10.1161/CIRCRESAHA.107.158659
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(Circulation Research. 2007;101:e32.)
© 2007 American Heart Association, Inc.

UltraRapid Communication

Impaired Heart Contractility in Apelin Gene–Deficient Mice Associated With Aging and Pressure Overload

Keiji Kuba, Liyong Zhang, Yumiko Imai, Sara Arab, Manyin Chen, Yuichiro Maekawa, Michael Leschnik, Andreas Leibbrandt, Mato Markovic, Julia Schwaighofer, Nadine Beetz, Renata Musialek, G. Greg Neely, Vukoslav Komnenovic, Ursula Kolm, Bernhard Metzler, Romeo Ricci, Hiromitsu Hara, Arabella Meixner, Mai Nghiem, Xin Chen, Fayez Dawood, Kit Man Wong, Renu Sarao, Eva Cukerman, Akinori Kimura, Lutz Hein, Johann Thalhammer, Peter P. Liu, Josef M. Penninger

From the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (K.K., Y.I., A.L., G.G.N., V.K., H.H., A.M., R.S., J.M.P.), Vienna; Medical Top Track Program (K.K.) and Department of Molecular Pathogenesis (A.K.), Medical Research Institute, Tokyo Medical and Dental University, Japan; Heart and Stroke/Richard Lewar Centre of Excellence and Toronto General Research Institute (L.Z., S.A., M.C., Y.M., M.N., X.C., F.D., K.M.W., R.S., E.C., P.P.L.), University Health Network, Ontario, Canada; Veterinary University of Vienna (M.L., M.M., U.K., J.T.), Austria; Division of Cardiology (J.S., B.M.), Medical University Innsbruck, Austria; and Institute for Experimental and Clinical Pharmacology and Toxicology (N.B., L.H.), University of Freiburg, Germany; Swiss Federal Institute of Technology (R.M., R.R.), Zurich, Switzerland.

Correspondence to Josef Penninger, MD, PhD, Institute of Molecular Biotechnology, Dr Bohr-Gasse 3, Vienna 1030, Austria. E-mail josef.penninger{at}oeaw.ac.at


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin-converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene–targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload–induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin–/y and Apelin+/y mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging.


Key Words: aging • angiotensin • cardiac failure • cardiac function • pressure overload


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Protease-mediated activation and inactivation of cardiovascular peptides, such as the renin–angiotensin system or the kinin–kallikrein system, play central roles in the homeostasis of the cardiovascular system.1,2 The angiotensin converting enzyme (ACE)2 was shown to negatively regulate the renin–angiotensin system by catalyzing the C terminus of the angiotensin II peptide to generate angiotensin 1 to 7.3,4 ACE2 was also identified as the key severe acute respiratory syndrome (SARS) coronavirus receptor5,6 and negatively regulates heart failure,7,8 diabetic nephropathy,9,10 and acute lung injury.11 Importantly, in vitro substrate screening for ACE2 has identified Apelin as an additional endogenous substrate, and ACE2 cleaves Apelin with a similar efficiency as its prominent substrate angiotensin II.12 Apelin is a newly identified endogenous peptide ligand that binds to APJ, a G protein–coupled receptor that shares significant homology with the angiotensin II type 1 receptor.13–17 The Apelin gene encodes a 77-aa preproApelin polypeptide that is processed into the C-terminal fragments Apelin-36 (amino acids 42 to 77), Apelin-17 (amino acids 61 to 77), and Apelin 13 (amino acids 65 to 77).14

Accumulating experimental evidence has implicated Apelin in multiple organ functions, such as diuresis,18 fluid intake,19,20 obesity,21 food intake,22,23 body temperature control,24 and blood vessel formation.25,26 In Xenopus, antisense oligo-mediated downregulation of Apelin expression results in impaired development of the cardiovascular system.27,28 In rodents, Apelin treatment lowers blood pressure19,29 and exerts a potent positive inotropic action in hearts.30–32 In addition, correlative clinical studies in heart failure patients suggest a possible role of Apelin in heart failure.33,34 In mammals, however, gene targeting of APJ did not reveal any overt phenotypes in mice.35 APJ knockout mice grow normally, look apparently healthy, and show normal blood pressure and normal behavior for water intake. Functionally, APJ mutant mice have a slightly increased vasopressor response to angiotensin II administration, suggesting that Apelin may counterbalance angiotensin II–mediated pressor effects.35 Whereas infusion of exogenous Apelin can exert multiple effects, including heart function and blood pressure homeostasis, the physiological in vivo role of endogenous Apelin is virtually unknown.

To address the in vivo function of Apelin, we generated Apelin gene–targeted mice. Apelin knockout mice display normal heart structures and exhibit normal drinking behavior, food intake, and body weight as compared with controls. Interestingly, Apelin knockout mice display aging-associated reduced cardiac contractility, which becomes evident at {approx}6 months of age. Furthermore, in a pressure overload–induced heart failure model, Apelin knockout mice develop severe impairment in heart contractility. These genetic data indicate that Apelin maintains cardiovascular homeostasis on pressure overload and aging.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Generation of Apelin Knockout Mice
For gene targeting of Apelin in mice, a targeting vector was constructed to replace exons 2 and 3 of the murine Apelin gene, which encode the entire coding region of Apelin, by using the self-excision ACN-Neo cassette.36 The ACN-Neo cassette contains a testis-specific ACE promoter–driven Cre recombinase and a PGK promoter–driven neomycin resistance gene flanked by loxP sites.36 In mouse testis, the whole ACN-Neo cassette is excised through Cre-lox–mediated recombination (Figure 1A). The linearized construct was electroporated into CMT-I embryonic stem (ES) cells (Specialty Media) derived from the 129/Ola strain. Approximately 1 in 100 ES cell clones were identified as correctly targeted by genomic Southern blotting. Chimeric mice from 2 independent clones transmitted the mutant allele through the germ line, and the targeted allele was confirmed by Southern blot (Figure 1B). Because the Apelin genes maps to the X chromosome, mutant female mice are termed Apelin–/– and mutant male mice Apelin–/y. F1 mice were backcrossed once into a C57BL/6 background followed by intercrossing for more than 5 generations. F5–F7 intercrossed mice were used for all experiments reported in this study. Mice were genotyped by PCR and Southern blotting and maintained at the animal facilities of the Institute of Molecular Biotechnology and the Toronto General Hospital, University Health Network. All animal experiments were performed in accordance with institutional guidelines.


Figure 1
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  		Figure 1. Gene targeting of Apelin in mice. A, Gene-targeting strategy. Exons 2 and 3 of the Apelin gene spanning the coding sequence were replaced with the ACN-Neo self-excision cassette by homologous recombination in ES cells. Self-excision of the ACN-Neo cassette in mouse testis results in the removal of the ACN-cassette in F1 mice. All 3 Apelin exons (Ex1 to -3) and the diphtheria toxin A (DTA) negative-selection cassette are indicated. B, Southern blotting of genomic DNA in Apelin wild-type female (+/+), Apelin heterozygous female (+/–), Apelin mutant female (–/–), Apelin wild-type male (+/y), and Apelin mutant male (–/y) mice. Following SacI (S) digestion, the wild-type allele runs as a 8.0-kb band and the knockout allele as a 2.7-kb band by using the probe indicated in A (magenta). C and D, Loss of Apelin mRNA expression (C) and preproApelin protein expression (D) in hearts of Apelin–/y mice as detected by RT-PCR and Western blotting. Amplification of plasmid DNA and a sample without reverse transcription (-RT) are shown as controls. β-actin mRNA and protein expression are shown as loading controls.

Feeding and Drinking Behaviors
For high-fat diet feeding, normal chow (6% fat; M-Z, energy rich) and high-fat (30% fat; EF1/22) diets were obtained from ssniff GmbH (Soest, Germany). Mice were fed the high-fat diet starting from 3 weeks of age until termination of the experiment. Drinking behavior was measured as described previously.37 Briefly, body weight loss of mice was measured after dehydration for 40 hours, followed by assessment of water consumption over a subsequent period of 3 hours.

mRNA and Protein Expression Analyses
Western blotting was performed as described previously with antibodies to the Apelin amino acids 42 to 77 (Phoenix Pharmaceuticals). Anti–β-actin (A-2066; Sigma) was used as a loading control. For real-time PCR analysis of atrial natriuretic factor (ANF), B-type natriuretic peptide (BNP), skeletal muscle actin, β-myosin heavy chain, ACE, ACE2, and angiotensinogen, 3 µg of DNase-treated total RNA extracted from hearts was reverse transcribed using First-Strand Beads (GE Healthcare) with random primers. SYBR green real-time PCR reactions were performed in 96-well plates using an iQ-Cycler (Bio-Rad).

Blood Pressure Measurements
Mice were anesthetized with pentobarbital (50 mg/kg IV) and tracheotomized.11 A catheter was then inserted into the right carotid artery to monitor blood pressures. All blood pressures were recorded (CardioMaxII, Harvard Apparatus) after 1 hour of equilibration. Conscious blood pressures were measured using telemetry; TA-PAC20 transmitters (DSI) were implanted into the right carotid artery, and blood pressures were measured by telemetry starting at 1 week after the surgery.38

Echocardiography and Invasive Hemodynamics
Echocardiographic and hemodynamic measurements were performed as described previously.39 Briefly, mice were anesthetized with isoflurane (1%)/O2, and echocardiography was performed using an Acuson Sequoia C256 equipped with a 15-MHz linear transducer. Fractional shortening (FS) was calculated as follows: FS=[(LVEDD–LVESD)/LVEDD]x100, where LVEDD is left ventricular end-diastolic diameter, and LVESD is left ventricular end-systolic diameter. Velocity of circumferential fiber shortening was calculated as FS per ejection time corrected for heart rate. We used 2D-guided M-mode measurements to determine percentage of FS. The heart was first imaged in 2D mode in the parasternal short-axis view. From this view, an M-mode cursor was positioned perpendicular to the interventricular septum and posterior wall of the LV at the level of the papillary muscles, and M-mode images were obtained for measurement of wall thickness and chamber dimensions with the use of the leading-edge convention adapted by the American Society of Echocardiography. For rescue experiments, osmotic minipumps (Alzet 1002) were loaded with Apelin 13 peptide (1 mg/kg per 24 hours) in saline or saline alone and implanted subcutaneously into the dorsal region of control wild-type or Apelin knockout mice. Mice were infused continuously for 2 weeks before echocardiography measurements were performed. For hemodynamic assessment, the right carotid artery was cannulated with a 1.4 French Millar catheter (Millar Inc, Houston, Tex) connected to an amplifier (TCP-500; Millar Inc). After insertion of the catheter into the carotid artery, the catheter was advanced into the aorta and then into the left ventricle to record aortic and ventricular pressures as well as volume conductance.

Aortic Banding
Eight- to 10-week-old (body weight, 24 to 26 g) control wild-type littermates and Apelin knockout mice were subjected to pressure overload by aortic banding (AB) through constriction of the descending aorta as described.39 The systolic pressure gradient was determined and was comparable between wild-type and Apelin knockout mice. The mice were monitored up to 12 weeks after surgery, and their heart functions were determined by echocardiography and invasive hemodynamics. For heart histology, hearts were arrested with 1 mol/L KCl, fixed with 10% formalin, and embedded in paraffin. For mRNA and protein analyses, hearts were snap-frozen in liquid nitrogen and stored at –80°C. For histological analysis, 5-µm-thick sections were cut and stained with hematoxylin/eosin. For detection of fibrotic areas, sections were stained with Masson trichrome.

Microarray Gene Expression Analysis
Total RNA was isolated from hearts of sham and aortic-banded control wild-type and littermate Apelin knockout mice. Three different heart samples for each genotype were analyzed. mRNA samples were prepared for hybridization according to standard protocols and performed at the Genomic Core Facility at the Hospital for Sick Children in Toronto using the MOE-430–2 mouse GeneChip set (Affymetrix, Santa Clara, Calif). Experimental design and data analysis were done in compliance with the Minimum Information About a Microarray Experiment guidelines.40

Hierarchical Clustering of Gene Expression
Scanned raw data were processed with Affymetrix GeneChip Operating software (GCOS) version 1.4. The intensity value for each probe set was calculated as the 75th percentile. To monitor the expression of genes, data obtained from GCOS absolute analyses of all the individual arrays were analyzed and clustered using GeneSpring software 7.0 (http://www.agilent.com). For statistical comparison, the parametric comparison for multiple groups (ANOVA) was performed to find the set of genes for which the specified comparison showed statistically significant differences in the mean normalized expression levels across all the groups. Calculations without the assumption of equality of variances are done using Welch’s approximate t test, and ANOVA. probability values were adjusted for multiple testing by controlling for false-discovery rate using the Benjamini–Hochberg method. For hierarchical clustering, an average linkage algorithm was used. Briefly, the expression values for a gene across all samples were standardized to have mean 0 and standard deviation 1 by linear transformation, and the distance between 2 genes was defined as 1–r, where r is the standard correlation coefficient between the standardized values of 2 genes. Two genes with the closest distance were first merged into a supergene and connected by branches with length representing their distance and were then deleted for future merging. The expression level of the newly formed supergene is the average of standardized expression levels of the 2 genes (average linkage) for each sample. Then the next pair of genes (supergene) with the smallest distance was merged, and the process was repeated to cover all genes.

Quantitative Real-Time PCR
RNA was extracted using TRIzol reagent (Invitrogen) and cDNA synthesized using SuperscriptII reverse transcriptase (Invitrogen). Sequences of the forward and reverse primers of the genes studied are as follows: Tgfb2 forward, 5'-AGAAGCGCGCTTTGGATGCTGC-3'; reverse, 5'-TGGGACACACAGCAAGGGGAAG-3'; Postn forward, 5'-TGCTCTGCTGCTGCTGTTCCTG-3'; reverse 5'-TGCTGGAGGGCACAGACGTTTG-3'; Ltbp2 forward, 5'-AAATGGCCAGCTGGAGTGTCCC-3'; reverse, 5'-TTCACGCACTCCGAGTCCTTGC-3'; Lox forward, 5'-ACTTCTTACCAAGCCGCCCTCG-3'; reverse, 5'-TGTGCAGTGCAGCCAAAGCG-3'; Col8a1 forward, 5'-TCAGACTCATTCAGGCCGGTGC-3'; reverse, 5'-CGCGCAAACTGGCTAACGGTAC-3'. Htrp was used as loading control. PCR was run in 384-well plates using a LightCycler 480 SYBR Green I Master (Roche) according to the instructions of the manufacturer. Relative gene expression was quantified using the standard curve with the LightCycler 480 Relative Quantification Software Version 1.0 (Roche).

Statistical Analyses
Data are presented as mean values±SEM and were analyzed for differences between wild-type and Apelin knockout mice at discrete time points. Normally distributed data were analyzed by an unpaired t test. Data not normally distributed were analyzed using the Mann–Whitney test. P<0.05 was considered significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Generation of Apelin-Deficient Mice
To investigate the physiological role of Apelin, we generated Apelin gene–deficient mice. The Apelin gene was disrupted in murine ES cells using a targeting approach that deletes the entire Apelin coding region (Figure 1A). Southern blotting of genomic DNA confirmed specific targeting and germline transmission of the targeted Apelin allele (Figure 1B). Deletion of Apelin was further confirmed at the mRNA and protein levels using RT-PCR and Western blotting, respectively (Figure 1C and 1D). Both Apelin–/y male and Apelin–/– female mice are viable, exhibit normal fertility, and appear healthy.

Apelin Has No Apparent Role in Feeding Behaviors and Obesity
Multiple experiments using exogenous administration of Apelin peptides have implicated the Apelin system in various organ functions such as diuresis, fluid and food intake, and obesity.15–17 To test these proposed functions genetically in Apelin mutant mice, we first performed water restriction experiments followed by fluid intake. Neither weight loss attributable to water restriction (Figure 2A) nor the subsequent fluid intake (Figure 2B) was altered in Apelin mutant mice as compared with wild-type controls. In addition, the body weights and weight gain of Apelin mutant and wild-type control mice were comparable at normal diet (Figure IA in the online data supplement) and at a high-fat diet (Figure 2C). Thus, in our experimental paradigms, genetic inactivation of Apelin has no apparent effect on feeding behaviors or diet-induced obesity.


Figure 2
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  		Figure 2. Body weight and heart morphologies. A, Body weight loss in Apelin-deficient mice (KO) and wild-type littermates (WT) after water restriction for 40 hours (n=6 per group). B, Water intake during 3 hours after 40 hours of dehydration (n=6 per group). C, Body weight increases under high-fat diet in Apelin–/y and control wild-type male mice (n=6 to 8 per group). D, HW/BW ratios in 12-month-old Apelin–/y and control male mice (n>10 per group). E, Histology of hearts stained with hematoxylin/eosin. F, Mean arterial blood pressure in anesthetized 3-month-old Apelin–/y and wild-type male mice determined by invasive hemodynamics (n=5 per group). All values in A through D and F are means±SEM. There were no statistical differences between the wild-type and knockout cohorts in all in vivo assays shown.

Normal Heart Development in Apelin-Deficient Mice
In Xenopus, Apelin has been implicated in development of the cardiovascular system.27,28 Because both Apelin and APJ are highly expressed in the vasculature of the heart and cardiomyocytes, we analyzed hearts of Apelin-deficient mice. Heart weights and heart to body weight (HW/BW) ratios were comparable between age-matched 3-, 6-, and 12-month-old Apelin–/y and Apelin+/y mice (Figure 2D and data not shown). Echocardiography confirmed that the left ventricular mass and its ratio to body weight were normal (Table 1). Moreover, we failed to observe overt structural changes (Figure 2E), and there was no indication of prototypical changes in ANF, BNP, β-myosin heavy chain, and skeletal muscle actin gene expression in the hearts of Apelin mutant mice (supplemental Figure IB). Thus, loss of Apelin expression had no apparent defect on heart development or heart structures.


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  		Table 1. Echocardiography Data of Apelin-Deficient Mice

Loss of Apelin Results in Impaired Heart Contractility in Aging Mice
Pharmacological treatment of rodents with Apelin peptides has suggested a role for the Apelin–APJ system in the regulation of heart function and cardiac hypertrophy.30–32 Moreover, previous reports show that the infusion of Apelin peptide decreases blood pressure in rodents.19,29 Loss of Apelin, however, did not result in alteration of blood pressure in 3-month-old Apelin–/y mice as compared with their control littermates (Figure 2F). We next performed telemetry in 6-month-old mice to measure blood pressure in awake mice under physiological conditions. Whereas heart rates were comparable between mutant and control mice at different times of the day and night (Figure 3A), Apelin mutant mice exhibited a tendency for reduced blood pressure (Figure 3B). This trend for reduction in blood pressure, however, did not reach statistical significance.


Figure 3
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  		Figure 3. Reduced heart contractility in aged Apelin knockout mice. A and B, Telemetry measurements of heart rate (A) and blood pressure (B) in awake 6-month-old Apelin–/y and wild-type littermate male mice. Note the decrease in systolic blood pressure in Apelin mutant mice (n=7 per group). C and D, Echocardiographic measurements of heart function. Apelin–/y male mice (KO) started to display a significant reduction in percentage of FS (%FS) at 6 months of age, which became more pronounced in 12-month-old mice (C). The reduced contractility is mainly attributable to an increased left ventricular end-systolic diameter (LVESD) (D), whereas the left ventricular end-diastolic diameter was not changed significantly (see Table 1) (n=5 to 10 per group). E, Rescue of reduced heart contractility in Apelin knockout mice by Apelin 13 peptide treatment. Continuous, 2-week infusion of Apelin 13 peptide to 6-month-old Apelin–/y male mice reversed the decreased contractility to basal levels seen in wild-type mice. The same treatment had no apparent effect in wild-type littermates (n=6 per group). All values in A through E are means±SEM. *P<0.05, **P<0.01.

Because heart morphology appeared normal but blood pressures tended to be reduced in aged mice, we analyzed heart functions in Apelin mutant animals. Assessment of cardiac function by echocardiography revealed normal heart functions in 3-month-old Apelin mutant mice (Figure 3C), which is in line with normal blood pressure at that age (Figure 2F). However, at 6 months of age, all Apelin–/y male and Apelin–/– female mice exhibited a reduction in cardiac contractility, as determined by decreased left ventricle FS and increased left ventricular diameter in systole (Table 1 and Figure 3C and 3D). The decrease in heart function was found to be more severe in 6-month-old male than in age-matched female mice. In addition, 12-month-old mice had a more pronounced phenotype than 6-month-old male animals (Table 1 and Figure 3C), suggesting an age-related progression of the phenotype. To address whether the echocardiographic defects in cardiac function were attributable to developmental defects or to impairment of contractility per se, Apelin 13 peptide was continuously infused for 2 weeks into 6-month-old Apelin-null mice by using osmotic minipumps (Figure 3E). Infusion of Apelin 13 peptides had no apparent effect on FS in wild-type mice. However, the impaired cardiac contractility in Apelin–/y mice was entirely restored to wild-type levels by Apelin 13 peptide infusion (Table 1 and Figure 3E). Thus, the phenotype of reduced contractility in aged Apelin knockout mice was not caused by a secondary developmental defect but constituted an intrinsic and progressive impairment in heart function.

Severe Heart Failure in Apelin Knockout Mice in Response to Pressure Overload
The reduced heart contractility in aged Apelin knockout mice suggested a role of Apelin in heart failure. Indeed, exposure of wild-type mice to chronic pressure overload by surgical constriction of aorta (AB) resulted in the upregulation of Apelin expression in hearts of wild-type mice at 1 and 2 weeks after AB, whereas at 12 weeks after AB, Apelin expression levels went down to baseline levels (Figure 4A and data not shown). The mRNA expression of the APJ receptor was not affected (Figure 4A). After AB, HW/BW ratio increased in both Apelin+/y and Apelin–/y mice (Figure 4B). Of note, HW/BW ratio was always slightly higher in aortic-banded Apelin knockout mice than litter mate wild-type mice, but this difference did not reach statistic difference at any of the time points of analysis (Figure 4B). Expression of the cardiac hypertrophy markers ANF, BNP (Figure 4C), β-myosin heavy chain, and skeletal muscle actin (not shown) was also similar between Apelin knockout and littermate wild-type mice. Moreover, histologically, hearts from wild-type and Apelin mutant mice exhibited a comparable hypertrophic response (Figure 4D). Although we cannot exclude differences in the hypertrophic response, these data indicate that loss of Apelin has no marked effect on pressure overload–induced adaptive cardiac hypertrophy.


Figure 4
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  		Figure 4. Cardiac hypertrophy in response to pressure overload. A, Real-time PCR analyses for Apelin and APJ mRNA expression. Apelin but not APJ mRNA was upregulated in the hearts of wild-type mice (WT) 2 weeks after AB compared with control sham-operated mice (sham) (n=5 to 7 per group). **P<0.01. B, HW/BW ratios in Apelin–/y (KO) and control wild-type male mice. Baseline levels (0 weeks) and time courses after AB are shown. Note that there was a tendency of increased HW/BW ratio in Apelin–/y mice, but this difference was not statistically significant (n=6 to 10 per group and time point). C, Real-time PCR analyses for BNP and ANF mRNA expression. Total RNA was isolated from hearts 2 weeks after AB or sham surgery, and BNP and ANF levels were normalized to GAPDH mRNA (n=5 to 7 per group). All values in A through C are means±SEM. D, Histology of hearts from Apelin KO and WT mice analyzed 12 weeks after AB or sham surgery. Hematoxylin/eosin staining.

Next, we assessed the heart function of wild-type and Apelin knockout mice following AB. In line with previous data,39 banded littermate wild-type mice showed a decrease in heart contractility (Table 2 and Figure 5A through 5C). Intriguingly, AB of Apelin–/y mice resulted in severe heart failure characterized by decreased FS and dilated left ventricle in echocardiography (Table 2 and Figure 5A through 5C). Invasive hemodynamics further demonstrated that both the maximum and minimum rates of change of left ventricular pressure, (dP/dt)max, and (dP/dt)min, as the ejection fraction, and the left ventricular end-systolic pressure were significantly reduced in Apelin–/y mice (Table 2 and Figure 5D). In line with this, we also observed a significant increase in left ventricular end-diastolic pressure in Apelin–/y mice (Table 2), thus confirming the impaired heart function in aortic-banded Apelin–/y mice.


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  		Table 2. Echocardiography and Invasive Hemodynamics Data of Aortic-Banded Apelin-Deficient Mice


Figure 5
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  		Figure 5. Severe heart failure in Apelin mutant mice after pressure overload. A through C, Echocardiography of Apelin–/y (KO) and wild-type control (WT) male mice 12 weeks after AB. Representative pictures of the echocardiography show reduced contractility in Apelin–/y male mice (knockout) with AB compared with Apelin+/y mice (wild type) with AB (A). Apelin knockout mice with AB show decreased FS (%FS) (B) and increased left ventricular diameter in systolic phase (LVESD) (C) compared with wild-type mice that received AB. Animals receiving sham surgery are shown as controls (n=5 to 10 per group). All values are means±SEM. *P<0.05, **P<0.01. D, Microarray analyses. Hierarchical clustering depicting expression profiles of differentially expressed genes in Apelin knockout and wild-type mice that received AB or sham surgery. Data from individual mice (n=3 per group) at 12 weeks after AB or sham surgery are shown. Gene expression levels are shown as color variations (red, high expression; blue, low expression). The microarray raw data has been submitted to the National Center for Biotechnology Information under the accession no. GSE7781. E, Enhanced interstitial fibrosis in the hearts of Apelin–/y mice with AB. Histology of hearts are shown for mutant and control wild-type mice analyzed 12 weeks after AB or sham surgery. Masson trichrome staining.

To further assess the molecular evidence of contraction impairment in Apelin–/y mice, we performed gene arrays experiments to analyze the effects of Apelin deficiency on a global genome basis (National Center for Biotechnology Information series entry GSE7781). Hierarchical clustering of differentially expressed genes in heart samples of Apelin–/y and Apelin+/y mice 12 weeks post AB showed concerted upregulation of defined sets of genes involved in extracellular matrix remodeling and fibrosis or genes regulating muscle contraction and stress fibers in Apelin–/y mice (Table 3Down and Figure 5D). Upregulation of some of these genes, such as collagen type VIII {alpha}1 (Col8a), latent transforming growth factor β–binding protein 2 (Ltbp2), periostin (Postn), lysyl oxidase (Lox), and transforming growth factor β2 (Tgfb2) was confirmed using real-time PCR (Figure 6). In addition, genes involved in angiogenesis, blood pressure regulation, actin cytoskeleton, and muscle ankyrin repeat proteins were upregulated in banded Apelin–/y mice (Table 3Down). On the other hand, the downregulated genes included mitogen-activated protein kinase, specifically Map4K4 and Mapk14, ion transporters, and metabolic genes including several phospholipases. Importantly, as predicted from our hierarchical clustering of extracellular matrix remodeling genes, Masson trichrome staining confirmed increased interstitial fibrosis in Apelin–/y mice following AB (Figure 5E). Therefore, AB of Apelin knockout mice results in marked alterations in the cardiac gene expression profile and impaired heart function.


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  		Table 3. Genes Dysregulated in Apelin–/y Mice vs Wild-Type Controls Twelve Weeks Post Aortic Banding


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  		Table 3A. (Continued)


Figure 6
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  		Figure 6. Real-time PCR expression analysis for upregulated genes in Apelin knockout (KO) and control wild-type (WT) mice after AB. A, Relative mRNA expression analysis in Apelin–/y (knockout) and wild-type control mice 12 weeks after AB or follow sham surgery was assayed using real time PCR. B, Fold changes in gene expression in banded and sham-operated Apelin–/y (knockout) as compared with their respective wild-type controls. The same samples as for the microarray data were used. Collagen type VIII {alpha}1 (Col8a), latent transforming growth factor β binding protein 2 (Ltbp2), periostin (Postn), lysyl oxidase (Lox), and transforming growth factor β2 (Tgfb2). Data from individual mice (n=3 per group) at 12 weeks after AB or sham surgery are shown.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study provides the first genetic evidence on the in vivo function of Apelin. Apelin does not have an apparent role in feeding behaviors, water intake, or diet-induced obesity. Moreover, Apelin is not involved in the development of the cardiovascular system. Of particular interest was our finding that Apelin knockout mice exhibit defects in heart contractility in aging and following AB. Thus, Apelin acts as a positive inotropic peptide crucial for maintaining heart contractility in aging and pressure overload.

ACE2 is a key enzyme that proteolytically processes Apelin peptides with an efficacy as high as for angiotensin II.12 Previous analyses of C-terminal Apelin peptide deletions or mutations showed that such peptides lost their blood pressure effects, which leads to the assumption that ACE2 inactivates Apelin peptides.41,42 One might therefore speculate that Apelin knockout mice show the opposite phenotype of Ace2-deficient animals. However, our genetic results show that Apelin-deficient mice recapitulate some cardiac phenotypes observed in Ace2 mutant mice. For instance, similar to our Apelin mutant mice, Ace2 knockout mice have an aging-associated defect in heart contractility.7 Moreover, Yamamoto et al demonstrated in an independent Ace2 mutant mouse line that loss of ACE2 results in severe heart failure and lung congestion in pressure overload.8 Thus, in contrast to the predictions based on biochemistry, our genetic data in vivo indicate that ACE2 might be a positive regulator of Apelin peptide functions in vivo, at least in terms of cardiac functions. Importantly, our study suggests, for the first time, that the ACE2 in vivo functions might not be solely dependent on the renin–angiotensin system but ACE2 might actively influence other peptide systems. For instance, based on our results, one could speculate that Apelin might be involved in the inotropic effects of ACE2. Further studies are required to understand the relationships between Apelin and ACE2.

In APJ knockout mice, the only reported defect was increased ({approx}10 mm Hg) blood pressure responses to angiotensin II infusion under conditions of ACE inhibition or an angiotensin II type 1 receptor mutant background.35 Moreover, several studies have shown blood pressure–lowering effects of Apelin peptide in rats and mice rodents.29,43 However, our Apelin mutant mice did not show an apparent increase in blood pressure; rather these mice exhibited a minor decrease in blood pressure under awake conditions, a phenomenon that appears to be secondary to the impaired heart function in aged knockout mice. We also performed similar angiotensin II infusion experiments in Apelin mutant mice but failed to observe a further increase in blood pressure (not shown). However, we cannot exclude that loss of Apelin affects blood pressure homeostasis under defined experimental conditions. Moreover, it would be interesting to test whether APJ mice develop heart failure in response to pressure overload and age-dependent contractility defects. Differences in blood pressure control and cardiac homeostasis between Apelin and APJ mutant mice could point to different ligands for APJ and/or additional Apelin peptide receptors.

In human patients, APJ, as well as Apelin, expression was found to be significantly upregulated after placement of a left ventricular assist device in 11 patients with heart failure.33 Increased levels of Apelin mRNA expression were also reported in patients with heart failure caused by both coronary heart disease and idiopathic dilated cardiomyopathy.34 Our data now show that Apelin is upregulated at 1 and 2 weeks after AB and that Apelin expression is normalized at 12 weeks. In line with our results in banded mice, in human patients, it has been reported that Apelin expression is normalized/decreased at later stages of heart failure.33 Thus, upregulation of Apelin expression might compensate for early cardiac stress, whereas reduced Apelin expression at later stages could contribute to the development of heart failure. This scenario is in line with previous data that infusion of Apelin has in vivo inotropic effects on failing hearts after myocardial infarction, as well as on normal hearts in rats.31 Furthermore, chronic infusion of Apelin into normal mice resulted in significant increases in the velocity of circumferential fiber shortening and cardiac output by echocardiography.44 Most importantly, our genetic data show that endogenous Apelin is indeed crucial to maintain cardiac contractility.

In this study, we report the first Apelin mutant mouse generated by homologous recombination. We show that Apelin knockout mice display aging-associated reduced cardiac contractility, which becomes evident at {approx}6 months of age. Furthermore, in a pressure overload–induced heart failure model, Apelin knockout mice develop severe impairment in heart contractility. These genetic data indicate that Apelin maintains cardiovascular homeostasis on pressure overload and aging. Thus, Apelin might be a promising therapeutic target for the treatment of hypertension and aging-related heart failure.


*    Acknowledgments
 
We thank Christian Theussl for blastocyst injection, Mario Capecchi for the ACN-Neo self-excision cassette, and all members of our laboratories for helpful discussions.

Sources of Funding

J.M.P. is supported by Institute of Molecular Biotechnology of the Austrian Academy of Sciences, the Austrian National Bank, the Austrian Ministry of Science and Education, and EuGeneHeart. K.K. is supported by the European Union network grant (EuGeneHeart) and the Special Coordination Funds for Promoting Science and Technology commissioned by the Ministry of Education, Culture, Sports, Science and Technology of Japan. P.P.L. is supported by the Heart and Stroke Foundation of Ontario, the Canadian Institutes of Health Research (CIHR), and the Team Research Program and Group Program from of the CIHR. P.P.L. is the Heart & Stroke/Polo Chair Professor of Medicine and Physiology (University of Toronto) and scientific director (Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research).

Disclosures

None.


*    Footnotes
 
Original received February 24, 2007; first resubmission received May 14, 2007; second resubmission received June 26, 2007; accepted July 19, 2007.


*    References
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 up arrowMaterials and Methods
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 *References
 

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