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European Journal of Heart Failure 2008 10(4):343-351; doi:10.1016/j.ejheart.2008.02.002
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© 2008 European Society of Cardiology

Tissue kallikrein deficiency aggravates cardiac remodelling and decreases survival after myocardial infarction in mice

Sandrine Ponsa,b, Violaine Griol-Charhbilia,b, Christophe Heymesc, Paul Fornesa,b, Didier Heudesa,d, Albert Hageged,e, Xavier Loyerc, Pierre Menetona, Jean-François Giudicellib, Jane-Lise Samuelc, François Alhenc-Gelasa,d and Christine Richera,b,*

a INSERM U 652/872, CRC, IFR 58 Paris, F-75006, France
b Université Paris-Sud, Le Kremlin-Bicêtre F-94276, France
c INSERM U689, IFR 139 Paris, F-75011, France
d Université Paris-Descartes Paris, F-75006, France
e INSERM U633, Hôpital Broussais Paris, F-75014, France

* Corresponding author. INSERM U652/872, 15 rue de l'Ecole de Médecine, 75270 Paris, France. Tel.: +33 1 44 07 90 38; fax: +33 1 44 07 90 40. E-mail address: christine.richer-giudicelli{at}u-psud.fr (C. Richer).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Tissue kallikrein (TK) is a major kinin-releasing enzyme present in arteries. TK is involved in cardioprotection in the setting of acute myocardial ischaemia but its role in post-ischaemic heart failure (HF), a major cause of delayed mortality after myocardial infarction (MI), is unknown.

Aim: To determine whether TK deficiency in the mouse influences survival and cardiac remodelling after MI.

Methods: MI was induced in 10 week-old male TK-deficient mice and wild-type littermates. Survival was assessed up to 14 months. Cardiac morphological and functional parameters were serially measured by echocardiography. In another experiment, myocardial capillary density and NOS content were evaluated at 3 months.

Results: Infarct size was similar in both genotypes. MI resulted in severe cardiac dysfunction. Up to 12 months after MI, TK–/– mice displayed an increased mortality rate (P < 0.05, relative risk of death = 3.41) and aggravation of left ventricular hypertrophy and dilatation by comparison with TK+/+ (+18% and +27% respectively, both P < 0.05). NOS1 and NOS3 were abnormally regulated in the heart of TK+/+ mice after MI.

Conclusions: TK exerts a protective role in HF in mice. Coronary effects are probably involved. As partial genetic deficiency in TK activity occurs in humans, TK-deficient subjects may be at increased risk of mortality in HF.

Key Words: Kallikrein–kinin system • Myocardial infarction • Mortality • Cardiac remodelling • Nitric oxide synthase • Genetically modified mice

Received June 6, 2007; Revised November 30, 2007; Accepted February 4, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Post-ischaemic heart failure (HF) is a major cause of delayed mortality after myocardial infarction (MI). Vasoactive peptide systems can play a role in the evolution of post-ischaemic HF. There is strong evidence to suggest that the activation of the renin angiotensin system contributes to the deterioration of the condition and that inhibition of angiotensin I-converting enzyme improves remodelling, cardiac function and survival [1]. Conversely, the kallikrein-kinin system (KKS) may exert a protective role in HF. However, although the cardioprotective role of the KKS has been well documented in the setting of acute myocardial ischaemia [2,3], the role of this system in the progression of post-ischaemic HF and in long term survival after myocardial necrosis is not well established, in part because of the lack of orally active chronically administrable KKS inhibitors. Short term studies, of up to 12 weeks post-MI, performed with kinin receptor antagonists or by using genetically modified mice with kinin receptor deficiency suggest aggravation of ventricular remodelling by suppression of kinin action [4-7]. However, not all studies are consistent. Whereas mice with dual genetic and pharmacological blockade of the B1 and B2 kinin receptors developed exaggerated cardiac remodelling, rats genetically deficient in kininogen and kinins did not display aggravation of HF [6-8]. In addition no data on KKS's role in long term evolution of HF and in survival are available.

A related question that remains unresolved is the nature of the enzyme(s) responsible for the release of kinins in the normal and the failing heart. The two major kinin forming enzymes in mammals are plasma prekallikrein and tissue kallikrein. These two enzymes differ in their structure and have different activation and inhibition processes. Tissue kallikrein (TK) is a serine protease synthesized in many organs including the heart and the arteries [9-11]. It plays an important role through kinin release in endothelial function in arteries, and participates in arterial adaptation to blood flow [10,12]. We recently showed that TK contributes to the cardioprotective effect of early ischaemic preconditioning in acute myocardial ischaemia [2]. Using genetically engineered mice deficient in TK, we performed this study to ascertain the role of TK and kinins in post-ischaemic HF. We focused on long term evolution of HF and survival by following the mice for up to 14 months.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animals and surgery
The TK+/+ and TK–/– mice were generated in our laboratory as previously described [9,13]. The mouse mutants had been backcrossed on a C57BL/6 genetic background for over 10 generations before generation by heterozygous crossing of the mutated TK–/– and their wild-type littermates TK+/+ used in the experiments.

All experiments were performed in accordance with the 1996 Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health.

Ten week-old male mice were anesthetized (mixture of pentobarbital, 40 mg/kg and ketamine, 60 mg/kg, ip). After thoracotomy, the heart was exposed and the left coronary artery was ligated proximally to its main bifurcation as previously described [14,15]. Sham-operated mice underwent a similar surgical procedure, but no coronary artery ligation was performed.

2.2. Experimental protocols
In the first set of experiments designed to follow survival, fifty-four TK+/+ mice were operated: 30 underwent coronary artery ligation, 5 of these mice died during surgery or within 1 week after MI; 24 were subjected to sham surgery, and 6 of these mice died during surgery or within 1 week after surgery. Sixty TK–/– mice were operated: 38 mice underwent coronary artery ligation, 10 of these mice died during surgery or within 1 week after MI; 22 mice were subjected to sham surgery, and 3 of them died during surgery or within 1 week after surgery. All mice were observed daily for survival in the 4 experimental groups: MI-TK+/+, n=25; Sham-TK+/+, n=18, MI-TK–/–, n=28, Sham-TK–/–, n=19, for 14 months after MI. Blood pressure was measured at 6 months after MI and echocardiography was performed at 1, 3, 6, 9 and 14 months after MI. At the end of the study, at 14 months post-MI, randomly selected surviving animals (n=9 to 14 in each group) were placed in individual metabolic cages. Urinary kallikrein activity, creatinine and urinary cGMP were measured on the 24-hour urine samples. Blood samples were collected under anaesthesia from the retro-orbitary plexus into heparinized Eppendorf tubes (n=9 to 17 per group). All animals were then killed in diastole with iv 1M KCl injection, the hearts and kidneys were excised and weighed. Kidneys were frozen in liquid nitrogen and kept at –80 °C until RNA extraction. The hearts were fixed in Bouin's solution.

In a second set of experiments focused on measurement of NOS and cardiac capillary density, additional groups of MI-TK+/+, n=15; Sham-TK+/+, n=13, MI-TK–/–, n=20, Sham-TK–/–, n=11, were studied. Echocardiographic parameters were measured at 2 months after MI. All animals were sacrificed at 3 months after MI and used either for quantification of cardiac NOS by Western blotting (n=6-12 per group), or for immunohistochemical study of capillary density and NOS (n=5-9 per group).

2.3. Blood pressure measurement
Systolic arterial pressure was measured in trained, conscious and warmed (30 °C) mice using a tail-cuff piezoelectric sensor and a PowerLab/S system connected to Chart system (A.D. Instruments, Milford, MA, USA) as previously reported [9].

2.4. Echocardiographic measurements
Transthoracic two-dimensional (2D) echocardiography was performed under light anaesthesia (xylazine, 10 mg/kg and ketamine, 70 mg/kg ip) using a 15 MHz linear-array probe (15L8) especially designed for cardiac ultrasonic studies in murine models and connected to a numeric ultrasound device (Sequoia 512®, Acuson Corp, Mountain View, CA, USA), as previously described [14]. Parasternal 2D echocardiographic views of the heart were obtained with the following machine settings: space time T1, contour 3, and delta 5. End-diastolic and end-systolic long-axis views of the LV were standardized as follows: inclusion of the apex, the posterior papillary muscle, the mitral valve, and the aortic root. Two-dimensional echocardiographic measurements were performed with the cine-loop feature to retrospectively obtain adequate visualization of these fast-beating hearts. End-diastolic and end-systolic areas (A) were obtained by hand-tracings of the LV endocardial contours on the frame showing the largest (and the smallest) LV cavity size by using the cine-loop acquisition, according to the American Society of Echocardiography (ASE) leading edge method [16]. On these frames, end-diastolic (or end-systolic) lengths (L) of the LV were obtained by tracing a line connecting the more distal part of the apex and the centre of a line connecting the mitral annular hinge points. End-diastolic and end-systolic volumes (LVEDV and LVESV, respectively) were then calculated by means of the single-plane area-length method (volume=8xA2 / 3{pi}xL). Left ventricular ejection fraction (EF, %) was calculated as [(LVEDV–LVESV) / LVEDV]x100. Fractional shortening (FS, %), an estimation of LV systolic function, was calculated from the M-mode LV dimensions as [(LVEDD–LVESD) / LVEDD]x100 (LVEDD and LVESD: left ventricular end-diastolic and -systolic diameter, respectively). Left ventricular mass was calculated using the M-mode feature of the system, from end-diastolic septal (S) and posterior wall thicknesses (P) and LV internal dimensions using the leading edge method according to the ASE recommendations with a myocardial density of 1.055, LVM=1.055x[(S+LVEDD+P)3–LVEDD3]. All measurements were averaged on 3 consecutive cardiac cycles and analyzed by a single observer who was blinded to the status of the animals. Intra-observer variability in echocardiographic measurements was assessed from 2 sets of baseline measurements in 10 randomly selected mice. Results were very close for linear (r>0.95, SEE <0.1 mm), volume (r>0.93, SEE <0.08 ml), and EF (r>0.95, SEE=4%) measurements.

2.5. Cardiac morphological parameters
Heart weight (HW), LV weight (LVW) and right ventricular weight (RVW) of sacrificed animals were measured and normalized to body weight (BW). Infarct size (IS) was measured in all animals whether they died spontaneously or were sacrificed. Hearts were cut into 3 slices from the base (starting under the fibrotic area surrounding the ligation site) to the apex and embedded in paraffin. Sections (3 µm) were cut from each slice and stained with Sirius red. Infarct size was expressed as the ratio of the scar perimeterx100 to the sum of external and internal perimeters of the LV and is expressed as the percentage of infarcted tissue within the left ventricle placed at risk by ligation [14]. LV internal perimeter (LVIP, mm) was measured. LV subendocardial collagen in the viable LV (magnificationx250) was calculated as the ratio of collagen area to myocardial area [17]. All morphometrical analyses were performed using computer-assisted image analyzer (NS 15000, Microvision, Evry, France).

2.6. Quantification of NOS isoforms and Akt in the heart
Left ventricular tissues were homogenized with a Triton X100 buffer as previously described [18]. Proteins (20 {varepsilon}g) were separated by electrophoresis (9% SDS-PAGE gels), then transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). After Ponceau red staining, membranes were incubated with antibodies against NOS1 (1:1000, Affinity Bioreagents) or phospho-NOS1 (Ser1417, 1:1000, 07-544, Cell Signaling, P-NOS1), NOS3 (1:1000, SC654, Santa Cruz Biotechnology, Santa Cruz) or phospho-NOS3 (Ser1177, 1:1000, Cell Signaling, P-NOS3), Akt (1:1000, Cell Signaling) or phospho-Akt (Ser473, 1:1000, Cell Signaling, P-Akt) and anti-mouse or anti-rabbit IgG-conjugated to horseradish peroxydase (1:5000, Amersham, Orsay, France). Immunoreactive bands were visualized by chemiluminescence (ECL+, Amersham) and quantified by densitometry using a computer-based imaging system (Fujifilm Las-3000; Raytest, Paris, France) and normalized to actin signal [18].

2.7. Immunohistology and analysis of cardiac capillary density
Cryostat sections (7 {varepsilon}m thick) of heart tissue were incubated with polyclonal antibodies (1:100) to Caveolin-1{alpha} (SC-894, Santa Cruz) or NOS3 (SC-654, Santa Cruz) (Transduction Laboratories) 1 h at 21 °C, and then with fluorescein-conjugated sheep anti-rabbit IgG or Texas-Red-conjugated donkey anti-rabbit IgG (1:40) (Amersham). Sections were mounted in Vectashield medium (Vector Laboratories, Burlingame CA, USA) with DAPI (4',6 diamidino-2-phenylindole) that produces a blue fluorescence when bound to DNA, to show nuclei localization. Mounted sections were analyzed using a DMRB Leica microscope equipped with epifluorescence optics (Leica-Microsystems, Rueil-Malmaison, France) [18] and capillary density was evaluated by following Caveolin-1{alpha} staining using image analysis software [18].

2.8. Measurements of kallikrein, renin and cGMP
Urinary kallikrein activity (expressed in nmol of substrate hydrolyzed per second for total kallikrein excreted during 24 h) was assessed by quantifying the generation of p-nitroanilide (p-NA) with a spectrophotometer at 405 nm (Biorad 3550, Hercules, CA, USA) after incubation of urine with the substrate S 2266 (H-D-Val-Leu-Arg-p-NA, Chromogenix-Instrumentation Laboratory, Milan, Italy) [19].

Urinary cGMP (expressed in nmol/24 h) an indicator of the severity of congestive heart failure [20], was determined by radioimmunoassay (Amersham, Orsay, France).

Plasma renin concentration (PRC, expressed in ng Ang I/ml/h) was determined by radioimmunoassay of angiotensin I at pH 8.5 [21]. Renal renin mRNA levels were also determined (n=7 to 14 in each group) by northern blotting as previously described [13]. Results were expressed as the ratio of cpm for renin mRNA to cpm for GAPDH mRNA.

2.9. Statistical analysis
Results are expressed as mean±SEM. For all parameters, comparisons of means were assessed by a two-way ANOVA followed by a post hoc Student's t test. Comparison of survival among groups was made according to Mantel [22].The level of statistical significance was fixed at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Survival
Deaths during or immediately after surgery were not different in TK+/+ and TK–/– mice (20% and 22% respectively).

Survival rates were compared between TK+/+ and TK–/– mice in the four experimental groups up to 14 months after surgery (Fig. 1). Survival was identical between Sham-TK+/+ and Sham-TK–/– mice. Mortality was significantly increased in MI animals as compared to sham animals in the two genotypes. Time to reach 20% mortality was 210 days for MI-TK–/– and 413 days for MI-TK+/+. At 12 months post-MI, the survival rate of MI-TK–/– mice (64%) was significantly lower than that of MI-TK+/+ mice (88%, relative risk of death 3.41, P<0.05). At the end of the study, 14 months post-MI, the survival rate of MI-TK–/– (57%) was still lower than that of MI-TK+/+ (72%), but the difference was no longer significant (Fig. 1). No clinical signs of congestive HF, such as a loss of physical activity, oedema, ascities or respiratory distress were observed before death.


Figure 01
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  		Fig. 1 Survival after myocardial infarction in TK–/– and TK+/+ mice. Animals were followed from 7 days to 14 months after surgery. Survival was significantly decreased in MI-TK–/– (n=28) versus MI-TK+/+ (n=25, P<0.05) 12 months post-MI. There was no significant difference in survival after sham operation between Sham-TK–/– (n=19) and Sham-TK+/+ (n=18).

 
3.2. Blood pressure
Systolic arterial pressure, measured in conscious mice, 6 months post-surgery, was not different between Sham-TK+/+ (140±6 mm Hg) and Sham-TK–/– (147±6 mm Hg). Blood pressure tended to decrease after MI but to the same extent in both genotypes (MI-TK+/+: 131±5 mm Hg and MI-TK–/–: 134±4 mm Hg).

3.3. Echocardiography
There was no significant difference in any investigated echocardiographic parameters between sham groups of both genotypes (Fig. 2). In MI animals, as compared to sham animals, LVM/BW, LVESV and LVEDV were significantly increased and EF and SF significantly decreased throughout the study. At 1, 3 and 9 months post-MI, LVM/BW, LVESV and LVEDV were significantly higher in MI-TK–/– than in MI-TK+/+ mice. At 3 months LVM/BW, LVESV and LVEDV were 23, 43 and 34% higher respectively in MI-TK–/– compared to MI-TK+/+. At 9 months, the corresponding figures were 18, 36 and 27%.


Figure 02
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  		Fig. 2 LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), LV mass (corrected by body weight, LVM/BW) and ejection fraction (EF) measured at 1, 3, 9 and 14 months after surgery in surviving TK–/– and TK+/+ mice with MI (MI-TK–/–, MI-TK+/+) or sham operation (Sham-TK–/–, Sham-TK+/+). *P<0.05 compared with corresponding Sham value. {dagger}P<0.05 compared with corresponding MI-TK+/+ value.

 
At 14 months, these parameters were no longer different between the two genotypes (Fig. 2). There was no genotype effect on EF. FS values were 16% lower in MI-TK–/– than in MI-TK+/+ mice at 9 months (P<0.05). In the second series of animals studied 2 months after MI, worsening of LV remodelling in MI-TK–/– was also observed. LVM/BW was 4.11±0.24 in MI-TK–/–, versus 3.86±0.20 in MI-TK+/+ (+6.5%), LVESV was 85±11 mm3 in MI-TK–/– versus 60±7 mm3 in MI-TK+/+ (+42%, P<0.05) and LVEDV was 126±14 mm3 in MI-TK–/– versus 94±9 mm3 in MI-TK+/+ (+34%, P<0.05).

3.4. Cardiac morphology
Among all animals followed in the survival study, whether spontaneously deceased or sacrificed, infarct size was identical in both genotypes (MI-TK+/+: 37±2%, and MI-TK–/–: 38±1%).

Data obtained in animals sacrificed at 14 months after MI are presented in Table 1. There was no difference between Sham-TK+/+ and Sham-TK–/– mice regarding body weight, heart weight, LVIP and LV subendocardial collagen. MI caused a significant elevation in HW/BW, LVW/BW, RVW/BW, LVIP, and LV subendocardial collagen content but 14 months post-MI, there was no significant genotype-related difference for the investigated parameters (Table 1).


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  		Table 1 Morphological parameters at 14 months post-MI

 
In animals sacrificed at 3 months capillary density was 2608±303 c/mm2 in Sham-TK–/– and 3324±255 in Sham-TK+/+ (NS). Following MI, capillary density was reduced to 2365±273 in TK–/– and 2476±170 in TK+/+ mice. This effect was significant (P<0.05) in MI-TK+/+ only. There was no genotype-related difference in capillary density post-MI.

3.5. Cardiac NOS and Akt content and phosphorylation status
NOS3 was present in coronary and endocardial endothelial cells (not shown). Western blot analysis demonstrated that MI tended to decrease NOS3 in MI-TK+/+ (–18%, NS). The decrease in NOS3 was more pronounced in MI-TK–/– mice (–36%, P<0.05) (Fig. 3A). Cardiac NOS1 protein level was not altered by MI or TK deficiency (Fig. 3B). Since NOS activity is highly dependent on NOS phosphorylated state, Western blot was also used to determine whether MI was associated with alterations in the abundance of phosphorylated NOS3 and NOS1. As shown in Fig. 3A and B, the results demonstrated that phosphorylation of NOS3 and NOS1 were increased in MI-TK+/+ mice (P<0.001 versus Sham-TK+/+ mice), resulting in significantly increased P-NOS3/NOS3 and P-NOS1/NOS1 ratios. In contrast, both phosphorylation levels of NOS isoforms and ratio were significantly reduced in MI-TK–/– mice, as compared to MI-TK+/+ mice. Because it has been demonstrated that Akt was the main kinase involved in NOS phosphorylation, we studied changes in Akt and P-Akt protein levels in response to MI in the different groups. Both Akt and P-Akt protein levels increased in MI-TK+/+ mice. In MI-TK–/– mice, Akt was decreased as compared to MI-TK+/+ mice, although its phosphorylation level was increased (Fig. 4).


Figure 03
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  		Fig. 3 (A) Upper panel: representative Western blots of phosphorylated NOS3 (P-NOS3) and NOS3 in LV homogenates from TK–/– and TK+/+ mice, sacrificed 3 months after myocardial infarction (MI) or sham operation (Sham). Lower Panels: Mean±SEM of the densitometry (arbitrary units AU) of P-NOS3 and NOS3 blots and P-NOS3/NOS3 ratio from LV homogenates from TK–/– and TK+/+ mice, sacrificed 3 months after myocardial infarction (MI) or sham operation (Sham). (n=6-12, per group), *P<0.05, ***P<0.001 compared with Sham-TK+/+ value, {dagger}P<0.05, {dagger}{dagger}{dagger}P<0.001 compared with Sham-TK–/– value, $P<0.05, $$$P<0.001 compared with MI-TK+/+ value. (B) Upper panel: representative Western blots of phosphorylated NOS1 (P-NOS1) and NOS1 in LV homogenates from TK–/– and TK+/+ mice, sacrificed 3 months after myocardial infarction (MI) or sham operation (Sham). Lower Panels: Mean±SEM of the densitometry (arbitrary units A.U.) of P-NOS1 and NOS1 blots and P-NOS1/NOS1 ratio from LV homogenates from TK–/– and TK+/+ mice, sacrificed 3 months after myocardial infarction (MI) or sham operation (Sham). (n=6-12, per group), ***P<0.001 compared with Sham-TK+/+ value, {dagger}{dagger}{dagger}P<0.001 compared with Sham-TK–/– value, $$$P<0.001 compared with MI-TK+/+ value.

 


Figure 04
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  		Fig. 4 (A) Upper panel: representative Western blots of phosphorylated Akt (P-Akt) and Akt in LV homogenates from TK–/– and TK+/+ mice, sacrificed 3 months after myocardial infarction (MI) or sham operation (Sham). Lower panels: mean±SEM of the densitometry (arbitrary units A.U.) of P-Akt and Akt blots and P-Akt/Akt ratio from LV homogenates from TK–/– and TK+/+ mice, sacrificed 3 months after myocardial infarction (MI) or sham operation (Sham). (n=6-12, per group), *P<0.05, **P<0.01, ***P<0.001 compared with Sham-TK+/+ value, {dagger}P<0.05, {dagger}{dagger}P<0.001 compared with Sham-TK–/– value, $P<0.05, $$P<0.001 compared with MI-TK+/+ value.

 
3.6. Kallikrein, renin and cGMP
Urinary kallikrein activity was suppressed in TK–/– as compared to TK+/+ mice, and was not affected by MI (Table 2). Urinary cGMP, plasma renin concentration and renal renin mRNA level did not differ among sham groups of both genotypes. Urinary cGMP was strongly and significantly increased in MI animals as compared to sham-operated animals whereas plasma renin concentration and renal renin mRNA were not affected by MI. There was no difference in renin and cGMP among genotypes (Table 2).


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  		Table 2 Biological parameters at 14 months post-MI

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The major finding of this study is that infarcted TK-deficient mice demonstrated a higher mortality rate and greater left ventricular remodelling when compared with infarcted wild-type mice. This study provides direct evidence for a cardioprotective role of TK in post-ischaemic HF.

Survival rate, blood pressure, echocardiographic and cardiac morphological parameters of Sham-TK–/– C57BL/6J mice did not differ from those of Sham-TK+/+ mice at up to 14 months of age. These results confirm and extend previous observations made in younger TK–/– mice of the same C57BL/6J background that documented the lack of cardiac defects in TK–/– mice bred on this genetic background [13], and suggest that no pre-existing anatomical or haemodynamic abnormality could explain the data obtained after MI.

Myocardial infarction led to remodelling evidenced by LV cavity dilation, cardiac hypertrophy, elevated urinary cGMP excretion reflecting increased ANP and BNP secretion, and early deterioration of the LV function. In wild-type mice, the evolution of HF resulted in a significant reduction in the survival rate of infarcted animals to 70% at 14 months post-ligation. This figure is higher than that classically observed in MI-rats (20 to 30%) for an identical infarct size of 35 to 40%, but still allowed us to evidence a further increase in mortality in the MI-TK-deficient mice. In wild-type mice, mortality accelerated after 10 months post-coronary ligation confirming previous observations [14]. In contrast, in MI-TK-deficient mice, mortality increased steadily over the 14 month observation period, despite a similar mean infarct size (Fig. 1).

The mechanisms whereby a deletion of the TK gene results in greater long term mortality after MI is probably linked to the exaggerated ventricular remodelling observed in TK-deficient animals. After MI, an adaptive process, involving LV dilatation and hypertrophy and interstitial myocardial fibrosis accumulation, develops and helps to preserve cardiac output. However, in the long term, this process can lead to increased oxygen consumption, deterioration of cardiac contractile function, impairment of ventricular relaxation and the occurrence of arrhythmias [1,23]. Death can occur as a consequence of arrhythmias or of haemodynamic decompensation, which usually evolves acutely in laboratory mice. Because of the importance of ventricular dilatation in HF evolution, LVESV and LVEDV are better predictors of long term mortality than EF [24], a concept supported by the present data. An interesting finding of the present study is that cardiac remodelling was significantly aggravated in TK-deficient mice up to 10 months post-MI, but at 14 months, at the time of sacrifice, the effect of TK deficiency was no longer apparent. The fact that the effect of TK deficiency on mortality and remodelling was less pronounced at 14 months than at earlier observation times, is most probably explained by the early death of the most severely affected animals during the prolonged follow-up period and the progressive selection in each group of the less infarcted and most resistant animals. This interpretation is consistent with previous observations, made in both animals and humans, documenting a time-related effect of cardioprotective treatments on the mortality rate in HF. Indeed, whereas a relative reduction in mortality rate was observed in patients with severe heart failure [25] or in rats after experimental myocardial infarction [17], during the initial 6 months of treatment with an ACE inhibitor as compared with a placebo, in the long term, the survival curves of the treated and untreated groups converged and eventually no longer differed statistically.

The cardioprotective effect of TK is likely to be mediated by its kinin generating activity. TK has recently been shown to be the major enzyme involved in kinin generation in the ischaemic heart [2] and the present study demonstrates that it also plays an important role in HF. The beneficial effects of kallikrein and kinins on remodelling and survival in post-ischaemic HF may be related to cardiac or peripheral vascular actions of kinins. No difference in blood pressure was detected between the TK–/– and TK+/+ mice after MI. This suggests that in TK–/– mice, no increase in afterload has occurred and contributed to the worsening of HF. No difference in plasma renin concentration and renal renin mRNA levels were found between the two genotype groups, whether subjected to MI or not. These data indicate that in the C57BL/6J strain with 1-renin gene, the renin angiotensin system was not activated in MI-mice 14 months after coronary artery ligation. They also suggest that no activation of the renin angiotensin system contributed to the worsening of HF in TK–/– mice. However, one cannot exclude the possibility that activation at an earlier stage could have occurred and that the evolution of HF would have been more severe in a 2-renin gene strain. The above observations suggest that kallikrein and kinins exert their beneficial effects during HF directly on the heart.

Some beneficial effects of kallikrein and kinins in the development of HF can be related to preserved coronary endothelial function and NO release capacity [26,27]. Coronary endothelial dysfunction might contribute to the worsening of HF observed in TK-deficient mice. Endothelial dysfunction has been well documented in TK-deficient mice [9,10]. For this reason, we investigated NOS isoforms in the heart at an early stage during remodelling, i.e. 3 months after MI. We observed a defect in NOS3 phosphorylation in MI-TK–/– mice, suggesting that NOS3-derived NO production by endothelial cells may be decreased in the failing heart of TK-deficient mice. This effect seems to be independent of genotype-related differences in capillary density. Similar results were observed for NOS1 phosphorylation post-MI. A decrease in Akt in MI-TK –/– mice may be involved in this effect. It cannot however be ascertained from the present data whether the defect in cardiac NOS3 and NOS1 observed in MI-TK–/– mice is a causal event in the exaggerated LV remodelling of these mice or is secondary to this remodelling. It has recently been documented that NOS3 or NOS1 deficiency exacerbates pathological left ventricular remodelling and functional deterioration after myocardial infarction [28,29]. These observations suggest that a decrease in NOS activity may be causally involved in the worsening of HF in TK-deficient mice. However, other pathogenic mechanisms may also be involved. Sham-TK–/– mice have increased cardiac NOS1 phosphorylation compared to Sham-TK+/+ for unknown reasons. It can be speculated that this is a compensatory effect to alteration in coronary or cardiac nitroso-redox equilibrium in these mice.

Genetic deficiency in TK activity occurs in man as a result of an amino-acid mutation and is present, in the heterozygous state, in 5 to 7% of white subjects and 14% of black subjects [12,30].The present data in TK-deficient mice suggest that TK-deficient subjects might be at increased risk of lethal heart failure after myocardial infarction, a hypothesis that should be confirmed by clinical studies.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This study was supported by INSERM, the Faculté de Médecine Paris-Sud 11, the National Research Agency (Grant ANR-05-PCOD-027), and a grant from the Bristol Myers Squibb Pharmaceutical Research Institute (Princeton, NJ, USA). This study was performed in the European Vascular Genomics Network, a network of excellence supported by the European Community's sixth framework Program for Research Priority 1 "Life sciences, genomics and biotechnology for health" (contract No LSHM-CT-2003-503254). S. Pons and V. Griol-Charhbili were supported by a fellowship from the Ministère de la Recherche et de la Technologie.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Pfeffer M.A., Braunwald E., Moye L.A., et al. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators. N Engl J Med (1992) 327:669–677.[Abstract]
  2. Griol-Charhbili V., Messadi-Laribi E., Bascands J.L., et al. Role of tissue kallikrein in the cardioprotective effects of ischemic and pharmacological preconditioning in myocardial ischemia. FASEB J (2005) 19:1172–1174.[Abstract/Free Full Text]
  3. Pinto Y.M., Bader M., Pesquero J.B., et al. Increased kallikrein expression protects against cardiac ischemia. FASEB J (2000) 14:1861–1863.[Abstract/Free Full Text]
  4. Tschope C., Spillmann F., Altmann C., et al. The bradykinin B1 receptor contributes to the cardioprotective effects of AT1 blockade after experimental myocardial infarction. Cardiovasc Res (2004) 61:559–569.[Abstract/Free Full Text]
  5. Wollert K.C., Studer R., Doerfer K., et al. Differential effects of kinins on cardiomyocyte hypertrophy and interstitial collagen matrix in the surviving myocardium after myocardial infarction in the rat. Circulation (1997) 95:1910–1917.[Abstract/Free Full Text]
  6. Xu J., Carretero O.A., Sun Y., et al. Role of the B1 kinin receptor in the regulation of cardiac function and remodeling after myocardial infarction. Hypertension (2005) 45:1–7.[Free Full Text]
  7. Yang X.P., Liu Y.H., Mehta D., et al. Diminished cardioprotective response to inhibition of angiotensin-converting enzyme and angiotensin II type 1 receptor in B(2) kinin receptor gene knockout mice. Circ Res (2001) 88:1072–1079.[Abstract/Free Full Text]
  8. Liu Y.H., Yang X.P., Mehta D., Bulagannawar M., Scicli G.M., Carretero O.A. Role of kinins in chronic heart failure and in the therapeutic effect of ACE inhibitors in kininogen-deficient rats. Am J Physiol Heart Circ Physiol (2000) 278:H507–H514.[Abstract/Free Full Text]
  9. Meneton P., Bloch-Faure M., Hagege A.A., et al. Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice. Proc Natl Acad Sci U S A (2001) 98:2634–2639.[Abstract/Free Full Text]
  10. Bergaya S., Meneton P., Bloch-Faure M., et al. Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice. Circ Res (2001) 88:593–599.[Abstract/Free Full Text]
  11. Nolly H., Carbini L.A., Scicli G., Carretero O.A., Scicli A.G. A local kallikrein-kinin system is present in rat hearts. Hypertension (1994) 23:919–923.[Abstract/Free Full Text]
  12. Azizi M., Boutouyrie P., Bissery A., et al. Arterial and renal consequences of partial genetic deficiency in tissue kallikrein activity in humans. J Clin Invest (2005) 115:780–787.[CrossRef][Web of Science][Medline]
  13. Trabold F., Pons S., Hagege A.A., et al. Cardiovascular phenotypes of kinin B2 receptor- and tissue kallikrein-deficient mice. Hypertension (2002) 40:90–95.[Abstract/Free Full Text]
  14. Pons S., Fornes P., Hagege A.A., Heudes D., Giudicelli J.F., Richer C. Survival, haemodynamics and cardiac remodelling follow up in mice after myocardial infarction. Clin Exp Pharmacol Physiol (2003) 30:25–31.[CrossRef][Web of Science][Medline]
  15. Patten R.D., Aronovitz M.J., Deras-Mejia L., et al. Ventricular remodeling in a mouse model of myocardial infarction. Am J Physiol (1998) 274:H1812–H1820.[Web of Science][Medline]
  16. Schiller N.B., Shah P.M., Crawford M., et al. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms. J Am Soc Echocardiogr (1989) 2:358–367.[Medline]
  17. Richer C., Mulder P., Fornes P., Domergue V., Heudes D., Giudicelli J.F. Long-term treatment with trandolapril opposes cardiac remodeling and prolongs survival after myocardial infarction in rats. J Cardiovasc Pharmacol (1992) 20:147–156.[Web of Science][Medline]
  18. Damy T., Ratajczak P., Shah A.M., et al. Increased neuronal nitric oxide synthase-derived NO production in the failing human heart. Lancet (2004) 363:1365–1367.[CrossRef][Web of Science][Medline]
  19. Jenzano J.W., Coffey J.C., Heizer W.D., Lundblad R.L., Scicli A.G. The assay of glandular kallikrein and prekallikrein in human mixed saliva. Arch Oral Biol (1988) 33:641–644.[CrossRef][Web of Science][Medline]
  20. Michel J.B., Mercadier J.J., Galen F.X., et al. Urinary cyclic guanosine monophosphate as an indicator of experimental congestive heart failure in rats. Cardiovasc Res (1990) 24:946–952.[Abstract/Free Full Text]
  21. Menard J., Catt K.J. Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I. Endocrinology (1972) 90:422–430.[Abstract/Free Full Text]
  22. Mantel N. Chi-square tests with one degree of freedom: extension of the Mantel-Haenszel procedure. J Am Stat Assoc (1963) 58:690–700.[CrossRef][Web of Science]
  23. Tomaselli G.F., Marban E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res (1999) 42:270–283.[Free Full Text]
  24. White H.D., Norris R.M., Brown M.A., Brandt P.W., Whitlock R.M., Wild C.J. Left ventricular end-systolic volume as the major determinant of survival after recovery from myocardial infarction. Circulation (1987) 76:44–51.[Abstract/Free Full Text]
  25. Torp-Pedersen C., Kober L. Effect of ACE inhibitor trandolapril on life expectancy of patients with reduced left-ventricular function after acute myocardial infarction. TRACE Study Group. Trandolapril Cardiac Evaluation. Lancet (1999) 354:9–12.[CrossRef][Web of Science][Medline]
  26. Kichuk M.R., Seyedi N., Zhang X., et al. Regulation of nitric oxide production in human coronary microvessels and the contribution of local kinin formation. Circulation (1996) 94:44–51.[Abstract/Free Full Text]
  27. Agata J., Chao L., Chao J. Kallikrein gene delivery improves cardiac reserve and attenuates remodeling after myocardial infarction. Hypertension (2002) 40:653–659.[Abstract/Free Full Text]
  28. Scherrer-Crosbie M., Ullrich R., Bloch K.D., et al. Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice. Circulation (2001) 104:1286–1291.[Abstract/Free Full Text]
  29. Dawson D., Lygate C.A., Zhang M.H., Hulbert K., Neubauer S., Casadei B. nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction. Circulation (2005) 112:3729–3737.[Abstract/Free Full Text]
  30. Slim R., Torremocha F., Moreau T., et al. Loss-of-function polymorphism of the human kallikrein gene with reduced urinary kallikrein activity. J Am Soc Nephrol (2002) 13:968–976.[Abstract/Free Full Text]

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V. Griol-Charhbili, L. Sabbah, J. Colucci, M.-P. Vincent, V. Baudrie, D. Laude, J.-L. Elghozi, P. Bruneval, N. Picard, P. Meneton, et al.
Tissue kallikrein deficiency and renovascular hypertension in the mouse
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2009; 296(5): R1385 - R1391.
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