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Am J Physiol Heart Circ Physiol 295: H352-H360, 2008. First published May 16, 2008; doi:10.1152/ajpheart.01101.2007
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Chronic doxycycline exposure accelerates left ventricular hypertrophy and progression to heart failure in mice after thoracic aorta constriction

Laurent Vinet,1 Patricia Rouet-Benzineb,1 Xavier Marniquet,1 Noémie Pellegrin,1 Laurence Mangin,2,5 Liliane Louedec,1 Jane-Lise Samuel,3 and Jean-Jacques Mercadier1,2,4,5

1Institut National de la Santé et de la Recherche Médicale (INSERM) U698, F-75018, 2Centre d'Explorations Fonctionnelles Intégré-Institut Fédératif de Recherche 02, F-75018, 3INSERM U689, F-75010, 4Université Paris Diderot, and 5Assistance Publique, Hôpitaux de Paris, Paris, France

Submitted 21 September 2007 ; accepted in final form 6 May 2008


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tetracycline is a powerful tool for controlling the expression of specific transgenes (TGs) in various tissues, including heart. In these mouse systems, TG expression is repressed/enhanced by adding doxycycline (Dox) to the diet. However, Dox has been shown to attenuate matrix metalloproteinase (MMP) expression and activity in various tissues, and MMP inactivation mitigates left ventricular (LV) remodeling in animal models of heart failure. Therefore, we examined the influence of Dox on LV remodeling and MMP expression in mice after transverse aortic constriction (TAC). One month after TAC, cardiac hypertrophy (99% vs. 67%) and the proportion of mice exhibiting congestive heart failure (CHF, 74% vs. 32%) were higher in the TAC + Dox group than in the TAC group (P < 0.05). These differences were no longer seen 2 mo after TAC, although LV was more severely dilated in TAC + Dox mice than in TAC mice (P < 0.05). One month after TAC, the increase in brain natriuretic peptide and β-myosin heavy chain mRNA levels was 1.6 and 1.7 times higher, respectively, in TAC + Dox mice than in TAC mice (P < 0.01). MMP-2 gelatin zymographic activity increased 1.9- and 2.4-fold in TAC and TAC + Dox mice, respectively (P < 0.01 and P < 0.05 relative to respective sham-operated animals), but the difference between TAC + Dox and TAC mice did not reach statistical significance. Dox did not significantly alter TAC-associated perivascular and interstitial myocardial fibrosis. These findings demonstrate that Dox accelerates the onset of cardiac hypertrophy and the progression to CHF following TAC in mice. Accordingly, care should be taken when designing and interpreting studies based on TG mouse models of LV hypertrophy using the tetracycline-regulated (tet)-on/tet-off system.

pressure overload; matrix metalloproteinase; heart catheterization; echocardiography

CONDITIONAL TRANSGENE (TG) expression is a powerful tool for controlling the expression of specific TGs in various tissues, including heart. Conditional TG expression using a tetracycline-regulated system (tet system), has been widely used to study the roles of numerous genes in the heart (4, 7, 14, 22, 23, 26, 31, 32, 39, 40). The main advantage of the tet system is its versatility: it offers the possibility of activating or inactivating TG expression by turning on or off a transactivator (TA) by adding or withdrawing tetracycline in the animal diet. Transactivation of a responsive promoter placed upstream of the TG of interest by tet-off (tTA) or tet-on (rtTA) is inhibited or enhanced, respectively, by tetracycline, which prevents (tTA) (8) or permits (rtTA) (11, 35) binding to the corresponding promoter. One of the tetracyclines most commonly used in this system is doxycycline (Dox) (14, 22, 23, 26, 31, 32, 39, 40), which is added to the drinking water or chow. Therefore, the most appropriate control animals for TG expression in this system are double transgenic mice in their repressed state (+Dox in the tet-off system and –Dox in the tet-on system), which are compared with double transgenic mice in their activated state (–Dox in the tet-off system and +Dox in the tet-on system). However, this raises questions as to intrinsic effects of Dox on the heart, especially in experimental models of left ventricular (LV) hypertrophy (LVH). Moreover, the dynamic synthesis and breakdown of extracellular matrix proteins plays an important role in myocardial remodeling, and Dox attenuates matrix metalloproteinase (MMP) expression and/or activity in various tissues (6, 10). Dox also mitigates inflammation by inhibiting phospholipase A2 (21) or decreasing the expression of nitric oxide synthase (1, 2). Finally, Dox is an ionophore able to bind divalent cations like Ca2+ and Mg2+, which may have important biological implications.

MMP-2 and -9 expression is enhanced in pressure overload (PO)-induced LVH in spontaneously hypertensive rats (18) and in patients with aortic stenosis (20). The increased MMP expression and activation has been shown to be involved in heart failure (HF) progression in various experimental models and in humans (28). Data regarding MMP inhibition appear somewhat controversial. Several studies have shown that MMP inhibition or gene inactivation prevents LV remodeling and dysfunction (5, 12, 16, 29). Villarreal et al. (37) showed that rat LV structure and function were preserved after brief Dox treatment early after myocardial infarction. Conversely, Tessone et al. (34) showed in the same model that MMP inhibition by long-term Dox treatment did not prevent LV remodeling. Finally, Spinale et al. (30) showed that MMP inhibition improved mouse survival early after myocardial infarction, whereas prolonged MMP inhibition was associated with higher mortality and adverse LV remodeling.

The aim of this study was to determine whether Dox treatment at a dose that both induces/represses TG expression in the tet system and inhibits MMPs influences the development of cardiac hypertrophy, the MMP expression, and the progression to HF in mice. We used transverse aortic constriction (TAC), the most widely used model of maladaptive LVH.


   METHODS
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 ABSTRACT
 METHODS
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 DISCUSSION
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Study design. Mice were housed in a specific pathogen-free facility and handled in accordance with European Union directives (86/609/EEC) on the care and use of laboratory animals. The investigation was allowed by the Animal Ethics Committee of the Institut Nacional de la Santé et de la Recherche Médicale. The review and approval of the study was obtained by the Local Animal Ethics Committee (No. B 75 18 03). Mice were assigned to standard mouse chow (St) or to St + Dox (1 g/kg) 1 wk before surgery. Dox intake was about 5 mg/day per mouse (160 mg·kg–1·day–1). Mice were then randomly subjected to TAC or sham operation (Sham). Seventeen Sham, 21 Sham + Dox, 58 TAC, and 57 TAC + Dox mice completed the study and were randomly selected to be euthanized 1 or 2 mo after surgery. Specific mouse subgroups underwent echocardiography and/or hemodynamic measurements.

TAC surgery. Five-week-old adult mice (B6D2F1/J strain from Janvier) weighing 20–22 g were anesthetized with a mixture of ketamine (100 mg/kg ip) and xylazine (8 mg/kg ip). TAC was performed as described by Hu et al. (13). In brief, the aortic arch was exposed under the thymus, without thoracotomy, in spontaneously breathing mice. A 5-0 silk thread was passed under the aorta between the origin of the right innominate and left common carotid arteries and snared around the aorta, and a bent 27-gauge needle was placed alongside the aortic arch. After ligation, the needle was quickly removed, the skin was closed, and the mice were allowed to recover under infrared light until they were fully awake. The sham operation was identical except that the thread was not ligated.

Echocardiography and heart catheterization. Echocardiography was performed under light isoflurane (1% to 2%) anesthesia with a Toshiba Power Vision 6000 device (SSA 370A) equipped with a linear 8- to 14-MHz transducer, as previously described (17). LV dimensions were obtained from the long-axis view by two-dimensional-guided M-mode imaging. LV mass was calculated as LV mass = {[(LVEDD + IVSTd + PWTd)3] – (LVEDD3)} x 1.055, and the LV ejection fraction (EF) was calculated as EF = [(LVEDD3 – LVESD3)/LVEDD3] x 100, where LVEDD is the LV end-diastolic diameter and LVESD is LV end-systolic diameter (19). Pressure-volume catheterization was performed under the same anesthesia. Body temperature was maintained at 37°C with a heating pad (Harvard Apparatus). A microtip pressure-volume catheter (SPR-839; Millar Instruments) was inserted into the right carotid artery and advanced into the LV.

Mouse killing and sampling. One or two months after surgery, mice were euthanized by an intraperitoneal pentobarbital sodium injection. The heart and lungs were rapidly excised and rinsed in cold saline. The atria and large vessels were removed, and the right ventricle (RV) was dissected from the LV mass. The tissue samples were weighed and then either immediately frozen in liquid nitrogen and stored at –80°C for quantitative (q)PCR and Western blot analysis or placed in 4% paraformaldehyde for histological analysis.

Immunoblot analysis. LV samples from TAC and sham-operated mice were homogenized in 50 mM Tris·HCl (pH 7.4), 0.9% NaCl, 0.25% Triton X-100, 1% SDS, and protease inhibitor (Complete, Roche). The crude homogenates were centrifuged at 10,000 g for 10 min, and the supernatant was aliquoted and kept at –80°C until use. Immunoblots were performed using anti-ryanodine receptor 2 (RyR2) (C3-33, 1:1,000), anti-phospholamban (PLB) (1:5,000), anti-calsequestrin (1:2,500), and anti-sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) (N-19, 1:200) antibodies. All antibodies were from Affinity Bioreagents, except for anti-SERCA2a that was from Santa Cruz. After being washed, the membranes were incubated with peroxidase-conjugated goat anti-rabbit, goat anti-mouse, or donkey anti-goat IgG antiserum (Jackson) for 60 min at room temperature, then washed three times with TBS and 0.1% Tween 20, and developed using enhanced chemiluminescence (ECL Plus, Amersham). RyR2, PLB, and SERCA2a levels were normalized to the calsequestrin level.

qPCR analysis. Total RNA was extracted in TRIzol (Invitrogen) as recommended by the manufacturer. RNA was quantified by UV spectrophotometry, and its quality was checked by electrophoresis on agarose gels. Brain natriuretic peptide (BNP), {alpha}- and β-myosin heavy chain (MHC), and GAPDH mRNA expression in LV samples was determined by using real-time RT-PCR (LC fast start DNA Master Plus S.G.S+E-20, Roche).

Zymography. Approximately 25 mg of LV were crushed in liquid nitrogen and homogenized with a Tenbroeck homogeneizer in 0.25 ml of buffer containing 20 mmol Tris·HCl (pH 7.5), 250 mmol sucrose, and 1% Triton X-100, supplemented with a complete EDTA-free protease inhibitor cocktail (Complete, Roche). Gel zymography was performed as described elsewhere (25). In brief, homogenates were loaded on 8% SDS-polyacrylamide gel and separated by electrophoresis in nonreducing conditions in the presence of 1 mg/ml porcine skin gelatin. After electrophoresis, the gel was washed for 30 min in 2.5% Triton X-100 at room temperature and then incubated overnight at 37°C in reaction buffer containing 100 mmol/l Tris·HCl (pH 7.4) and 10 mmol/l CaCl2. After being stained by Coomassie brilliant blue R250, gelatin-degrading enzymes were identified as clear zones on a blue background. Gelatinase activities in the gel slabs were quantified using ImageJ 1.35r image analysis software, which quantifies both the surface area and the intensity of lysis bands. The results are expressed in arbitrary units.

Histological study. Ventricular sections (8 µm thick) were incubated with a saturated solution of picric acid containing 0.1% Sirius red for collagen staining. For each animal, five randomly chosen high-power fields were averaged. The relative collagen content was quantified using IPLab software and is expressed as a percentage of the myocardial surface area in the field.

Statistical analysis. Data are reported as means ± SE. Parametric tests were used to compare normally distributed variables (unpaired t-test and ANOVA for multiple comparisons). The {chi}2 test was used to analyze the percentage of nonfailing and failing hearts in each group. A P value of <0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The post-TAC mortality rates were 17% and 41% in the St and St + Dox groups, respectively (P = 0.08) and 0% in the sham-operated groups. Because no difference in any of the study parameters was found between the sham-operated groups 1 and 2 mo after surgery, they were pooled as a single group.

Echocardiography and hemodynamics. LV mass increased by 48% in TAC mice at 1 mo (P < 0.01 vs. Sham; Table 1), with a further increase (109% vs. Sham) at 2 mo (P < 0.05 vs. Sham and TAC 1 mo). This was associated with marked LV chamber dilation at 1 mo (P < 0.01 vs. Sham) and 2 mo (P < 0.01 vs. Sham). In addition, TAC mice at 2 mo had increased posterior wall thickness (PWTd) and interventricular septum thickness (IVSTd) at end diastole compared with Sham and TAC 1 mo (P < 0.01 vs. Sham; P < 0.05 vs. TAC 1 mo). These results indicated that TAC mice developed eccentric LVH 1 and 2 mo post-TAC, with a late increase in wall thickness. EF was reduced by 30% in TAC 1 mo (P < 0.01 vs. Sham) and by 41% in TAC 2 mo (P < 0.05 vs. TAC 1 mo). As expected, hemodynamic measurements showed that LV peak systolic pressure (LVPSP) was markedly increased in TAC 1 mo (P < 0.01 vs. Sham), but it was lower in TAC 2 mo than in TAC 1 mo (P < 0.05). In parallel, the rise of the first derivative of pressure (+dP/dt) did not change 1 mo after TAC but fell 2 mo after TAC (P < 0.05 vs. TAC 1 mo). LVEDP increased 1 mo after TAC (P < 0.05) with no change in the decline of the first derivative of pressure (–dP/dt) and further increased 2 mo after TAC (P < 0.05 vs. TAC 1 mo) in parallel with a reduction in –dP/dt (P < 0.05 vs. TAC 1 mo). Together, these results were consistent with isolated diastolic dysfunction and with combined systolic and diastolic dysfunction, respectively, 1 and 2 mo after TAC.


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  		Table 1. Echocardiographic and hemodynamic data

 
Dox treatment had no effect on any echographic or hemodynamic parameters in sham-operated mice. Echographic parameters were similar in TAC and TAC + Dox mice at 1 mo, except that the latter had higher IVSTd and PWTd values than the former (P < 0.05). At 2 mo, compared with TAC mice, TAC + Dox mice had a more strongly dilated LV at both end diastole and end systole (P < 0.05 for both comparisons). No hemodynamic differences were observed between TAC and TAC + Dox mice. Together, these results indicated that Dox accelerated the development of TAC-induced LVH and progression to HF. To determine whether this was due to a higher pressure gradient in TAC + Dox mice, we measured proximal systolic pressure (PS) and the systolic pressure gradient (SPG) in the two groups. PS and SPG were similar in TAC and TAC + Dox mice at 1 mo [PS: TAC = 191 ± 12 mmHg, TAC + Dox = 170 ± 13 mmHg, not significant (NS); SPG: TAC = 45 ± 4 mmHg, TAC + Dox = 42 ± 3 mmHg, NS], indicating a similar hemodynamic load in the two groups.

Gravimetric analysis. Body weights (BWs) were similar in the Sham and TAC groups, regardless of Dox treatment (data not shown). One and two months after surgery, TAC and TAC + Dox mice both showed a marked increase in HW-to-BW ratio (HW/BW) and LVW/BW compared with the Sham group (P < 0.01; Fig. 1). Interestingly, at 1 mo the HW/BW was higher in TAC + Dox mice than in TAC mice (P < 0.01), whereas the LVW/BW was not significantly increased. This was associated with higher RV weight (RVW)/BW and lung weight (LungW)/BW values (P < 0.01 vs. TAC), suggesting that Dox may have a direct effect on the lung and RV. To confirm this, we measured RVW in a subgroup of mice 2 wk after TAC. We found that Dox treatment was associated with an increased RVW/BW as early as 2 wk post-TAC (P < 0.01 vs. Sham and P < 0.01 vs. TAC, Fig. 1C), whereas the same ratio was significantly increased only 2 mo after TAC in mice not treated with Dox (P < 0.05 vs. Sham), supporting a specific effect of Dox on the lung and RV. Two months after TAC, HW/BW, LVW/BW, and LungW/BW were all higher than at 1 mo (P < 0.01 for all comparisons), but there were no differences between TAC and TAC + Dox mice.


Figure 1
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  		Fig. 1. Heart weight (HW)-, left ventricle weight (LVW)-, and lung weight (LungW)-to-body weight (BW) ratios in transverse aortic constriction (TAC)- and sham-operated (Sham) mice with or without doxycycline (Dox) treatment 1 and 2 mo after surgery. A: data 1 mo after surgery: n = 20 for Sham, n = 17 for Sham + Dox, n = 22 for TAC, and n = 18 for TAC + Dox mice. B: data 2 mo after surgery: n = 20 for Sham, n = 17 for Sham + Dox, n = 19 for TAC, and n = 14 for TAC + Dox mice. C: right ventricle weight (RVW)-to-BW ratio in Sham, TAC, and TAC + Dox, 2 wk, 1 mo, and 2 mo after surgery. Values are means ± SE. **P < 0.01 vs. Sham; §§P < 0.01 vs. TAC.

 
qPCR. At 1 mo, the LV BNP mRNA level increased 2.4-fold in TAC mice (P < 0.001 vs. Sham, Fig. 2) and further increased in TAC + Dox mice (3.7-fold vs. Sham; 1.6-fold vs. TAC, P < 0.01). Similarly, the β-MHC mRNA level increased 4.2-fold in TAC + Dox mice compared with the Sham group (P < 0.001) and 1.7-fold compared with TAC mice (P < 0.01), indicating that Dox treatment also accelerated PO-induced LV molecular remodeling. At 2 mo, BNP and β-MHC mRNA levels were further enhanced in TAC mice compared with TAC mice at 1 mo (3.7- and 7.5-fold vs. Sham, respectively, P < 0.001; and 1.6- and 3.1-fold vs. TAC 1 mo, respectively, P < 0.01) with no differences between TAC and TAC + Dox mice. In parallel, the {alpha}-MHC mRNA level decreased in TAC mice compared with the Sham group at 1 and 2 mo (P < 0.001 for the two comparisons), irrespective of Dox treatment.


Figure 2
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  		Fig. 2. Left ventricular (LV) brain natriuretic peptide (BNP), β-myosin heavy chain (MHC), and {alpha}-MHC mRNA levels in Sham- and TAC-operated mice, with or without Dox treatment, 1 and 2 mo after surgery (n = 9 for Sham and Sham + Dox, n = 9 for TAC and TAC + Dox 1 mo, and n = 8 for TAC and TAC + Dox mice at 2 mo). Values are means ± SE. ***P < 0.001 vs. Sham; §§P < 0.01 vs. TAC 1 mo; #P < 0.05 vs. TAC 1 mo + Dox.

 
Rate of HF. Given the variable rates of HF in the various TAC groups, we classified mice retrospectively according to the presence and absence of pulmonary oedema (15, 27). We considered that TAC mice with LungW/BW > Sham LungW/BW (+3 SD) had congestive HF (CHF, Fig. 3A) (15). On this basis, twice as many TAC + Dox mice than TAC mice had CHF at 1 mo (74% vs. 32%; P < 0.02; Fig. 3). At 2 mo, however, the proportion of TAC and TAC + Dox mice with CHF was similar (TAC = 67%, TAC + Dox = 65%, NS). Consistent with this classification, TAC mice with CHF at 1 mo had markedly increased time constant of relaxation ({tau}) and LVEDP values but similar +dP/dt and LVPSP values compared with nonfailing TAC mice (P < 0.05; Table 2), confirming the diastolic HF in these mice. At 2 mo, TAC mice with CHF showed a further increase in {tau} and LVEDP, whereas LVPSP was lower than in TAC mice with CHF at 1 mo (P < 0.05), indicating a worsened diastolic dysfunction accompanied by systolic dysfunction. Whereas Dox treatment had no significant effects on hemodynamic parameters in nonfailing and failing TAC mice at 1 mo, it was associated with decreased LVPSP and –dP/dt values in failing mice at 2 mo. Together, these data confirmed that Dox treatment facilitated the progression to CHF in TAC mice.


Figure 3
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  		Fig. 3. Classification of mice according to the absence/presence of congestive heart failure (CHF). A: TAC mice with LungW/BW > LungW/BW in Sham (+2 SD) were considered to have CHF. Values are means ± SE. **P < 0.01 vs. Sham and nonfailing mice. B: percentage of mice in CHF 1 mo (left) and 2 mo (right) after TAC, with or without Dox treatment.

 

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  		Table 2. Hemodynamic data

 
Expression of SERCA2, PLB, and RYR2. Because of the difference in the HF rates in TAC and TAC + Dox mice at 1 mo, we determined RyR2, SERCA2a, and PLB levels in the corresponding LV samples. A typical Western blot is shown in Fig. 4A. Whereas no significant differences were seen among the various mice groups (Fig. 4B), except for a 22% reduction in RyR2 expression in TAC + Dox vs. Sham + Dox mice (P < 0.05), a 38% reduction in RyR2 expression was found in nonfailing TAC/TAC + Dox mice, along with marked decreases in RyR2, SERCA2a, and SERCA2a/PLB in failing TAC/TAC + Dox mice (Fig. 4C).


Figure 4
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  		Fig. 4. Quantification of the main sarcoplasmic reticulum proteins in mouse left ventricles 1 mo after TAC. A: typical Western blot of ryanodine receptor 2 (RYR2), sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), phospholamban (PLB), and calesquestrin (CSQ). B: RYR2, SERCA2a, and PLB protein levels in the 4 mouse groups. C: RYR2, SERCA2a, and PLB levels in mice with and without CHF. F, failing; NS, not significant. Values are means ± SE. *P < 0.05 vs. Sham + Dox; **P < 0.01 vs. Sham; §P < 0.05 vs. TAC nonfailing (NF).

 
MMP-2 and MMP-9 expression/activity in ventricular homogenates and histological findings. MMP-9 signals at 92 kDa were weak and MMP-9 at 83 kDa was undetectable in all cardiac samples. Dox treatment reduced 92-kDa MMP-9 levels in sham-operated mice (1.7-fold, P < 0.01; Fig. 5). In contrast, no such Dox-induced decrease was found 1 and 2 mo after TAC. The 92-kDa MMP-9 level was 2.3-fold higher in TAC + Dox mice than in The Sham + Dox group at 1 mo (P < 0.05), reaching a value similar to that in TAC mice at 1 mo. At 1 mo, 72-kDa MMP-2 gelatinolytic activity increased 2.4- and 2.9-fold in TAC and TAC + Dox mice, respectively, and fell to intermediate levels in TAC + Dox mice at 2 mo (P < 0.01, Fig. 5B). In parallel, 68-kDa MMP-2 increased by 1.9- and 2.4-fold at 1 mo in TAC and TAC + Dox mice compared with the respective sham-operated groups (P < 0.01, Fig. 5B), and increased further in TAC mice at 2 mo (P < 0.05). However, long-term Dox treatment failed to inhibit the increase observed 1- and 2-mo post-TAC.


Figure 5
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  		Fig. 5. Gelatin zymographic activity of matrix metalloproteinase (MMP)-9 and MMP-2, 1 and 2 mo after TAC, with or without Dox treatment. A: typical gel showing MMP-9 (92 kDa), MMP-2 (72 kDa), and MMP-2 (68 kDa) activities in LV homogenates of the various mouse groups. B: quantification of relative band density (in arbitrary units in % of Sham) of MMP-9 (92 kDa), MMP-2 (72 kDa), and MMP-2 (68 kDa) (n = 4 for Sham and Sham + Dox, and n = 6 for TAC and TAC + Dox mice at 1 and 2 mo). Values are means ± SE. **P < 0.01 vs. Sham; §P < 0.05 vs. Sham + Dox; #P < 0.05 vs. TAC + Dox 1 mo; ¥P < 0.05 vs. TAC 1 mo.

 
Histological analysis showed marked perivascular fibrosis in TAC mice that reached statistical significance at 2 mo only. In TAC + Dox mice, significant perivascular fibrosis was seen as early as 1 mo (Fig. 6, A and C). A slight but nevertheless significant interstitial fibrosis was seen in TAC mice at 1 and 2 mo compared with the Sham group (Fig. 6). Dox treatment did not modify this fibrosis.


Figure 6
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  		Fig. 6. A and B: LV sections stained with Sirius red. Increased collagen staining (red) was observed in TAC mainly in perivascular areas (A), with more diffuse interstitial staining (B). C and D: quantification of the relative perivascular (C) and interstitial (D) collagen content in LV sections of TAC and TAC + Dox mice (n = 5 for all groups). Values are means ± SE. *P < 0.05 vs. Sham; **P < 0.01 vs. Sham; §§P < 0.01 vs. TAC 1 mo.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study yielded two main findings. First, in the model of PO-induced LVH, Dox accelerates the onset of hypertrophy and progression to HF. Second, Dox does not prevent the MMP-2 and -9 associated with PO-induced LVH and maladaptive LV remodeling.

We chose to investigate the effects of Dox in TAC-mediated PO-induced LVH because this model of LVH is the model most commonly used to study molecular and cellular mechanisms of maladaptive versus adaptive LV remodeling (38). In this context, transgenic and knockout models have proven invaluable for demonstrating the role of specific proteins and signaling pathways. Following TAC, the onset of LVH and progression to HF depend on the degree of aortic constriction (24) and, for a given degree of constriction, on the mouse strain (3). We used B6D2 mice generated by crossing C57BL/6J and DBA2/J mice because we had already generated a conditional tet-off transgenic mouse line in this background (9) and because, contrary to other backgrounds, it offers the chance, when using a 27-gauge needle to calibrate aortic constriction in mice weighing 20–22 g, to observe the sequential onset of compensated LVH and progression to HF within a 2–8-wk time span (3). The use of a 28-gauge needle is associated with increased short-term mortality due to acute HF, whereas the use of a 26-gauge needle results in only moderate LVH with no progression to HF.

One month after TAC, an echographic examination revealed cardiac morphometric and functional alterations typical of eccentric LVH with preserved systolic function and moderately altered diastolic function, reflected only by a rise in LVEDP. Two months after TAC, eccentric LVH had progressed further, whereas systolic function had declined and diastolic dysfunction had also deteriorated further. This time-dependent development of LVH resembles that reported by Barrick et al. (3) in C57BL/6J mice versus 129S1 mice; the latter mice developed concentric LVH with preserved LV systolic function when exposed to the same mechanical stress. Our B6D2 mice already exhibited significant LV dilation at 1 mo, as seen in C57BL/6J mice in the study of Barrick et al. (3), possibly owing to their partial C57BL/6J genetic background. Nevertheless, the degrees of aortic constriction, mouse age, and mouse weight at the time of surgery are not stated in many study reports, ruling out meaningful comparisons with our data.

Importantly, these morphofunctional changes coincided with a time-dependent hypertrophic mRNA phenotype in the LV of TAC mice characterized by marked accumulation of BNP and β-MHC mRNAs at 1 mo, with further accumulation at 2 mo and a decrease in {alpha}-MHC mRNA levels. Interestingly, at 1 mo, RyR2, SERCA2a, and PLB LV protein levels were not significantly altered (Fig. 4B), in keeping with grossly intact systolic and diastolic (except for a moderate increase in LVEDP) LV function at this stage. Taken together, these results indicated that, at 1 mo, TAC mice were in an intermediate stage between sham-operated and TAC mice at 2 mo, already exhibiting marked morphofunctional and molecular LV remodeling but still showing a large potential for progression of the remodeling to HF.

Morphofunctional and molecular parameters summarized in Table 1 and Figs. 1 and 2 clearly indicate that Dox accelerated the TAC-induced LV remodeling process, with more hypertrophy at 1 mo and more dilation at 2 mo than in untreated mice. However, to our surprise, Dox treatment was associated with increased HW/BW and LungW/BW at 1 mo, whereas LVW/BW was not significantly increased, suggesting that Dox aggravated LV diastolic dysfunction at this stage and/or had a specific effect on the lungs and, in turn, on the RV. Although the degree of LV PO was similar in the two groups, Dox-treated mice showed a nonsignificant increase in LVEDP and LV mass on echography but a significant increase in IVSTd and PWTd consistent with decreased LV diastolic compliance compared with TAC mice. A direct effect of Dox on the lungs and RV seems unlikely, since all the study parameters were similar in the Sham and Sham + Dox groups. More likely, Dox sensitized the lungs to hemodynamic stress resulting from increased LVEDP and, thus, pulmonary venous pressures. Indeed, in rats submitted to chronic hypoxia, Dox treatment (aimed at inhibiting MMPs) was associated with a significant increase in distal pulmonary artery muscularization and an increase in periadventitial collagen content, resulting in increased pulmonary artery pressure (36). The effect of Dox, relative to hypoxic stress, on pulmonary vessels submitted to increased postcapillary (venous) pressures remains to be determined, but a similar sensitizing effect may occur.

Two months after TAC, most morphofunctional parameters were similar in TAC and TAC + Dox mice, except for increased LVEDD and LVESD in the latter, in keeping with a worsening effect of Dox on remodeling. Because of the different proportions of mice with HF in the various TAC and TAC + Dox groups, we classified mice retrospectively according to the absence/presence of pulmonary edema. This approach has proven useful for revealing the effect of gene deletion or overexpression on the time-course of LV remodeling and on the occurrence of HF following TAC (15, 27). We found that ~2/3 of the mice had compensated LVH, whereas the other 1/3 had HF at 1 mo. The reverse situation was seen at 2 mo (Fig. 3A). More interestingly, mice with HF at 1 mo mainly belonged to the TAC + Dox group, whereas the situation was balanced at 2 mo, even though TAC + Dox mice had worsened systolic and diastolic LV functions compared with TAC mice (Table 2). Importantly, alterations in the expression of sarcoplasmic reticulum proteins at 1 mo appeared closely linked to cardiac function, with significant decreases in SERCA2a and SERCA2a/PLB levels in failing ventricles only, whereas Dox treatment had almost no specific effect (Fig. 4C vs. 4B). Taken together, these data support the conclusion that Dox accelerates LV remodeling and progression to HF following TAC in mice, although the effect of Dox on the lungs and, in turn, on the RV cannot be neglected. The molecular mechanisms and signaling pathways by which Dox exerts these accelerating effects remain to be identified.

Although it may appear paradoxical, given that MMPs participate in extracellular matrix breakdown, increased MMP expression and myocardial fibrosis are common features of PO-induced LVH (16, 18, 20). We found that MMP-2 expression in TAC mice was twice that in sham-operated mice at 1 and 2 mo, in agreement with previous reports (16, 33). Dox treatment failed to prevent this increase, since it failed to prevent LVH (Dox even worsened it slightly). Regarding MMP-9 expression, it was not significantly increased in TAC mice, in agreement with Matsusaka et al. (16) who used the same model as we did. In contrast, it was increased in TAC + Dox mice compared with the Sham + Dox group, essentially because of a 50% decrease in the latter compared with the Sham group (Fig. 5B, left). It must be pointed out that these measurements, although indicative of LV remodeling, do not reflect exactly the actual MMP activities and their degree of inhibition by Dox in vivo.

In TAC mice, myocardial fibrosis predominated around intramyocardial arteries, likely because of the marked increase in pressures in the systemic circulation proximal to the aortic constriction. Dox failed to prevent TAC-associated perivascular and interstitial fibrosis. It even worsened perivascular fibrosis at 1 mo (Fig. 6C). Taken together, our results suggest that TAC-induced maladaptive LV remodeling predominated over potential inhibitory effects of Dox. It is possible that higher doses of Dox would have had inhibitory effects on maladaptive LV remodeling.

In conclusion, at doses used to control TG expression in the tet system, Dox has no significant influence on MMP-2/9 expression or myocardial fibrosis in the TAC model of PO-induced LVH. In contrast, it markedly accelerates the onset of hypertrophy and progression to HF. This has to be taken into account when considering the use of the tet system to switch cardiac TG expression on or off in mouse models of chronic biomechanical cardiac stress.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by a grant from Programme National de Recherche Cardiovasculaire 2004 of the Institut National de la Santé et de la Recherche Médicale (INSERM); by the Ministère de l'Enseignement Supérieur et de la Recherche and by Institut de Recherches Servier (Suresnes, France) (to L. Vinet); and by INSERM, Université Paris Diderot, Association Française du COEur, Fondation de France, and EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart (to L. Vinet, P. Rouet-Benzineb, N. Pellegrin. and J.-J. Mercadier).


   ACKNOWLEDGMENTS
 
We thank Dr. G. Caligiuri for help with the experimental model and Drs. J.-B. Michel and Dr. J.-P. Vilaine for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Vinet, INSERM U698, G. H. Bichat-Claude Bernard, 46 rue Henri Huchard, 75877 Paris Cedex 18, France (e-mail: laurent.vinet{at}inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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