Protective role of AT₂ and B₁ receptors in kinin B₂-receptor-knockout mice with myocardial infarction

Abstract

AT₂Rs [AngII (angiotensin II) type 2 receptors] contribute to the cardioprotective effects of angiotensin II receptor blockers, possibly via kinins acting on the B₁R (B₁ receptor) and B₂R (B₂ receptor). Recent studies have shown that a lack of B₂R up-regulates B₁R and AT₂R; however, the pathophysiological relevance of such an event remains unclear. We hypothesized that up-regulation of AT₂R and B₁R compensates for the loss of B₂R. Blockade of AT₂R and/or B₁R worsens cardiac remodelling and dysfunction following MI (myocardial infarction) in B₂R^−/− (B₂-receptor-knockout mice). B₂R^−/− mice and WT (wild-type) controls were subjected to sham MI or MI and treated for 4 weeks with (i) vehicle, (ii) a B₁R-ant (B₁R antagonist; 300 μg/kg of body weight per day), (iii) an AT₂R-ant [AT₂ receptor antagonist (PD123319); 20 mg/kg of body weight per day], or (iv) B₁R-ant + AT₂R-ant. B₂R^−/− mice had a greater MCSA (myocyte cross-sectional area) and ICF (interstitial collagen fraction) at baseline and after MI compared with WT controls. Cardiac function and increase in macrophage infiltration, TGFβ₁ (transforming growth factor β₁) expression and ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation post-MI were similar in both strains. Blockade of AT₂R or B₁R worsened cardiac remodelling, hypertrophy and dysfunction associated with increased inflammation and ERK1/2 phosphorylation and decreased NO excretion in B₂R^−/− mice, which were exacerbated by dual blockade of B₁R and AT₂R. No such effects were seen in WT mice. Our results suggest that, in the absence of B₂R, both B₁R and AT₂R play important compensatory roles in preventing deterioration of cardiac function and remodelling post-MI possibly via suppression of inflammation, TGFβ₁ and ERK1/2 signalling.

INTRODUCTION

CHF (chronic heart failure) due to myocardial ischaemia is a major cause of death in the U.S.A. and worldwide. Activation of the RAS (renin–angiotensin system) and release of AngII (angiotensin II) acting on the AT₁Rs (AngII type 1 receptors) both play a crucial role in the pathophysiology of CHF. Blockade of AT₁R with ARBs (angiotensin receptor blockers) has been shown to improve LV (left ventricular) function, regress LV remodelling and prolong survival in patients with CHF [1,2]. We and others [3–5] have demonstrated that the cardioprotective effects of ARBs are mediated in part via activation of AT₂Rs (AngII type 2 receptors) due to increased circulating AngII during AT₁R blockade. AngII in turn acts on AT₂R and affords cardioprotection via a kinin-dependent pathway [6,7].

Two types of kinin receptors have been identified: B₁R (B₁ receptor) and B₂R. Most known cardiovascular actions of kinins are generally attributed to the constitutively expressed B₂R, whereas the role of B₁R is not well defined. Although B₁R is weakly expressed under physiological conditions, it is markedly increased in response to pathological stimuli such as myocardial ischaemia [8,9]. We have reported previously that both B₁R and B₂R contribute to the cardioprotective effects of ARBs [3,8,10]. Up-regulation of the B₁R is also demonstrated when the B₂R is absent [11], which may compensate for the loss of B₂R. Indeed, we have shown that blockade or deletion of B₂R alone does not have a significant impact on cardiac morphology and function either under basal conditions or in animals with MI (myocardial infarction); however, blocking both B₂R and B₁R exacerbates cardiac remodelling and dysfunction post-MI [8]. Kakoki et al. [12] have also shown that renal ischaemia/reperfusion injury becomes more severe in mice lacking both B₁R and B₂R compared with a lack of B₂R alone. Taken together, these studies suggest that up-regulation of B₁R during deletion or blockade of B₂R may represent a compensatory mechanism.

A lack of the B₂R also reportedly up-regulates the AT₂R. Shariat-Mader et al. [13] showed that, in B₂R^−/− mice, AT₂R expression increased 8-fold in association with increased B₁R. However, the functional or pathophysiological relevance of B₁R and AT₂R up-regulation in B₂R^−/− mice is not well understood. The present study is designed to test the hypothesis that, in the absence of B₂R, AT₂R and B₁R are up-regulated, which compensates for the loss of B₂R. Using B₂R^−/− mice subjected to MI, we studied whether blockade of AT₂R and/or B₁R in the absence of B₂R increases inflammation and aggravates cardiac hypertrophy, fibrosis, dilatation and dysfunction. The roles of TGFβ₁ (transforming growth factor β₁) and ERK (extracellular-signal-regulated kinase)1/2 signalling were also investigated.

MATERIALS AND METHODS

Animals

Male B₂R^−/− mice (B6; 129S7-bdkrb2^tm1Jfh/J; stock number 002641) and WT (wild-type) controls (B6129SF2/J; stock number 101045) were purchased from Jackson Laboratories. Both strains were on a mixed genetic background of C57BL/6J and 129S7/SvEv. Animals were housed in an air-conditioned room with a 12 h light/12 h dark cycle, received standard chow and drank tap water. This study was approved by the IACUC (Institutional Animal Care and Use Committee) of the Henry Ford Health System. All studies were conducted in accordance with the NIH (National Institutes of Health) Guidelines for the Care and Use of Laboratory Animals.

Induction of MI and experimental protocols

Mice 8–10 weeks of age were anaesthetized with sodium pentobarbital (50 mg/kg of body weight, intraperitoneal). An analgesic, buprenorphine (2.5 mg/kg of body weight, subcutaneous), was given 1 h before the operation and 6 h afterward. MI was induced by ligating the coronary artery [3,14]. Briefly, a left thoracotomy was performed via the fourth intercostal space, the heart exposed and the pericardium opened. The left anterior descending coronary artery was ligated with an 8-0 silk suture. Acute myocardial ischaemia was deemed successful when the anterior wall of the LV became cyanotic and the ECG showed obvious ST segment elevation. The lungs were then inflated by increasing positive end-expiratory pressure and the thoracotomy site closed in layers. Mice were kept on a heating pad until they recovered. After the MI or sham MI, each strain was divided into (i) sham MI (n=13 WT, n=12 B₂R^−/−); (ii) MI+vehicle (saline) (n=9 WT, n=9 B₂R^−/−); (iii) MI+AT₂ receptor antagonist (AT₂R-ant, PD123319; 20 mg/kg of body weight per day) (n=10 WT, n=9 B₂R^−/−); (iv) MI+B₁ receptor antagonist (B₁R-ant, R892, 300 μg/kg of body weight per day) (n=7 WT, n=7 B₂R^−/−); and (v) MI+B₁R-ant+AT₂R-ant (n=8 WT, n=9 B₂R^−/−). Doses of the antagonists were based on previously published work of ourselves and others [10,15–18]. All drugs were infused via an osmotic mini-pump intraperitoneally and started on the same day as coronary ligation and continued for 4 weeks.

Blood pressure and cardiac function and remodelling

SBP (systolic blood pressure) and HR (heart rate) were measured in conscious mice using a non-invasive computerized tail-cuff system (BP-2000; Visitech) [19]. LV dimension and function were evaluated in awake mice using a Doppler echocardiograph equipped with a 15-MHz linear transducer (Acuson c256) as described previously [19,20].

Histopathological study

At 4 weeks after MI, the heart was stopped at diastole by intraventricular injection of 15% KCl (50 μl). The heart, lungs and liver were weighed, corrected by BW (body weight) and expressed as mg/10 g of BW to assess LV hypertrophy and lung congestion. The LV was sectioned transversely into three slices for assessment of infarct size, MCSA (myocyte cross-sectional area) and ICF (interstitial collagen fraction) as previously described [19]. Images were taken at ×400 magnification and analysed with a MicroSuite computerized image analysis system (Olympus).

Immunohistochemical staining for macrophages

Frozen sections of the left ventricle (6 μm) were stained with a rat anti-(mouse CD68) monoclonal antibody (1:200 dilution; AbD Serotec), a marker for macrophages, as described previously [19,21]. CD68-positive cells (reddish/brown staining) were recognized as macrophages and are expressed as number of cells per mm² of myocardium.

ERK1/2 phosphorylation and TGFβ₁ protein expression

LV tissue was homogenized in lysis buffer with protease inhibitors. pERK1/2 (phosphor-ERK1/2) and TGFβ₁ protein expression was analysed by Western blot [19,22]. pERK1/2 was normalized by total ERK1/2 and TGFβ₁ proteins by GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Results were expressed as the ratio of the density of specific bands to the corresponding internal controls.

Urinary NO and creatinine

Urine was collected from the bladder under anaesthesia 4 weeks after MI to measure NO_x (combined nitrate + nitrite), which are stable end products of NO [23]. NO_x was measured with a colorimetric assay kit (Cayman Chemical). Urinary creatinine was measured using a commercial kit (Sigma). Urinary NO_x excretion was corrected by creatinine and expressed as the ratio of NO_x to creatinine.

Real-time PCR of B₁R, B₁R₂ and AT₂R mRNA expression

Additional sham-operated mice (six WT and six B₂R^−/−) and mice with MI (seven WT and five B₂R^−/−) were used to determine B₁R, B₁R₂ and AT₂R mRNA expression. Total RNA was isolated from mouse hearts using an RNeasy fibrous tissue kit (Qiagen). TaqMan gene expression assays from Applied Biosystems were used to assess expression of B₁R (Mm04207315), B₂R (Mm00437788), AT₂R (Mm01341373) and GAPDH (Mm99999915). Real-time PCR conditions were: one cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min. Gene expression was quantified and analysed using the comparative C_t (cycle threshold value) method as described in the Applied Biosystems user bulletin. All values were corrected to GAPDH as an internal control and the value in sham-operated WT mice was set to a value of 1.0. Changes in mRNA expression after MI were expressed as 2^−ΔΔC_t.

Data analysis

All results are expressed as means ± S.E.M. A two-sample Student’s t test was used to compare differences between strains or between treatments within strains. When multiple comparisons were performed, Hochberg’s step-up procedure was used to adjust P values. The family-wise type I error rate was set at 0.05.

RESULTS

SBP, HR, BW and tissue weight and infarct size

SBP, HR and BW were similar among groups with or without MI. In the sham-operated mice, LV and total heart weight were heavier in B₂R^−/− than in WT and MI increased heart weight the same in both strains. Lung weight was similar among groups with or without MI in either strain. However, liver weight tended to be higher in all B₂R^−/− groups, although the increase reached statistical significance only in mice treated with B₁R-ant with or without AT₂R-ant. Infarct size was similar between strains with or without treatment (Table 1).

Table 1. Effect of blockade of AT₂R and/or B₁R on SBP, HR, tissue weight and infarct size in WT and B₂R^−/− mice post-MI.

*P<0.05, **P<0.01, compared with sham MI within strain; †P<0.05 and ††P<0.01 compared with WT; #P<0.05 compared with vehicle within strain. LVW, LV weight corrected by body weight; THW, total heart weight corrected by body weight; lung and liver weight were corrected by body weight; IS, infarct size.

	WT					B2R−/−
	MI					MI
Parameter	Sham MI (n=13)	Vehicle (n=9)	AT2R-ant (n=10)	B1R-ant (n=7)	AT2Rant+ B1R-ant (n=8)	Sham MI (n=12)	Vehicle (n=9)	AT2R-ant (n=9)	B1R-ant (n=7)	AT2Rant+ B1R-ant (n=9)
BW (g)	31±2	31±2	31±1	31±1	31±1	33±11	32±1	31±1	35±1	33±2
SBP (mmHg)	111±4	106±5	110±4	108±5	106±4	123±5	120±7	111±3	119±4	117±5
HR (beats/min)	673±12	642±25	670±14	646±20	673±23	654±18	618±19	601±29	661±15	626±31
LVW (mg/10 g)	31±1	41±1**	38±2	39±2	38±2	35±2†	42±2**	41±3	44±3	43±3
THW (mg/10 g)	42±2	53±2**	51±3	52±2	51±3	47±2†	58±4*	56±4	60±4	58±4
Lungs (mg/10 g)	60±4	61±4	70±6	60±7	61±5	60±2	67±7	71±7	68±4	70±4
Liver (mg/10 g)	392±18	381±14	409±11	364±22	384±15	443±18	420±15	454±25	469±10††#	463±14††#
IS (%)	–	34±3	32±3	34±3	32±2	–	37±3	38±3	36±4	36±3

Cardiac function and remodelling

Knockout of B₂R had no effect on basal LV chamber size and function. MI increased LVDd (LV diastolic dimension) and decreased EF (ejection fraction) similarly in both strains. Blocking AT₂R or B₁R further increased LVDd and decreased EF in B₂R^−/− mice, and these changes were exaggerated when both AT₂R and B₁R were blocked. None of these effects were seen in the WT mice (Figure 1).

Figure 1. Effect of AT₂R and/or B₁R blockade on LV EF and LVDd in WT and kinin B₂R^−/− mice post-MI.

(A) Representative two-dimensional M-mode ECGs of conscious WT and B₂R^−/− mice with sham MI or MI treated with vehicle, the AT₂R antagonist PD123319 (AT₂R-ant), the kinin B₁ receptor antagonist R892 (B₁R-ant) or AT₂R-ant plus B₁R-ant for 4 weeks. (B) Quantitative analysis of LVDd (top) and EF (bottom). ***P < 0.001 compared with sham within strain; †P < 0.05 and ††P < 0.01 compared with vehicle within strain; #P < 0.05 compared with AT₂R-ant or B₁R-ant alone within strain.

Myocyte hypertrophy and cardiac interstitial fibrosis

Under basal conditions (sham MI), B₂R^−/− mice exhibited increased MCSA and ICF compared with WT. MI increased MCSA and ICF in both strains and this increase was greater in B₂R^−/− mice. AT₂R-ant, B₁R-ant or the combination of these two had no significant effect on MCSA and ICF in WT. However, in B₂R^−/− mice both AT₂R-ant and B₁R-ant alone increased MCSA and ICF further and these responses were exacerbated by dual blockade of AT₂R and B₁R (Figure 2).

Figure 2. Effect of AT₂R and/or B₁R blockade on MCSA and ICF in WT and B₂R^−/− mice post-MI.

(A) Representative images showing MCSA (delineated by the interstitial space) and interstitial collagen deposition (green staining) in WT and B₂R^−/− given vehicle, AT₂R-ant, B₁R-ant or AT₂R-ant + B₁R-ant. (B) Quantitative analysis of MCSA (top) and ICF (bottom). ***P < 0.001 compared with sham within strain; †P < 0.05 and †††P < 0.001 compared with vehicle within strain; #P < 0.05 compared with AT₂R-ant or B₁R-ant alone within strain.

Macrophage infiltration, TGFβ₁ protein expression and ERK1/2 phosphorylation in the heart

A few macrophages were seen infiltrating the myocardium in both WT and B₂R^−/− mice with sham MI. Macrophage infiltration was increased after MI and no strain difference was noticed. Blocking either AT₂R or B₁R increased the number of infiltrated macrophages only in B₂R^−/− mice. Dual blockade of AT₂R and B₁R markedly elevated macrophage infiltration, which was also only seen in B₂R^−/− (Figure 3). Cardiac TGFβ₁ protein expression did not differ between sham-operated WT and B₂R^−/− animals and rose similarly in response to MI in both strains. AT₂R-ant or B₁R-ant significantly enhanced TGF-β₁ protein expression in B₂R^−/− mice but not in WT mice, whereas dual blockade of AT₂R and B₁R markedly enhanced TGF-β₁ expression in both strains, but the response was greater in B₂R^−/− mice (Figure 4). ERK1/2 phosphorylation was similar between sham-operated groups and increased similarly after MI in both strains. AT₂R-ant or B₁R-ant significantly increased ERK1/2 phosphorylation further only in B₂R^−/− mice, while blocking both AT₂R and B₁R enhanced this effect in B₂R^−/− but not in WT mice (Figure 5).

Figure 3. Effect of AT₂R and/or B₁R blockade on macrophage infiltration in the LV myocardium in WT and B₂R^−/− post-MI.

(A) Representative images showing infiltrating macrophages (reddish-brown stained) in WT and B₂R^−/− given vehicle, AT₂R-ant, B₁R-ant or AT₂R-ant + B₁R-ant. (B) Quantification of the number of macrophages infiltrating the LV myocardium. *P < 0.05 compared with sham within strain; †P < 0.05 and ††P < 0.01 compared with vehicle within strain; #P < 0.05 compared with AT₂R-ant or B₁R-ant alone within strain.

Figure 4. Effect of AT₂R and/or B₁R blockade on TGFβ₁ protein expression in WT and B₂R^−/− mice post-MI.

(A) Representative Western blots of TGFβ₁ and GAPDH. (B) Semi-quantitative analysis of TGFβ₁ protein corrected by GAPDH and expressed as fold change relative to sham controls within strain. *P < 0.05 and **P < 0.01 compared with sham within strain; †P < 0.05 and †††P < 0.001 compared with vehicle within strain; ##P < 0.01 compared with AT₂R-ant or B₁R-ant alone within strain.

Figure 5. Effect of AT₂R and/or B₁R blockade on ERK1/2 phosphorylation in WT and B₂R^−/− mice post-MI.

(A) Representative Western blots of pERK1/2 and total ERK1/2 (tERK1/2). (B) Semi-quantitative analysis of pERK1/2 corrected by tERK1/2 and expressed as fold change relative to sham within strain. *P<0.05 compared with sham within strain; †P < 0.05 and ††P < 0.01 compared with vehicle within strain; #P < 0.05 compared with AT₂R-ant or B₁R-ant alone within strain.

Urinary NO_x

Urinary NO_x concentration was not different in sham-operated or vehicle-treated groups between strains. AT₂R-ant and B₁R-ant significantly decreased urinary NO_x only in B₂R^−/− mice. Dual inhibition of AT₂R and B₁R only lowered urinary NO_x further in B₂R^−/− mice (Figure 6).

Figure 6. Effect of AT₂R and/or B₁R blockade on urinary NO excretion in WT and B₂R^−/− mice post-MI.

††P < 0.01 and †††P < 0.001 compared with vehicle within strain; #P < 0.05 compared with AT₂R-ant or B₁R-ant alone within strain.

Cardiac B₁R, B₂R and AT₂R mRNA expression

Basal B₁R and AT₂R mRNA expression were significantly higher in B₂R^−/− mice compared with WT. B₂R mRNA was undetectable in B₂R^−/− mice. MI increased B₁R, B₂R and AT₂R mRNA expression in WT mice, whereas in B₂R^−/− mice expression of B₁R and AT₂R tended to be higher in response to MI, although these changes did not reach statistical significance (Figure 7).

Figure 7. Effect of B₂R deletion on AT₂R and B₁R mRNA expression evaluated by real-time RT (reverse transcription)–PCR and normalized to GAPDH.

Results are presented as the fold change relative to sham WT controls. *P < 0.05 and **P < 0.001 compared with sham within strain.

DISCUSSION

In the present study, we demonstrated that deletion of B₂R resulted in up-regulation of B₁R and AT₂R without altering SBP or cardiac function, although myocyte size and collagen deposition were slightly but significantly increased under basal conditions. Likewise B₂R deletion had no significant impact on MI-induced cardiac hypertrophy, fibrosis or dysfunction compared with WT mice. These findings may suggest that up-regulation of B₁R and AT₂R in B₂R^−/− mice may have compensated for the loss of B₂R, maintaining cardiac and haemodynamic homoeostasis and preventing deterioration of cardiac dysfunction and remodelling post-MI. This hypothesis is supported by our observation that blocking either AT₂R or B₁R exacerbated cardiac hypertrophy and dysfunction in B₂R^−/− mice, which was worsened by concomitant blockade of AT₂R and B₁R. Moreover, these detrimental cardiac effects were associated with increased macrophage infiltration, cardiac TGFβ₁ protein expression and ERK1/2 phosphorylation as well as decreased urinary NO_x excretion. Together these findings suggest that up-regulation of AT₂R and B₁R plays an important cardioprotective role in compensating for the loss of B₂R, particularly in mice with myocardial ischaemia. Suppressing this compensation promotes exacerbation of MI-induced cardiac hypertrophy, fibrosis and dysfunction, possibly via an enhanced inflammatory response and ERK1/2 activation.

The effects of B₂R deletion on regulation of SBP and cardiac structure and function remain in question. We and others reported that lack of B₂R (B₂R^−/− mice on a C57BL/6J background) did not alter either SBP or cardiac phenotype under basal conditions, nor did it affect cardiac remodelling and function post-MI or in response to DOCA (deoxycorticosterone acetate)-salt-induced hypertension, although the therapeutic effects of ACEi (angiotensin-converting enzyme inhibitor) and ARB on cardiac remodelling and dysfunction were diminished [3,24,25]. In contrast, others have reported that lack of B₂R (B₂R^−/− mice on a 129/J background) increased SBP and caused cardiac hypertrophy and dilated cardiomyopathy by 40 weeks of age [11,26,27]. Thus the discrepancy observed in B₂R^−/− mice might be attributed to differences in genetic background, age and/or the use of WT controls. In the present study, we compared B₂R^−/− mice on a mixed background of C57BL/6J and 129S7/SvEv with B6129SF2/J mice (WT) and found that the B₂R^−/− tended to have a higher SBP associated with a greater heart weight, MCSA and ICF under basal conditions, suggesting that B₂R may be involved in regulation of BP and cardiac homoeostasis, which may be largely dependent on their genetic background.

Recent studies suggested that deletion of B₂R up-regulates B₁R and/or AT₂R [11,13,28,29], which is confirmed by our current observation that both B₁R and AT₂R mRNA expression were increased in B₂R^−/− mice. However, the functional significance of B₁R and/or AT₂R up-regulation is not fully understood. In our study, a lack of B₂R did not have a significant impact on cardiac remodelling and dysfunction post-MI, which may be partially due to up-regulation of B₁R and AT₂R that may assume some effects of B₂R. However, blocking B₁R and/or AT₂R in B₂R^−/− mice worsened LV chamber dilatation and cardiac hypertrophy, fibrosis and dysfunction, supporting our hypothesis that up-regulation of B₁ and AT₂ may compensate for loss of B₂R.

Whether the role of B₁R expression in the cardiovascular and renal system is beneficial or harmful remains inconclusive. We and others have shown that ACEi and ARB improved cardiac function and remodelling post-MI associated with up-regulation of B₁R expression and these effects were diminished in B₁R^−/− mice or in rats treated with a B₁R antagonist, although deletion or inhibition of B₁R did not have a significant impact on cardiac remodelling and dysfunction post-MI [30,31]. These findings may suggest that the cardioprotective actions of ACEi or ARB require B₁R. Previously, Schulze-Topphoff et al. [32] reported that in a mouse model of EAE (experimental autoimmune encephalomyelitis), activation of B₁R with the agonist, Sar-[d-Phe]des-Arg⁹-BK, reduced T-lymphocyte infiltration into the brain and improved the symptoms of EAE, whereas inhibition or deletion of B₁R enhanced the immune response and worsened severity of the disease [32]. Merino et al. [33] reported that B₁R deficiency aggravated atherosclerosis and promoted aneurysm formation in ApoE^−/− (apolipoprotein E-knockout) mice [33]. All together, these reports support the concept that B₁R plays a cardioprotective, anti-inflammatory and anti-atherogenic role. On the other hand, contradictory results have also been reported, showing that blocking or deleting B₁R protects the heart against injury and myocyte death [34,35]. At present, we have no good explanation for these discrepancies.

It has been suggested that activation of ERK1/2 promotes cardiac inflammation, hypertrophy and fibrosis [36]. Kinins acting on B₁R and/or B₂R reduced inflammation by inhibiting MAPK (mitogen-activated protein kinase) activation, including ERK1/2 [37,38], whereas blocking B₁R and/or B₂R increases ERK1/2 phosphorylation and promotes cardiac inflammation and remodelling. Activation of AT₂R also reportedly attenuated TGFβ₁-induced ERK activation [39]. In the present study, we found that dual blockade of B₁R and AT₂R enhanced ERK1/2 phosphorylation and inflammatory responses as indicated by increased macrophage infiltration and TGFβ₁ protein expression, suggesting that enhanced ERK1/2 phosphorylation and inflammation may contribute to the deterioration of cardiac remodelling and dysfunction observed in B₂R^−/− mice treated with both B₁R and AT₂R antagonists.

Studies have suggested that activation of eNOS (endothelial NO synthase) and enhanced NO production may be important mediators involved in signalling mechanisms responsible for B₁R-, B₂R- and/or AT₂R-induced cardioprotection [40,41]. We previously reported that overexpression and activation of AT₂R in cultured ECs (endothelial cells) stimulated release of NO and this stimulation was diminished by B₂R blockade [42], suggesting that the kinin/NO pathway plays an important role in mediating the cardioprotective effects of AT₂R. It has been suggested that NO inhibits TGFβ₁, thus suppressing ERK1/2 phosphorylation, reducing proliferation, inflammation, oxidative stress and improving cardiac remodelling and dysfunction [43–45]. In the present study, we found that the levels of urinary NO in B₂R^−/− mice were significantly lower than in WT post-MI when treated with AT₂R-ant and/or B₁R-ant. It is possible that reduced NO production due to blockade of AT₂R and B₁R in B₂R^−/− enhances TGFβ₁ expression and ERK1/2 signalling, which promotes inflammation and cardiac hypertrophy, fibrosis and dysfunction post-MI.

Limitations of the study

The present study has demonstrated that up-regulation of AT₂R and B₁R in B₂R^−/− mice may compensate for loss of B₂R and play an important role in the cardioprotective effect of ARB. Our end points focused on functional and pathophysiological and histological remodelling of the heart; thus the detailed molecular and cellular mechanisms and signalling pathways that lead to NO synthase phosphorylation and TGFβ₁ and ERK activation were not emphasized, which will be our future focus and tested in both in vivo and in vitro studies.

In summary, we have shown that deletion of B₂R unregulated cardiac AT₂R and B₁R expression, which may compensate for loss of the beneficial effects of B₂R on remodelling and dysfunction in mice with MI, since blocking both AT₂R and B₁R worsened cardiac remodelling, hypertrophy and dysfunction in association with enhanced inflammatory responses and ERK1/2 phosphorylation and decreased urinary NO excretion. Thus AT₂R, B₁R and B₂R may all play an important role in protecting the heart from MI-induced damage by increasing NO production and suppressing inflammatory cell infiltration, TGFβ₁ protein expression and ERK1/2 phosphorylation.

CLINICAL PERSPECTIVES.

• CVD is the leading cause of death in the U.S.A. and world-wide. Activation of the RAS plays a crucial role in the pathophysiology of CVD and up until now the standard therapy has been inhibition of the RAS using ACEi and/or ARB.

• We and others have demonstrated that activation of B₁R, B₂R and/or AT₂R plays an important role in the cardioprotective effects of ACEi and ARBs. The present study helps to clarify the interaction between B₂R, B₁R and AT₂R and the mechanisms by which these receptors offer cardioprotection.

• From a clinical perspective, understanding these receptor-mediated mechanisms may help us to develop new therapeutic strategies to treat CVD, such as targeted activation of AT₂R, B₂R and/or B₁R, or combining them with an ACEi or ARB.

Abbreviations

ACEi

angiotensin-converting enzyme inhibitor

AngII

angiotensin II

ARB

angiotensin receptor blocker

AT₁R

AngII type 1 receptor

AT₂R

AngII type 2 receptor

AT₂R-ant

AT₂R antagonist

B₁R

B₁ receptor

B₁R-ant

B₁R antagonist

B₂R

B₂ receptor

BW

body weight

CHF

chronic heart failure

CVD

cardiovascular disease

EAE

experimental autoimmune encephalomyelitis

EF

ejection fraction

ERK

extracellular-signal-regulated kinase

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HR

heart rate

ICF

interstitial collagen fraction

LV

left ventricular

LVDd

LV diastolic dimension

MCSA

myocyte cross-sectional area

MI

myocardial infarction

NO_x

combined nitrate + nitrite

pERK1/2

phospho-ERK1/2

TGFβ₁

transforming growth factor β₁

RAS

renin–angiotensin system

SBP

systolic blood pressure

WT

wild-type