Histamine H2 receptor (H2R) blockade has been reported to be beneficial for patients with chronic heart failure (CHF), but the mechanisms involved are not entirely clear. In the present study, we assessed the influences of H2R disruption on left ventricular (LV) dysfunction and the mechanisms involved in mitochondrial dysfunction and calcineurin-mediated myocardial fibrosis. H2R-knockout mice and their wild-type littermates were subjected to transverse aortic constriction (TAC) or sham surgery. The influences of H2R activation or inactivation on mitochondrial function, apoptosis and fibrosis were evaluated in cultured neonatal rat cardiomyocytes and fibroblasts as well as in murine hearts. After 4 weeks, H2R-knockout mice had higher echocardiographic LV fractional shortening, a larger contractility index, a significantly lower LV end-diastolic pressure, and more importantly, markedly lower pulmonary congestion compared with the wild-type mice. Similar results were obtained in wild-type TAC mice treated with H2R blocker famotidine. Histological examinations showed a lower degree of cardiac fibrosis and apoptosis in H2R-knockout mice. H2R activation increased mitochondrial permeability and induced cell apoptosis in cultured cardiomyocytes, and also enhanced the protein expression of calcineurin, nuclear factor of activated T-cell and fibronectin in fibroblasts rather than in cardiomyocytes. These findings indicate that a lack of H2R generates resistance towards heart failure and the process is associated with the inhibition of cardiac fibrosis and apoptosis, adding to the rationale for using H2R blockers to treat patients with CHF.

CLINICAL PERSPECTIVES

  • Mast cell degranulation and histamine release have been implicated in the development of HF, whereas clinical and animal experiments have indicated that H2R blockade could improve CHF, but the underlying mechanism is unclear.

  • The present study has shown that blocking of H2R activation reduced cardiac fibrosis through down-regulation of calcineurin expression in cardiac fibroblasts and reduced myocardial apoptosis through lowering of mitochondrial permeability in cardiomyocytes, and therefore improved CHF.

  • H2R blockers are widely used to treat patients with digestive ulcer, and their pharmacological features are well studied; thus, it is relatively forward-looking to add CHF as a new therapeutic indication. The genetic evidence obtained in the present study adds to the rationale for using H2R blockers to treat patients with CHF.

INTRODUCTION

Cardiac mast cells have been reported to be involved in various cardiovascular diseases, such as ischaemia/reperfusion injury [13], arrhythmias [2], atherosclerosis [4], diabetic mellitus [5] and graft rejection after heart transplant [6], all of which are closely associated with the development of heart failure (HF). Emerging evidence shows that mast cells play an important role in the pathophysiological process of chronic heart failure (CHF) [79]. Cardiac mast cells are known to release histamine [10], which is present in the human heart at high concentrations [11]. Moreover, activation of histamine H2 receptors (H2R) in the heart may influence heart function by coupling to Gs-proteins and consequently activating several intracellular signals [1214]. H2R activation is likely to facilitate the production of cAMP by activating adenylate cyclase (AC) in a similar way to the activation of β1-adrenergic receptors. It is well known that antagonism of β1-adrenergic receptors is beneficial for patients with CHF [15], and we have previously reported that the inhibition of AC by adenosine receptor A1 agonists reduced cardiovascular remodelling in rodents [16]. These findings suggest that the inhibition of AC through blocking of H2R might improve HF.

Using data-mining techniques, we previously predicted that H2R blockers might be beneficial for patients with CHF [17], which was validated in a retrospective case-controlled study and in a prospective investigation with small groups of patients with CHF [18]. However, these preliminary clinical findings have to be confirmed further, and the underlying mechanisms need to be clarified before the H2R blockers can be applied to treat CHF [19]. Indeed, we demonstrated that famotidine, an H2R blocker, improved CHF induced by rapid pacing in dogs, an effect associated with a decrease in the myocardial cAMP level and is additive to the β-adrenergic receptor blockade [20]. This suggests that other mechanisms independent of AC inhibition are also involved in the beneficial effects of H2R blockade for CHF.

Both apoptosis and fibrosis are known to play crucial roles in CHF. Our recent study has demonstrated that H2R activation contributes to the exacerbation of myocardial ischaemia/reperfusion injury by inducing cardiomyocyte apoptosis [3], which could also be one of the mechanisms for worsening of CHF. Although there is a paucity of information on the role of H2R activation in myocardial fibrosis, myocardial histamine is associated with myocardial fibrosis in the progression from autoimmune myocarditis to dilated cardiomyopathy or in post-transplant hearts [21,22]. Mast cell hyperplasia has been associated with diseases characterized by pathological tissue remodelling and fibrosis [23]. Histamine and renin released from mast cells were found to promote pulmonary fibrosis [24]. It is reported that calcineurin joins in angiotensin II signalling-induced cardiac fibrosis [25], whereas histamine has been shown to promote angiotensin II-induced fibrosis [24]. Ca2+-activated calcineurin-dependent cardiac remodelling is believed to be an important therapeutic target for CHF, but little is known about whether H2R activation exerts any effect on calcineurin expression.

Up until now, although both clinical and animal experiments have indicated that H2R blockade could improve CHF, there is still no genetic evidence from any experiments using H2R-knockout animals to prove the effect of this treatment. Besides, it is unknown whether H2R blockade could influence cardiac fibrosis and apoptosis in HF animals. The aim of the present study was to evaluate the effect of H2R disruption on the development of HF induced by haemodynamic overload in mice and the potential mechanisms related to cardiac apoptosis and fibrosis. We observed that H2R deficiency or pharmacologically blocking H2R improves HF through inhibiting myocardial apoptosis and fibrosis.

MATERIALS AND METHODS

Animal models

All procedures were performed in accordance with our institutional guidelines for animal research that conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Mice were kept at standard housing conditions with a light/dark cycle of 12 h and free access to food and water. H2R−/− mice and H2R+/+ littermates were used for the experimental study. Generation of mice lacking H2R was previously described by Fukushima et al. [26]. Heterozygous H2R mice were bred at our animal facility to produce H2R−/−, H2R−/+ and H2R+/+ offspring (Figure 1A). Genotyping primer sequences are shown in Supplementary Table S1 (http://www.clinsci.org/cs/127/cs1270435add.htm). Mice (7–8 weeks of age, male, body weight 20–28 g) were anaesthetized with a mixture of xylazine [5 mg/kg, injected intraperitoneally (i.p.)] and ketamine (100 mg/kg, injected i.p.), and the adequacy of anaesthesia was monitored by the disappearance of the pedal withdrawal reflex. Mice were intubated with PE-90 tubing, and ventilated using a mouse miniventilator with room air. Aortic stenosis was generated by transverse aortic constriction (TAC) to induce cardiac hypertrophy and HF as described previously [27,28]. After surgery in wild-type mice, the animals were assigned to a sham group (n=17) without aortic banding, a TAC group (n=20) with TAC only, and a famotidine group (n=17) with TAC and the daily oral administration of famotidine (10 mg/kg per day, suspended in 0.5% carboxymethylcellulose). The dose of famotidine was chosen on the basis of previous reports [3,29]. H2R-knockout mice were assigned to the sham (n=17) and TAC groups (n=17).

H2R deficiency attenuated cardiac remodelling induced by TAC for 4 weeks

Figure 1
H2R deficiency attenuated cardiac remodelling induced by TAC for 4 weeks

(A) Genotyping results. H2R−/− (HO) and H2R+/+ (WT) mice for experiments were generated by mating H2R+/− (HE) mice with each other. (B) Western blot of H2R in response to sham or TAC for 4 weeks. *P<0.01 compared with the sham group; n=5 in each group. (C) Cardiac mast cell density. Toluidine-Blue-positive mast cells are indicated by black arrows and the magnified pictures are shown beside; n=5 per group. *P<0.01 compared with the sham group. (D) Echocardiographic LVFS was larger in KO TAC mice than in WT TAC mice. (E) Lung weight/body weight (LW/BW) ratio was smaller in KO TAC mice than in WT TAC mice. The insets are representative pictures of lungs from KO and WT mice with TAC. (F) Heart weight/body weight (HW/BW) ratio in response to sham or TAC for 4 weeks. For (DF), *P<0.01 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group. The number of mice in each group was the same as in Table 1. HE, heterozygote; HO, homozygote; KO, knockout; WT, wild-type.

Figure 1
H2R deficiency attenuated cardiac remodelling induced by TAC for 4 weeks

(A) Genotyping results. H2R−/− (HO) and H2R+/+ (WT) mice for experiments were generated by mating H2R+/− (HE) mice with each other. (B) Western blot of H2R in response to sham or TAC for 4 weeks. *P<0.01 compared with the sham group; n=5 in each group. (C) Cardiac mast cell density. Toluidine-Blue-positive mast cells are indicated by black arrows and the magnified pictures are shown beside; n=5 per group. *P<0.01 compared with the sham group. (D) Echocardiographic LVFS was larger in KO TAC mice than in WT TAC mice. (E) Lung weight/body weight (LW/BW) ratio was smaller in KO TAC mice than in WT TAC mice. The insets are representative pictures of lungs from KO and WT mice with TAC. (F) Heart weight/body weight (HW/BW) ratio in response to sham or TAC for 4 weeks. For (DF), *P<0.01 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group. The number of mice in each group was the same as in Table 1. HE, heterozygote; HO, homozygote; KO, knockout; WT, wild-type.

Echocardiography and left ventricular (LV) haemodynamic studies were performed at 4 weeks after the surgery, and then the mice were killed by overdose anaesthesia with pentobarbital sodium (150 mg/kg, i.p.) and cervical dislocation, and their hearts and lungs were extracted for further analysis. For histological examinations, hearts were fixed in 10% formalin, whereas the hearts used for molecular analysis were snap-frozen in liquid nitrogen and stored at −80°C until use.

Echocardiography

Non-invasive transthoracic echocardiography was performed in mice using a Sequoia 512 system with a 17L-5 probe (Siemens). Mice were anaesthetized with isoflurane inhalation at a concentration of 1.5%. 2D short-axis views of the LV were obtained for guided M-mode measurement of the left ventricular posterior wall thickness in diastole (LVPWd), end-diastolic diameter (LVEDd) and end-systolic diameter (LVESd). Left ventricular fractional shortening (LVFS) was calculated as follows:

 
formula

Invasive haemodynamic study

LV haemodynamics were evaluated before killing. Mice from each group were anaesthetized with isoflurane inhalation at a concentration of 1.5% and were ventilated as mentioned above. A Millar catheter was inserted via the right carotid artery and carefully introduced into the left ventricle to measure left ventricular systolic pressure (LVSP) and end-diastolic pressure (LVEDP). Maximum and minimum LV pressure rise and fall rates (max dp/dt and min dp/dt respectively) as well as the contractility index (max dp/dt divided by the pressure at the time of max dp/dt) and the exponential time constant of relaxation (τ) were calculated using PowerLab software.

Cell culture

The neonatal Sprague–Dawley (SD) rats at 1–3 days after birth were anaesthetized by 2% isoflurane inhalation. Isolation and culture of ventricular cardiomyocytes and fibroblast cells were performed as described elsewhere [30]. Subsequently, primary cardiomyocytes and passage 1 fibroblasts were exposed to histamine, amthamine dihydrobromide (AD, a selective H2R agonist) or famotidine (a selective H2R antagonist—all were purchased from Sigma–Aldrich) for the indicated dose and time as described in the corresponding Figure legend, and then the related gene and protein expression levels, as well as mitochondrial permeability and apoptosis, were analysed.

Construction of recombinant lentivirus carrying shRNA of calcineurin

The shRNA cassettes of Ppp3ca (calcineurin-α, NM_017041.1) were cloned into the lentivirus vector pLVX-shRNA2 (632179, Clontech) to construct pLVX-shRNA-calcineurin (Lv-CN). The lentivirus construct of pLVX was used a control (Lv-NC). The generation of Lv-CN, the titre determination and cell infection were conducted according to the Lenti-X™ shRNA Expression Systems User Manual (PT5146-1, Clontech). Neonatal rat fibroblasts (passage 1) were infected with various multiplicities of infection (MOI=2.5, 5, 10) for 72 h, and the transfection efficiency was evaluated by the encoding GFP under a fluorescent microscope. We noted that the best MOI was 5 for fibroblast infection (see Figure 2C); therefore, we used MOI 5 for the related cell experiments.

Effect of H2R deficiency on myocardial fibrosis and calcineurin accumulation

Figure 2
Effect of H2R deficiency on myocardial fibrosis and calcineurin accumulation

(A) Representative pictures of Masson's stained heart slice of mice at 4 weeks after receiving sham or TAC operation. Blue staining indicates myocardial fibrosis. Scale bar=20 μm. (B) Semi-quantitative analysis of myocardial fibrosis in the different groups. *P<0.05 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group; n=5 in each group. (C) Western blot of calcineurin in different groups of mice. *P<0.01 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group; n=4 in each group. KO, knockout; WT, wild-type.

Figure 2
Effect of H2R deficiency on myocardial fibrosis and calcineurin accumulation

(A) Representative pictures of Masson's stained heart slice of mice at 4 weeks after receiving sham or TAC operation. Blue staining indicates myocardial fibrosis. Scale bar=20 μm. (B) Semi-quantitative analysis of myocardial fibrosis in the different groups. *P<0.05 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group; n=5 in each group. (C) Western blot of calcineurin in different groups of mice. *P<0.01 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group; n=4 in each group. KO, knockout; WT, wild-type.

Fibroblast identification and proliferation assay

Cardiac fibroblasts cultured on glass-bottomed dishes were fixed with 4% paraformaldehyde and permeabilized with 0.01% Triton X-100. After being blocked in 3% BSA, cells were incubated with the primary antibody (rabbit anti-vimentin, ab92547, Abcam) overnight at 4°C. Cell nuclei were stained with DAPI. The fluorescence images were obtained using a Nikon confocal microscope.

Fibroblast cells (1×105 cells per well) were subcultured into 96-well plates with 200 μl of the complete culture medium. The next day, lentiviruses (Lv-CN or Lv-NC) were added with serum-free medium (MOI=5), and the medium was changed after 24 h. Then at day 3 of infection, the cells were exposed to AD for 24 h. Finally, the supernatant was removed, and 100 μl of DMEM/F12 medium containing 10 μl of CCK8 (Dojindo) was added to each well and incubation for 4 h at 37°C. The absorbance values were read at 450 nm.

PCR

Total RNA was extracted from cultured cells (total RNA isolation system, Omega). Reverse transcription was carried out in a final volume of 20 μl using 0.5 μg of total RNA. The mRNA expression of procollagen I and procollagen III as well as GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in cardiac fibroblasts from neonatal rats was determined using the Quantitect SYBR Green real-time PCR method. Data are expressed as fold change after normalizing to GAPDH. The rat primers used for real-time PCR are shown in Supplementary Table S1.

Western blot analysis

Proteins were prepared from cultured cardiomyocytes, fibroblasts and whole heart homogenates. Immunoblotting was then performed using antibodies directed against calcineurin (ab3673, Abcam), nuclear factor of activated T-cell c3 (NFATc3) (sc-8321, Santa Cruz), cleavage caspase-3 (ab123114, Abcam), Bax (04-434, Millipore), H2R (sc-33974, Santa Cruz), fibronectin (ab6328, Abcam) or α-smooth muscle actin (α-SMA) (ab5694, Abcam). Blotting of β-actin (Santa Cruz), Cox IV (#4844, Cell Signaling) or histone H3 (#4499, Cell Signaling) was used as a loading control. Immunoreactive bands were visualized by the ECL method (GE Healthcare) and then were quantified by densitometry with Scion Image software.

Histological examinations and apoptosis assay

Hearts were fixed in 10% formalin, then dehydrated and embedded in paraffin, and 4-μm-thick sections were cut and stained with Masson's trichrome.

Toluidine Blue was used to stain myocardial mast cells, as described previously [31]. Briefly, paraffinized sections of myocardium were dewaxed using xylene and ethanol, then rehydrated and incubated with 0.05% Toluidine Blue for 30 min. Toluidine-Blue-positive mast cells observed in a high-power field of view (×400 magnification) were counted. At least 20 fields per slide were counted for each group.

Apoptosis in cultured cardiomyocytes, fibroblasts and myocardium was determined using the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay [3]. Briefly, apoptotic cells were detected with an in situ cell death detection kit, TMR red (Roche). The sections were treated with proteinase K for 20 min, incubated with TUNEL reaction mixture or negative control solution for 60 min at 37°C and then stained with the DAPI solution for 10 min. Slides were rinsed twice with PBS between each step. The positive rate of TUNEL-labelled nuclei was calculated from four different and randomly selected areas under confocal microscopy. Apoptosis in cultured cardiomyocytes was also detected using the Hoechst staining method as we have described previously [3].

Mitochondrial permeability and membrane potential (ΔΨm) assay

The openness of the mitochondrial permeability transition pore (mPTP) was evaluated by detecting calcein fluorescence intensity in mitochondria. Briefly, cardiomyocytes were loaded with 1.0 mM calcein-acetomethoxy ester (Molecular Probes). Fluorescence images were acquired for both calcein and MitoTracker under fluorescence microscopy.

Tetramethylrhodamine ethyl ester (TMRE) is a positively charged dye. When the ΔΨm collapses in cells, TMRE no longer accumulates inside the mitochondria and overall cellular fluorescence levels fall dramatically. Cardiomyocytes were loaded with 50 nM TMRE (Molecular Probes, Invitrogen) for 30 min at room temperature. TMRE fluorescence intensity representing ΔΨm was recorded with a fluorescence microscope and analysed by Scion Image software.

Statistical analysis

A Student's t test was used for comparisons between two groups, whereas one-way ANOVA with post-hoc analysis by the Tukey–Kramer test was employed for multiple comparisons. Results are expressed as the means±S.E.M, and P<0.05 was considered to indicate statistical significance.

RESULTS

Cardiac dysfunction induced by TAC was attenuated in H2R-knockout mice

The basic level of myocardial H2R protein expression in mice was relatively low, and it was significantly up-regulated in response to TAC for 4 weeks (Figure 1B). In addition, mast cell density in the myocardium detected by Toluidine Blue staining was also increased in TAC mice (Figure 1C), implying an increase in myocardial histamine in pressure-overloaded mice. Therefore, we used H2R-knockout mice and their wild-type littermates to investigate the influence of H2R inactivation on HF. During development and up to adulthood, there was no difference in growth rate, body weight, heart and lung weight, echocardiographic findings and LV haemodynamics between wild-type and knockout mice (data for adult mice are shown in the sham groups). (Table 1 and Figure 1).

Table 1
Invasive LV haemodynamic and echocardiographic results at 4 weeks

Data are shown as the means±S.E.M. Contractility index, max dp/dt divided by the pressure at the time of max dp/dt; *P<0.05; **P<0.01 compared with WT TAC; †P<0.05; ‡P<0.01 compared with the corresponding sham group. Fa, famotidine (10 mg/kg per day); KO, knockout; WT, wild-type.

Parameters WT sham (n=6) WT TAC (n=9) KO sham (n=7) KO TAC (n=7) WT TAC + Fa (n=6) 
LVSP (mmHg) 103.0±3.5 175.0±13.7‡ 107.0±5.3 203.0±8.9‡* 209.0±11.0‡ 
LVEDP (mmHg) 9.0±2.3 28.2±4.2‡ 5.0±0.5 16.5±2.9‡* 13.8±4.0* 
Max dp/dt (mmHg/s) 9816±1711 7037±790 11640±1926 8664±648 9337±634 
Min dp/dt (mmHg/s) 9532±1080 7271±929 10527±1735 9369±550* 10414±1122* 
Contractility index 181±21 123±12† 174±26 166±10* 159±14 
τ (ms) 8.0±1.6 13.0±0.9† 11.0±2.6 12.0±0.8 10.0±1.0 
Heart rate (beats/min) 582±31 508±15† 555±40 469±22†* 488±7† 
LVEDd (mm) 3.08±0.07 3.83±0.24† 2.92±0.09 3.26±0.03‡ 3.15±0.25 
LVESd (mm) 1.32±0.10 2.59±0.29‡ 1.12±0.05 1.82±0.04*,‡ 1.72±0.26 
LVPWd (mm) 0.69±0.02 0.78±0.02† 0.67±0.03 0.79±0.02‡ 0.75±0.03 
LVPWs (mm) 1.08±0.03 0.96±0.03† 1.03±0.05 1.12±0.04** 1.02±0.21 
Parameters WT sham (n=6) WT TAC (n=9) KO sham (n=7) KO TAC (n=7) WT TAC + Fa (n=6) 
LVSP (mmHg) 103.0±3.5 175.0±13.7‡ 107.0±5.3 203.0±8.9‡* 209.0±11.0‡ 
LVEDP (mmHg) 9.0±2.3 28.2±4.2‡ 5.0±0.5 16.5±2.9‡* 13.8±4.0* 
Max dp/dt (mmHg/s) 9816±1711 7037±790 11640±1926 8664±648 9337±634 
Min dp/dt (mmHg/s) 9532±1080 7271±929 10527±1735 9369±550* 10414±1122* 
Contractility index 181±21 123±12† 174±26 166±10* 159±14 
τ (ms) 8.0±1.6 13.0±0.9† 11.0±2.6 12.0±0.8 10.0±1.0 
Heart rate (beats/min) 582±31 508±15† 555±40 469±22†* 488±7† 
LVEDd (mm) 3.08±0.07 3.83±0.24† 2.92±0.09 3.26±0.03‡ 3.15±0.25 
LVESd (mm) 1.32±0.10 2.59±0.29‡ 1.12±0.05 1.82±0.04*,‡ 1.72±0.26 
LVPWd (mm) 0.69±0.02 0.78±0.02† 0.67±0.03 0.79±0.02‡ 0.75±0.03 
LVPWs (mm) 1.08±0.03 0.96±0.03† 1.03±0.05 1.12±0.04** 1.02±0.21 

The pressure gradients across the aortic stenosis were similar between the H2R-knockout and wild-type groups evaluated by randomly sampling at 3 days after TAC. At 4 weeks after TAC, LVSP was higher in H2R-knockout mice than in the wild-type mice because in more wild-type mice it developed into severe HF and consequently progressed to a decrease in systolic pressure (Supplementary Figure S1 at http://www.clinsci.org/cs/127/cs1270435add.htm). LVEDP and heart rate were lower, and contractility index was larger in H2R-knockout mice than in wild-type mice (Table 1), suggesting an improvement in diastolic and systolic dysfunction by deletion of H2R.

Echocardiographic examinations showed that LVESd (Table 1) was smaller and left ventricular posterior wall thickness in systole (LVPWs) (Table 1) and LVFS (Figure 1D) were larger in H2R-knockout TAC mice than in wild-type TAC mice, indicating a better systolic function in H2R-deficient mice. Similar results to H2R-knockout TAC mice were obtained when selective H2R blocker famotidine was administered in wild-type TAC mice (Table 1 and Supplementary Figure S2 at http://www.clinsci.org/cs/127/cs1270435add.htm).

Morphological results in response to TAC

At 4 weeks after TAC, H2R-knockout mice had a significantly smaller lung weight/body weight ratio when compared with wild-type mice (Figure 1E). There was no significant difference between the two groups in the heart weigh/body weight ratio (Figure 1F) and LVPWd (Table 1). Similar results to H2R-knockout TAC mice were obtained when the H2R blocker famotidine was administered in wild-type TAC mice (Supplementary Figure S2). Histological examination showed that there was no significant difference in cardiomyocyte size, but there was a significantly lower amount of fibrosis in H2R-knockout TAC mice than in wild-type TAC mice (Figures 2A and 2B). These results indicate that H2R deficiency attenuates pulmonary congestion and myocardial fibrosis.

H2R inactivation reduced calcineurin expression in cardiac fibroblasts

Calcineurin is believed to promote cardiac hypertrophy. We found that calcineurin protein expression in the myocardium was significantly increased in response to pressure overload, but the extent of the increase was lower in H2R-deficient TAC mice than in wild-type TAC mice (Figure 2C), which seems paradoxical to the morphological finding that the cardiac hypertrophy was similar between the two groups. We then tried to clarify this issue. In cultured cells, we observed that H2R was expressed in both fibroblasts and cardiomyocytes (Figure 3A), and it was in the cardiac fibroblasts (confirmed by vimentin staining, see Supplementary Figure S3A at http://www.clinsci.org/cs/127/cs1270435add.htm) rather than cardiomyocytes that histamine or the H2R-selective agonist AD increased the protein expression of calcineurin, which was abrogated by co-treatment with famotidine (Figures 3B and 3C, and Supplementary Figure S3B).

Effect of H2R activation on calcineurin expression and fibroblast proliferation

Figure 3
Effect of H2R activation on calcineurin expression and fibroblast proliferation

(A) Western blot analysis of H2R and calcineurin in neonatal rat fibroblasts and cardiomyocytes; β-actin served as loading control. *P<0.05 compared with control (Con). Ang II, angiotensin II; His, histamine. (B) Both His (1 μM) and AD increased calcineurin expression in neonatal fibroblasts, and co-treatment with famotidine (Fam; 1 μM) abrogated AD-induced up-regulation of calcineurin. #P<0.05 compared with control (Con). §P<0.05 compared with AD alone. In (A and B), experiments were repeated three times. (C) Microscopic images of GFP-positive fibroblasts infected by lentivirus carrying shRNA of calcineurin (Lv-CN) or negative control (Lv-NC) (×100) to show the infection efficiency (MOI=5). (D) Calcineurin protein expression in fibroblasts transfected with Lv-CN or Lv-NC (MOI=5) for 72 h. Lv-CN reduced calcineurin protein expression by 62%. (E) Cell proliferation assay in fibroblasts. Lv-CN significantly inhibited cell proliferation stimulated by AD (1 μM, 24 h). *P<0.05 compared with Con. #P<0.05 compared with Lv-NC. §P<0.01 compared with Lv-NC. &P<0.01 compared with Lv-NC+AD.

Figure 3
Effect of H2R activation on calcineurin expression and fibroblast proliferation

(A) Western blot analysis of H2R and calcineurin in neonatal rat fibroblasts and cardiomyocytes; β-actin served as loading control. *P<0.05 compared with control (Con). Ang II, angiotensin II; His, histamine. (B) Both His (1 μM) and AD increased calcineurin expression in neonatal fibroblasts, and co-treatment with famotidine (Fam; 1 μM) abrogated AD-induced up-regulation of calcineurin. #P<0.05 compared with control (Con). §P<0.05 compared with AD alone. In (A and B), experiments were repeated three times. (C) Microscopic images of GFP-positive fibroblasts infected by lentivirus carrying shRNA of calcineurin (Lv-CN) or negative control (Lv-NC) (×100) to show the infection efficiency (MOI=5). (D) Calcineurin protein expression in fibroblasts transfected with Lv-CN or Lv-NC (MOI=5) for 72 h. Lv-CN reduced calcineurin protein expression by 62%. (E) Cell proliferation assay in fibroblasts. Lv-CN significantly inhibited cell proliferation stimulated by AD (1 μM, 24 h). *P<0.05 compared with Con. #P<0.05 compared with Lv-NC. §P<0.01 compared with Lv-NC. &P<0.01 compared with Lv-NC+AD.

H2R activation increased proliferation of fibroblasts mediated by calcineurin

We constructed recombinant lentivirus carrying shRNA of calcineurin and efficiently infected neonatal rat fibroblasts (Figure 3C). Protein levels of calcineurin were significantly silenced by Lv-CN (Figure 3D). The H2R agonist AD significantly increased fibroblast proliferation, which was abrogated by co-treatment with Lv-CN (Figure 3E). AD or Lv-CN did not influence the apoptosis of fibroblasts (Supplementary Figure S4 at http://www.clinsci.org/cs/127/cs1270435add.htm).

Effect of H2R activation on the calcineurin/NFAT signalling pathway

The H2R agonist AD markedly increased the nuclear translocation of NFATc3 and fibronectin protein in cultured fibroblasts, which was blocked by pre-treatment with Lv-CN (Figures 4A and 4B). AD also significantly up-regulated α-SMA, but this effect was not abolished by pre-treatment with Lv-CN (Figure 4B). Real-time quantitative PCR showed that pre-treatment with Lv-CN in cultured fibroblasts substantially blocked the AD-induced up-regulation of procollagen I and procollagen III (Figure 4C).

Effect of H2R activation on downstream calcineurin signalling in neonatal rat fibroblasts

Figure 4
Effect of H2R activation on downstream calcineurin signalling in neonatal rat fibroblasts

(A) Western blot analysis of NFATc3. AD (1 μM) treatment for 24 h increased nuclear NFATc3. Histone H3 expression was used as loading control. There was no change in NFATc3 expression in the total cell fraction. Effect of AD on NFATc3 was blocked by shRNA of calcineurin (Lv-CN). (B) Western blot analysis of fibronectin and α-SMA protein expression. (C) Real-time quantitative PCR for expression of procollagens I and III. *P<0.05 compared with Con. #P<0.05 compared with Lv-NC. §P<0.05 compared with Lv-NC + AD. Experiments were repeated three times. Con, control.

Figure 4
Effect of H2R activation on downstream calcineurin signalling in neonatal rat fibroblasts

(A) Western blot analysis of NFATc3. AD (1 μM) treatment for 24 h increased nuclear NFATc3. Histone H3 expression was used as loading control. There was no change in NFATc3 expression in the total cell fraction. Effect of AD on NFATc3 was blocked by shRNA of calcineurin (Lv-CN). (B) Western blot analysis of fibronectin and α-SMA protein expression. (C) Real-time quantitative PCR for expression of procollagens I and III. *P<0.05 compared with Con. #P<0.05 compared with Lv-NC. §P<0.05 compared with Lv-NC + AD. Experiments were repeated three times. Con, control.

Influence of H2R inactivation on apoptosis in vivo

The myocardial cell apoptosis detected by the TUNEL assay was significantly increased in TAC mice when compared with the corresponding sham group, but the amplitude of increase was markedly smaller in H2R-deficient TAC mice than in wild-type TAC mice (Figures 5A and 5B). Expression levels of myocardial Bax and cleavage caspase-3 protein were markedly up-regulated in pressure-overloaded mice, but the amplitude of increase was significantly smaller in H2R-deficient TAC mice than in wild-type TAC mice (Figure 5C).

H2R deficiency led to a decrease in myocardial apoptosis in response to TAC for 4 weeks

Figure 5
H2R deficiency led to a decrease in myocardial apoptosis in response to TAC for 4 weeks

(A) Representative images of the TUNEL assay in different groups. The nuclei were labelled with DAPI (blue staining), and apoptosis was labelled with TUNEL reaction mixture (red staining). The merged pink staining indicates apoptotic nuclei. Scale bar=100 μm. (B) Quantitative analysis of apoptotic cells. The positive rate of TUNEL-labelled nuclei was calculated from four different and randomly selected areas under confocal microscopy for each slide. Three hearts in each group were used. #P<0.05 compared with the WT TAC group. (C) Western blot of Bax and cleavage caspase-3. *P<0.05 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group. §P<0.01 compared with the WT sham group. #P<0.05 compared with the WT TAC group (n=5 in each group). KO, knockout; WT, wild-type.

Figure 5
H2R deficiency led to a decrease in myocardial apoptosis in response to TAC for 4 weeks

(A) Representative images of the TUNEL assay in different groups. The nuclei were labelled with DAPI (blue staining), and apoptosis was labelled with TUNEL reaction mixture (red staining). The merged pink staining indicates apoptotic nuclei. Scale bar=100 μm. (B) Quantitative analysis of apoptotic cells. The positive rate of TUNEL-labelled nuclei was calculated from four different and randomly selected areas under confocal microscopy for each slide. Three hearts in each group were used. #P<0.05 compared with the WT TAC group. (C) Western blot of Bax and cleavage caspase-3. *P<0.05 compared with the corresponding sham group. #P<0.05 compared with the WT TAC group. §P<0.01 compared with the WT sham group. #P<0.05 compared with the WT TAC group (n=5 in each group). KO, knockout; WT, wild-type.

H2R activation increased mitochondrial permeability and apoptosis in vitro

We measured the fluorescence intensity of merged calcein and MitoTracker to assess mitochondrial permeability in cultured cardiomyocytes. We observed that the fluorescence intensity of calcein in the mitochondria of cardiomyocytes was markedly reduced by stimulation with histamine or the H2R agonist AD, whereas this effect was significantly antagonized by pre-treatment with famotidine (Figure 6A). In addition, the mPTP inhibitor cyclosporin A (CsA) significantly blocked the decrease in the fluorescence intensity of calcein in the mitochondria induced by AD (Figure 6A). Mitochondrial depolarization was investigated by TRME staining. We found that TMRE fluorescence was markedly reduced in the cardiomyocytes exposed to AD, whereas co-treated with famotidine or CsA attenuated this effect (Figures 6A and 6B). We observed further that treatment with histamine for 24 h significantly up-regulated the level of the total Bax and cleavage caspase-3, both of which are important pro-apoptotic proteins, whereas pre-treatment with famotidine attenuated this effect (Figures 6C and 6E). Histamine or the selective H2R agonist AD increased the translocation of Bax to the mitochondria, whereas famotidine inhibited Bax translocation (Figure 6D). TUNEL assay and Hoechst staining showed that both histamine and AD increased cardiomyocyte apoptosis, and this effect was antagonized by co-treatment with famotidine (Figure 7).

H2R activation increased mitochondrial permeability and apoptotic signalling in neonatal rat cardiomyocytes

Figure 6
H2R activation increased mitochondrial permeability and apoptotic signalling in neonatal rat cardiomyocytes

(A) Confocal microscopic images of calcein (green) and MitoTracker (red) staining as well as TMRE staining to reflect ΔΨm. In the merged images, mitochondrial calcein was indicated by orange colour. (B) Intensity of TMRE fluorescence. *P<0.01 compared with Con. #P<0.05 compared with the corresponding Fam or CSA alone. (C) Western blot analysis of total Bax. (D) Western blot analysis of mitochondrial Bax. (E) Western blot analysis of cleavage caspase-3. *P<0.01 compared with Con. #P<0.05 compared with AD alone. For (CE), experiments were repeated three times. Con, control; Fam, famotidine; His, histamine. Scale bar=200 μm.

Figure 6
H2R activation increased mitochondrial permeability and apoptotic signalling in neonatal rat cardiomyocytes

(A) Confocal microscopic images of calcein (green) and MitoTracker (red) staining as well as TMRE staining to reflect ΔΨm. In the merged images, mitochondrial calcein was indicated by orange colour. (B) Intensity of TMRE fluorescence. *P<0.01 compared with Con. #P<0.05 compared with the corresponding Fam or CSA alone. (C) Western blot analysis of total Bax. (D) Western blot analysis of mitochondrial Bax. (E) Western blot analysis of cleavage caspase-3. *P<0.01 compared with Con. #P<0.05 compared with AD alone. For (CE), experiments were repeated three times. Con, control; Fam, famotidine; His, histamine. Scale bar=200 μm.

H2R activation increased apoptosis in cultured neonatal rat cardiomyocytes

Figure 7
H2R activation increased apoptosis in cultured neonatal rat cardiomyocytes

(A) TUNEL assay shows His or AD increased apoptosis of cultured neonatal rat cardiomyocytes (pink indicates apoptosis), whereas Fam abrogated AD-induced apoptosis. Scale bar=75 μm. (B) Similar results to (A) were obtained by Hoechst staining. Bright blue indicates apoptosis. Scale bar=200 μm. Quantitative analyses of TUNEL (C) and Hoechst (D) staining-positive nuclei were performed. *P<0.01 compared with control. #P<0.01 compared with AD alone. Con, control; Fam, famotidine; His, histamine.

Figure 7
H2R activation increased apoptosis in cultured neonatal rat cardiomyocytes

(A) TUNEL assay shows His or AD increased apoptosis of cultured neonatal rat cardiomyocytes (pink indicates apoptosis), whereas Fam abrogated AD-induced apoptosis. Scale bar=75 μm. (B) Similar results to (A) were obtained by Hoechst staining. Bright blue indicates apoptosis. Scale bar=200 μm. Quantitative analyses of TUNEL (C) and Hoechst (D) staining-positive nuclei were performed. *P<0.01 compared with control. #P<0.01 compared with AD alone. Con, control; Fam, famotidine; His, histamine.

DISCUSSION

This research provides genetic evidence to prove that blocking H2R activation could reduce cardiac fibrosis through down-regulation of calcineurin expression in cardiac fibroblasts, as well as reducing myocardial apoptosis through lowering mitochondrial permeability in cardiomyocytes, finally achieving the goal of improving HF (Figure 8).

Overview of the signalling pathways of H2R activation influencing HF

Figure 8
Overview of the signalling pathways of H2R activation influencing HF

Pressure overload increases myocardial density of mast cells and promotes histamine production and up-regulation of H2R. Activation of H2R by histamine exerts distinct roles on fibroblasts and cardiomyocytes. In fibroblasts, H2R activation stimulates the pathway of calcineurin/nuclear translocation of NFAT/fibronectin/procollagen/cell proliferation/fibrosis, whereas in cardiomyocytes, H2R activation stimulates the pathway of mitochondrial translocation of Bax/mitochondrial permeability increase/cleavage caspase-3/apoptosis. Both fibrosis and cardiomyocyte apoptosis promote the progression of HF. αq, Gq-protein α subunit; PLC, phospholipase C; PLP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol trisphosphate; CaM, calmodulin; αs, Gs-protein α subunit.

Figure 8
Overview of the signalling pathways of H2R activation influencing HF

Pressure overload increases myocardial density of mast cells and promotes histamine production and up-regulation of H2R. Activation of H2R by histamine exerts distinct roles on fibroblasts and cardiomyocytes. In fibroblasts, H2R activation stimulates the pathway of calcineurin/nuclear translocation of NFAT/fibronectin/procollagen/cell proliferation/fibrosis, whereas in cardiomyocytes, H2R activation stimulates the pathway of mitochondrial translocation of Bax/mitochondrial permeability increase/cleavage caspase-3/apoptosis. Both fibrosis and cardiomyocyte apoptosis promote the progression of HF. αq, Gq-protein α subunit; PLC, phospholipase C; PLP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol trisphosphate; CaM, calmodulin; αs, Gs-protein α subunit.

We observed in the pressure-overload model that the increasing extent of cardiac calcineurin in H2R-knockout mice was markedly lower than that in the wild-type mice; however, it is well known that calcineurin is a vital signalling molecule inducing cardiac hypertrophy [32]. The paradox here is that knocking out or blocking H2R did not reduce pressure-overload-induced cardiac hypertrophy. In order to solve this paradox, we performed cell culture experiments. As a result, we found that basic myocardial H2R expression was relatively low and H2R activation only increased calcineurin expression in fibroblasts but not in cardiomyocytes, which could at least partly explain the fact that H2R blockade reduced cardiac fibrosis without having an influence on cardiac hypertrophy. This research provides evidence indicating that histamine or AD-stimulated H2R activation could up-regulate calcineurin in cardiac fibroblasts, whereas it has been widely shown that calcineurin accelerates cardiac fibrosis [25,3336]. Although myocardial H2R activation is likely to be an auxiliary regulator in the induction of calcineurin after TAC because many other important stimuli such as activation of the angiotensin system and sympathetic system also contribute greatly to the induction of calcineurin, it has been reported that histamine can lead to the activation of calcium/calmodulin-dependent protein kinase II [37], an upstream signalling molecule of calcineurin. In addition, there are several lines of evidence for histamine accelerating tissue fibrosis in studies from various laboratories [2123]. In agreement with our findings, a significant correlation between myocardial fibrosis and cardiac mast cell density has been reported [38]. Increased mast cell density in the pressure-overloaded heart would lead to degranulation of mast cells and the release of histamine and then activates H2R [9]. In our present research, the reduction in pressure-overload-induced cardiac fibrosis in H2R-knockout mice may have contributed to the improvement in cardiac diastolic function, manifested as the lowering of LVEDP and/or the increase in min dp/dt. Since cardiac fibrosis is one of the main causes of diastolic HF [39], searching for molecules to target for antifibrosis has clinical significance.

Previous clinical observations have shown a marked increase in plasma histamine concentrations in patients with variant angina [40]. Mast cells and histamine release have been implicated in the development of HF [18,20,41]. Our recent research found that H2R activation worsened mitochondrial dysfunction in ischaemic cardiomyocytes and could therefore enhance myocardial apoptosis [3]. Thus, the finding of the present research that myocardial apoptosis under pressure overload in H2R-knockout mice was lower than that in the wild-type mice makes perfect sense. It has been reported that histamine-mediated H2R activation increases the expression of tumour necrosis factor-α (TNF-α) [3,42], and this cytokine plays an important role in enhancing myocardial apoptosis, therefore worsening HF [43]. The present research also showed that H2R activation increased mitochondrial permeability, and accelerated the up-regulation of the apoptotic signalling molecules Bax and caspase-3. The intrinsic apoptotic signalling pathway can be initiated by mitochondrial dysfunction, whereas the extrinsic apoptosis pathway can be initiated by the TNF-α. Thus, both the extrinsic and intrinsic apoptosis pathways induced by H2R activation might have contributed to the enhanced cardiomyocyte apoptosis detected in the present study.

Abbreviations

     
  • AC

    adenylate cyclase

  •  
  • AD

    amthamine dihydrobromide

  •  
  • CHF

    chronic heart failure

  •  
  • CsA

    cyclosporin A

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • H2R

    histamine H2 receptor

  •  
  • HF

    heart failure

  •  
  • i.p.

    intraperitoneally

  •  
  • LV

    left ventricular

  •  
  • Lv-CN

    lentivirus-shRNA-calcineurin

  •  
  • LVEDd

    left ventricular end-diastolic diameter

  •  
  • LVEDP

    left ventricular end-diastolic pressure

  •  
  • LVESd

    left ventricular end-systolic diameter

  •  
  • LVFS

    left ventricular fractional shortening

  •  
  • LVPWd

    left ventricular posterior wall thickness in diastole

  •  
  • LVPWs

    left ventricular posterior wall thickness in systole

  •  
  • LVSP

    left ventricular systolic pressure

  •  
  • max

    dp/dt, maximum LV pressure rise rate

  •  
  • min

    dp/dt, minimum LV pressure fall rate

  •  
  • MOI

    multiplicities of infection

  •  
  • ΔΨm

    mitochondrial membrane potential

  •  
  • mPTP

    mitochondrial permeability transition pore

  •  
  • NFATc3

    nuclear factor of activated T-cell c3

  •  
  • α-SMA

    α-smooth muscle actin

  •  
  • TAC

    transverse aortic constriction

  •  
  • TMRE

    tetramethylrhodamine ethyl ester

  •  
  • TNF-α

    tumour necrosis factor-alpha

  •  
  • τ

    the exponential time constant of relaxation

  •  
  • TUNEL

    terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling

AUTHOR CONTRIBUTION

Yulin Liao, Masafumi Kitakaze and Zhi Zeng conceived and designed the study; Zi Zeng, Yulin Liao, Liang Shen, Xixian Li and Tao Luo performed the experiments and collected the data; Zhi Zeng, Yulin Liao, Xuan Wei, Jingwen Zhang, Shiping Cao, Xiaobo Huang, Yasushi Fukushima, Jianping Bin, Masafumi Kitakaze and Dingli Xu analysed and interpreted the results; Yulin Liao, Jingwen Zhang and Zhi Zeng drafted, edited and revised the paper before submission; and all authors read and approved the final version of the paper.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant numbers 81170146 and 31271513 (to Y.L.)]; and the Team Program of Natural Science Foundation of Guangdong Province, China [grant number S2011030003134 (to Y.L. and J.B.)]

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Supplementary data