Mol Cell Biochem (2014) 391:259–266 DOI 10.1007/s11010-014-2012-8
Dynamic alterations of connexin43, matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 during ventricular fibrillation in canine Jing Wang • Jing-sha Li • Hong-zhen Liu Shao-lei Yi • Guo-ying Su • Yun Zhang • Jing-quan Zhong
•
Received: 10 January 2014 / Accepted: 28 February 2014 / Published online: 18 March 2014 Ó Springer Science+Business Media New York 2014
Abstract The aim of this study is to investigate the dynamic alterations of cardiac connexin 43 (Cx43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF) duration. In this study, thirtytwo dogs were randomly divided into sham control group, 8-min VF group, 12-min VF group, and 30-min VF group. Cx43 and phosphorylated Cx43 (p-Cx43) in tissues were detected by western blot and immunofluorescence analysis. MMP-2 and TIMP-2 were detected by western blot and immunohistochemistry analysis. The results showed that Cx43 levels in three VF groups were significantly decreased compared with sham control group. p-Cx43 levels in 12-min and 30-min VF groups were significantly reduced compared with sham control group. The ratio of p-Cx43/Cx43 was also decreased in VF groups. Compared with sham controls, no significant difference was observed between the sham control group and 8-min VF group in MMP-2 level, but MMP-2 level increased in 12-min and 30-min VF groups. The ratios of MMP-2/TIMP-2 were higher in VF groups, and were correlated with the duration of VF. A remarkable correlation was observed between the J. Wang J. Li H. Liu S. Yi G. Su Y. Zhang J. Zhong (&) Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Jinan 250012, Shandong, China e-mail: [email protected] J. Wang J. Li H. Liu S. Yi G. Su Y. Zhang J. Zhong Department of Cardiology, Qilu Hospital, Shandong University, Jinan 250012, Shandong, China J. Wang Department of Keshan Disease, Shandong Provincial Institute for Endemic Diseases Control, Jinan 250014, China
ratio of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P \ 0.01). In conclusion, the alteration of Cx43 and/or p-Cx43 levels and the imbalance of MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate prognosis of VF. Keywords Ventricular fibrillation Connexin Tissue inhibitor of metalloproteinase-2 Matrix metalloproteinase-2
Introduction Ventricular fibrillation (VF) is one of the most common causes of sudden cardiac death, and is related to abnormal impulse formation and/or circuit movement re-entry [1]. Reentry is an important electrophysiological mechanism of cardiac fibrillation in which a persisting electrical impulse reactivates an area of previously activated myocardial tissues which is no longer refractory, resulting in a circus movement of activation [2]. Nevertheless, the mechanisms involved in the initiation and persistence of re-entrant arrhythmias are still not fully elucidated. It was hypothesized that intercellular electrical coupling and communication mediated by gap junctions may determine conduction velocity, and the alterations in gap junction distribution and/or defective cell-to-cell coupling may contribute to abnormal conduction, and facilitate the occurrence of re-entrant arrhythmias such as VF [3– 5]. Gap junctions that link adjoining cardiomyocytes ensure cell-to-cell electrical coupling and direct communication via clusters of intercellular channels (connexons), which are composed of connexins. Several connexin isoforms are
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expressed in the heart that differ in conductance and connexin43 (Cx43) predominates in the ventricle [6]. The homogeneous conduction of electrical activity not only relies on cardiomyocytes integration but also is affected by extracellular matrix (ECM) among the cardiomyocytes [2]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that regulate the ECM turnover in a balance with tissue inhibitors of metalloproteinase (TIMPs) [7]. Cardiomyocytes can synthesize and release MMPs, in particular MMP-2 (gelatinase A) and MMP-9 (gelatinase B), which participate in the ventricular remodeling process [8, 9]. However, the expression patterns of MMPs and TIMPs during VF are also unclear. Therefore, in this study, we aimed to investigate the dynamic changes of cardiac Cx43, MMP-2, and TIMP-2 expression in the setting of different duration of VF, in order to reveal the possible initiation and/or persistence mechanisms of VF.
Mol Cell Biochem (2014) 391:259–266
was defined as a decrease in aortic blood pressure below 25 mm Hg and the presence of VF waveform on the ECG. Dogs in the sham control group were intubated tracheally. Untreated VF lasted for 8, 12, or 30 min in other groups. All measurements were performed by an investigator blinded to the experiment assignment. After finishing the experimental protocol, the animals were killed by infusing KCl. Transthoracic echocardiography (TTE) All animals before VF were examined by TTE (PHILIPS 7500 with a 2.5–3.5 MHz transducer) to measure the left atrium dimension (LAD), left ventricular diastolic dimensions (LVDd), and left ventricular ejection fraction (LVEF), respectively. All of the above examinations were performed in a parasternal long-axis and M-mode view. Hemodynamic measurement
Materials and methods Animal preparation All experimental protocols were approved by the Animal Care and Use Committee of Qilu Hospital, Shandong University, China. Thirty-two mongreal dogs (obtained from the Center for Experimental Animals of Qilu Hospital of Shandong University) of either sex (weight 11.5–17 kg) were randomly divided into four groups: sham control group (n = 8), 8-min VF group (n = 8), 12-min VF group (n = 8), and 30-min VF group (n = 8). The animals were anesthetized via intravenous injection of pentobarbital-Na (30 mg/kg), then placed in the supine position on the table and restrained at the four extremities. A 5.0 cuffed tracheal tube was inserted into the trachea for intubation. The tube was attached to a ventilator (Newport E-100 M, Newport Medical Instruments, Costa Mesa, CA, USA). Ventilation began at the tidal volume of 10–15 ml/kg, ventilator rate of 16–20 breaths/min, and inspiration to expiration ratio of 1:1.5–1:2.0. Three surface electrodes were placed under three limbs separately for standard lead II electrocardiogram (ECG). The right femoral artery and bilateral femoral veins were cannulated. 6-F catheters (Cordis Corp., Miami, FL, USA) were positioned in the intrathoracic ascending aorta and the right atrium under fluoroscopic guidance. The remaining cannulated femoral vein was used for drug infusion. Heparin was administered at 100 U/kg for anticoagulation, and added when necessary. Creation of VF model VF was induced by delivering a 5-s alternating current at 80 V across the thorax through two needles. Successful VF
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Catheter positions were confirmed by X-ray fluoroscopy. Both catheters in the intrathoracic ascending aorta and the right atrium were connected to the pressure transducers attached to a PRO EP recording system (PowerLab/16sp, AD Instruments, Australia), by which aortic systolic pressure (AOSP), diastolic aortic pressure (AODP), and right atrial pressure (RAP) could be recorded. Coronary perfusion pressure (CPP) was calculated as AODP minus RAP and mean aortic pressure (MAP) was calculated as 1/3 AOSP plus 2/3AODP. Tissue collection Tissue samples of the left ventricular myocardium were collected within 15 s following the death. Then, the samples were immediately snap-frozen in liquid nitrogen (stored at -80 °C) to minimize time-dependent effects of myocardial ischemia on the changes of connexins, MMPs and TIMPs. Western blot analysis Tissue samples (20 mg) were homogenized in 200 ll of icecold lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 15,000 r/min for 10 min at 4 °C. Supernatants were collected. Equal amounts of protein from each sample were subjected to SDS-PAGE (10 % polyacrylamide) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 2 h with 1 % bovine serum albumin, then incubated overnight at 4 °C with the antibodies, including rabbit polyclonal anti-Cx43, anti-pCx43 (Uscnlife, Wuhan, China), anti-MMP-2, anti-TIMP-2
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(Santa Cruz CA, USA), and b-actin (Sigma, USA). After being washed, the membrane was incubated with the corresponding secondary antibody (Santa Cruz, CA, USA) conjugated to horseradish peroxidase (HRP). Immunoreactive bands were visualized with the SuperSignal West Pico enhanced chemiluminescence kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. b-actin was used as loading control. Band intensities were quantified with Quantity One (Bio-Rad Laboratories, Inc).
overnight with primary antibody for MMP-2 or TIMP-2 (Santa Cruz, CA, USA) followed by incubation with HRPconjugated secondary antibody (1:500 dilution). Nuclei were counterstained with hematoxylin. For negative controls, PBS solution was used instead of primary antibody. The images were analyzed by Image-Pro plus 6.0 software (Media Cybernetics, Inc).
Immunofluorescence and confocal laser scanning microscopy
Data were presented as mean ± SEM and analyzed by SPSS13 software. All variables had normal distribution and were analyzed by ANOVA analysis. P \ 0.05 was considered significant.
Left ventricular preparations were fixed in 4 % paraformaldehyde for 24 h. Tissue samples were embedded with paraffin, cut into 4 lm sections, mounted on gelatin-coated slides, then dewaxed and rehydrated with graded alcohols. The slides were microwaved in boiling 0.01 M sodium citrate buffer for 15 min, then blocked in goat serum/PBS (1:20) solution for 20 min at room temperature. Next, the slides were incubated with primary antibody for Cx43 and p-Cx43 (1:100 dilutions in PBS) overnight at 4 °C. Finally, the slides were incubated with isothiocyanate (FITC)-conjugated secondary antibody (1:500 dilution) for 30 min at 37 °C. For negative controls, PBS solution was used instead of primary antibody. Samples were examined by laser scanning confocal microscope (TCS SP2, Leica, Germany). High-intensity signal was measured and analyzed with Image-Pro plus 6.0 software (Media Cybernetics, Inc).
Statistical analysis
Results Baseline characteristics of different groups Among 32 dogs employed in this study, one dog in the sham control group was excluded because of death during catheterization, and one dog in 30-min VF group died during VF. There were no significant differences among the animals in each group in body weight, heart rate, LAD, LVDd, LVEF, and hemodynamic variables at baseline (Table 1).
Immunohistochemistry staining
The expression of Cx43 and p-Cx43 in hearts of different groups
Five micrometer ventricular tissue sections were rehydrated and blocked with 3 % hydrogen peroxide followed by incubation in 20 % (v/v) normal goat serum (Santa Cruz, CA, USA). Tissue sections were incubated at 4 °C
Confocal microscopy showed that with the duration of VF, Cx43, and p-Cx43 signals became weaker and distributed in heterogeneity. The percentage area and the integral optical density (IOD) of Cx43 and p-Cx43 in VF groups
Table 1 Baseline characteristics of experimental groups
Contents
Sham group
8-minVF group
12-minVF group
30-minVF group
Subjects
8
8
8
8
Weight (kg)
13.36 ± 1.69
13.43 ± 1.72
13.01 ± 1.75
13.26 ± 1.67
Heart (bpm)
156 ± 9
154 ± 13
157 ± 11
150 ± 10
Echocardiography bpm beat per minute, LAD left atrial diameter, LVDd left ventricle diastolic diameter, LVEF left ventricular ejection fraction, AOSP systolic aortic pressure, AODP diastolic aortic pressure, MAP mean aortic pressure, RAP right atrial pressure, CPP coronary perfusion pressure Values were expressed as mean ± SEM
LAD (mm)
16.73 ± 0.83
15.92 ± 1.47
17.28 ± 1.09
16.83 ± 1.26
LVDd (mm)
24.83 ± 1.90
25.27 ± 1.75
24.36 ± 1.43
25.32 ± 1.69
0.67 ± 0.08
0.68 ± 0.06
0.69 ± 0.09
0.69 ± 0.07
LVEF Hemodynamics AOSP (mmHg)
137.67 ± 17.34
133.73 ± 16.96
135.12 ± 19.52
136.31 ± 18.43
AODP (mmHg)
109.47 ± 15.37
102.39 ± 24.16
114.15 ± 17.84
116.24 ± 16.27
MAP (mmHg)
118.87 ± 16.28
112.84 ± 19.62
121.14 ± 18.73
122.93 ± 17.53
RAP (mmHg)
8.26 ± 3.67
7.72 ± 2.68
7.46 ± 3.25
6.95 ± 2.37
CPP (mmHg)
101.21 ± 11.71
94.67 ± 21.48
106.69 ± 14.59
109.29 ± 14.25
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262 Fig. 1 Immunofluorescence analysis of Cx43 and p-Cx43 distribution in heart samples. With the duration of VF, Cx43, and p-Cx43 signals became weaker and distributed in heterogeneity. (Color figure online)
Mol Cell Biochem (2014) 391:259–266
Sham
8-min VF
12-min VF
30-min VF
Cx43
p-Cx43
were significantly decreased compared to the sham control group (Fig. 1). Western blot analysis further confirmed that compared to the sham control group, Cx43 level in the heart was significantly decreased in three VF groups and Cx43 level declined with the duration of VF (0.83 ± 0.04 vs. 0.71 ± 0.06 vs. 0.66 ± 0.05 vs. 0.52 ± 0.07, P \ 0.05) (Fig. 2a). p-Cx43 levels in 12-min VF group and 30-min VF group were significantly reduced (0.76 ± 0.07 vs. 0.56 ± 0.05 vs. 0.41 ± 0.03, P \ 0.05), while there was no significant difference between the sham control group and 8-min VF group (0.76 ± 0.07 vs. 0.69 ± 0.05, P [ 0.05) (Fig. 2b). In addition, the ratio of p-Cx43/Cx43 was decreased with the duration of VF (0.87 ± 0.03 vs. 0.85 ± 0.04 vs. 0.76 ± 0.06 vs. 0.64 ± 0.04) (Fig. 2c). The expression of MMP-2 and TIMP-2 in hearts of different groups Compared with sham controls, dogs under VF showed significantly decreased levels of TIMP-2 in the hearts, and the decline of TIMP-2 level was correlated to the duration of VF (0.86 ± 0.08 vs. 0.75 ± 0.06 vs. 0.68 ± 0.04 vs. 0.51 ± 0.06, P \ 0.05) (Figs. 3, 4). MMP-2 levels showed no significant difference between the sham control group and the 8-min VF group (0.51 ± 0.03 vs. 0.54 ± 0.04, P [ 0.05) (Fig. 4a), however, MMP-2 levels were increased in other two longer-duration VF groups (0.59 ± 0.07 and 0.71 ± 0.05, P \ 0.05 compared to sham control group) (Fig. 4b). The ratios of MMP-2/TIMP-2 were higher in VF groups, and increased gradually with the duration of VF (0.64 ± 0.08, 0.85 ± 0.09, 1.12 ± 0.07 and 1.39 ± 0.09 in four groups, respectively) (Fig. 4c).
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Correlation between p-Cx43/Cx43 and MMP-2/TIMP-2 To investigate the correlation between p-Cx43/Cx43 and MMP-2/TIMP-2, we performed correlation analysis. The results showed a remarkable correlation between the ratios of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P \ 0.01; Fig. 5).
Discussion Since the mechanisms implicated in the initiation and progression of VF are still controversial, fundamental knowledge about cellular basis of arrhythmia is crucial for the development of novel and effective approaches for the prevention and therapy of heart diseases. Cx43 is the predominant ventricular gap junction channel protein [4, 10]. Numerous studies have demonstrated that gating properties of Cx43-formed channels (hemichannels, gap junctions) play an essential role in normal rapid electrical signal propagation and cardiac muscle synchronization [10–12]. In turn, alterations of Cx43 expression are implicated in the development of ventricular tachyarrhythmias [13, 14]. Previous studies have shown that Cx43-deficient mice exhibited markedly slow myocardial conduction that facilitates re-entrant arrhythmias and sudden arrhythmic death [15, 16]. Down-regulation and/or abnormal distribution of myocardial Cx43 linked with increased susceptibility to hypokalemia induced VF has been reported in diabetic rats [17]. A peptide, ZP123 has been shown to increase gap junction conductance and prevent re-entrant ventricular tachycardia in a myocardial ischemia dog model [18]. In addition, ZP123 decreased defibrillation
Mol Cell Biochem (2014) 391:259–266 Sham
A
8-minVF 12-minVF 30-minVF
Sham
B
8-minVF 12-minVF 30-minVF p-Cx43
Cx43
β-actin
β-actin 1
p-Cx43/Cx43
#
*
*
0.6
**
0.4 0.2 0
C
#
0.8
Sham
8-minVF
12-minVF
Δ #
1
30-minVF
Relative value of p-Cx43
1
Relative value of Cx43
Fig. 2 Western blot analysis of Cx43 and p-Cx43 levels in the heart samples. a Cx43 levels and statistical analysis. b pCx43 levels and statistical analysis. c Analysis of p-Cx43/ Cx43 comparison. Data were shown as mean ± SEM. *P \ 0.05, **P \ 0.01 versus sham control; #P \ 0.05 versus 30-min VF; DP \ 0.05 versus 12-min VF. b-actin was loading control
263
Δ #
0.8
#
*
0.6
**
0.4 0.2 0
Sham
8-minVF
12-minVF
30-minVF
#
*
0.8
* 0.6 0.4 0.2 0
Fig. 3 Immunohistochemistry staining of MMP-2 and TIMP-2 in heart samples. Yellow staining indicated positive expression of MMP-2 and TIMP-2. They were mainly in the interstitium of the cardiomyocytes. Amplification: 940. (Color figure online)
Sham
Sham
8-minVF
12-minVF
8-min VF
30-minVF
12-min VF
30-min VF
MMP-2
TIMP-2
threshold in a VF rabbit model [19]. Furthermore, the change of phosphorylation status of Cx43 has been implicated in the regulation of the function of channel gating that determines channel conductance [20, 21]. Dephosphorylation could regulate Cx43 turnover, change channel properties and result in gap junction uncoupling [22, 23]. In present study, we first performed echocardiographic and hemodynamic measurements to ensure that every group had similar basic characteristics. VF was induced by delivering an alternating current on healthy animals. Thus, the potential confounders such as various chronic diseases
have been eliminated. Dynamic alterations in total amount, distribution, and phosphorylation status of Cx43 were observed within 30 min duration of VF. The results showed that the amount of Cx43 decreased significantly in VF groups compared with the sham control group and the decrease was correlated with the duration of VF. In addition, the distribution of Cx43 became confused and disordered in VF groups. These results were consistent with our previous findings [24]. Moreover, we observed that the level of p-Cx43 was decreased in VF groups, but it was not significant different between the 8-min VF group and the
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264 Sham
0.8
C
β-actin
β-actin
*
#
0.4
0.2
0
Sham
8-minVF
12-minVF
1.6
8-minVF 12-minVF 30-minVF TIMP-2
*
0.6
Sham
MMP-2
#
30-minVF
1
Δ #
*
0.8
#
*
0.6
**
0.4 0.2 0
Sham
8-minVF
12-minVF
30-minVF
**
1.4
MMP-2/TIMP-2
B
8-minVF 12-minVF 30-minVF
Relative value of TIMP-2
A
Relative value of MMP-2
Fig. 4 Western blot analysis of MMP-2 and TIMP-2 in heart samples. a MMP-2 expression and statistical analysis. b TIMP2 expression and statistical analysis. c Analysis of MMP-2/ TIMP-2 comparison. Data were shown as mean ± SEM. *P \ 0.05, ** P \ 0.01 versus sham control; # P \ 0.01 versus 30-min VF; DP \ 0.05 versus 12-min VF. b-actin was loading control
Mol Cell Biochem (2014) 391:259–266
1.2 1
#
Δ #
*
8-minVF
12-minVF
*
0.8 0.6 0.4 0.2 0
Sham
Fig. 5 Correlation between p-Cx43/Cx43 and MMP-2/TIMP-2
sham control group. This may imply that the phosphorylation effect is more powerful than dephosphorylation effect at the primary stage of VF. Although it had been proposed that homogeneous conduction of electrical activity can be affected by ECM [6], the role of ECM remodeling in the initiation and/or persistence of VF is also unclear. MMPs primarily degrade the components of ECM and are inhibited by TIMPs [25, 26]. MMPs and TIMPs play important roles in a variety of diseases, such as ventricular remodeling of acute myocardial infarction, chronic heart failure, hypertension, occurrence or maintenance of atrial fibrillation, and dilated cardiomyopathy [27, 28]. The imbalance between MMPs and TIMPs is involved in acute pathological or
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30-minVF
physiological processes, such as platelet aggregation, the regulation of vascular tone, inflammation, and ischemia– reperfusion injury [29–33]. In this study, we found that the level of TIMP-2 in heart samples decreased significantly in VF groups compared with sham control group, while the level of MMP-2 increased in VF groups. Consequently, the ratios of MMP2/TIMP-2 were higher in VF groups than in control. Furthermore, a remarkable correlation was observed between the ratio of p-Cx43/Cx43 and the ratio of MMP-2/TIMP-2. These results are consistent with previous study [34]. There are several limitations in the present study. The data were collected from dogs with physiological VF, which may have different properties from VF that occurs in human. In this study, we only examined the changes of the markers during VF, further investigations are needed to employ pharmacological intervention. In addition, we only studied Cx43, MMP-2, and TIMP-2, and did not investigate other isoforms of Cx, MMPs, and TIMPs. In conclusion, the alteration of Cx43 and/or p-Cx43 levels and the imbalance between MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Therefore, maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate the prognosis of VF. Acknowledgments This study was sponsored by the Natural Science Foundation of China (81270238) and supported by the Scientific Development Plan of Shandong Province of China (2012G0021850).
Mol Cell Biochem (2014) 391:259–266 Conflict of interest of interest.
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The authors declare that they have no conflict 17.
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Dynamic alterations of connexin43, matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 during ventricular fibrillation in canine Jing Wang • Jing-sha Li • Hong-zhen Liu Shao-lei Yi • Guo-ying Su • Yun Zhang • Jing-quan Zhong
•
Received: 10 January 2014 / Accepted: 28 February 2014 / Published online: 18 March 2014 Ó Springer Science+Business Media New York 2014
Abstract The aim of this study is to investigate the dynamic alterations of cardiac connexin 43 (Cx43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF) duration. In this study, thirtytwo dogs were randomly divided into sham control group, 8-min VF group, 12-min VF group, and 30-min VF group. Cx43 and phosphorylated Cx43 (p-Cx43) in tissues were detected by western blot and immunofluorescence analysis. MMP-2 and TIMP-2 were detected by western blot and immunohistochemistry analysis. The results showed that Cx43 levels in three VF groups were significantly decreased compared with sham control group. p-Cx43 levels in 12-min and 30-min VF groups were significantly reduced compared with sham control group. The ratio of p-Cx43/Cx43 was also decreased in VF groups. Compared with sham controls, no significant difference was observed between the sham control group and 8-min VF group in MMP-2 level, but MMP-2 level increased in 12-min and 30-min VF groups. The ratios of MMP-2/TIMP-2 were higher in VF groups, and were correlated with the duration of VF. A remarkable correlation was observed between the J. Wang J. Li H. Liu S. Yi G. Su Y. Zhang J. Zhong (&) Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Jinan 250012, Shandong, China e-mail: [email protected] J. Wang J. Li H. Liu S. Yi G. Su Y. Zhang J. Zhong Department of Cardiology, Qilu Hospital, Shandong University, Jinan 250012, Shandong, China J. Wang Department of Keshan Disease, Shandong Provincial Institute for Endemic Diseases Control, Jinan 250014, China
ratio of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P \ 0.01). In conclusion, the alteration of Cx43 and/or p-Cx43 levels and the imbalance of MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate prognosis of VF. Keywords Ventricular fibrillation Connexin Tissue inhibitor of metalloproteinase-2 Matrix metalloproteinase-2
Introduction Ventricular fibrillation (VF) is one of the most common causes of sudden cardiac death, and is related to abnormal impulse formation and/or circuit movement re-entry [1]. Reentry is an important electrophysiological mechanism of cardiac fibrillation in which a persisting electrical impulse reactivates an area of previously activated myocardial tissues which is no longer refractory, resulting in a circus movement of activation [2]. Nevertheless, the mechanisms involved in the initiation and persistence of re-entrant arrhythmias are still not fully elucidated. It was hypothesized that intercellular electrical coupling and communication mediated by gap junctions may determine conduction velocity, and the alterations in gap junction distribution and/or defective cell-to-cell coupling may contribute to abnormal conduction, and facilitate the occurrence of re-entrant arrhythmias such as VF [3– 5]. Gap junctions that link adjoining cardiomyocytes ensure cell-to-cell electrical coupling and direct communication via clusters of intercellular channels (connexons), which are composed of connexins. Several connexin isoforms are
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expressed in the heart that differ in conductance and connexin43 (Cx43) predominates in the ventricle [6]. The homogeneous conduction of electrical activity not only relies on cardiomyocytes integration but also is affected by extracellular matrix (ECM) among the cardiomyocytes [2]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that regulate the ECM turnover in a balance with tissue inhibitors of metalloproteinase (TIMPs) [7]. Cardiomyocytes can synthesize and release MMPs, in particular MMP-2 (gelatinase A) and MMP-9 (gelatinase B), which participate in the ventricular remodeling process [8, 9]. However, the expression patterns of MMPs and TIMPs during VF are also unclear. Therefore, in this study, we aimed to investigate the dynamic changes of cardiac Cx43, MMP-2, and TIMP-2 expression in the setting of different duration of VF, in order to reveal the possible initiation and/or persistence mechanisms of VF.
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was defined as a decrease in aortic blood pressure below 25 mm Hg and the presence of VF waveform on the ECG. Dogs in the sham control group were intubated tracheally. Untreated VF lasted for 8, 12, or 30 min in other groups. All measurements were performed by an investigator blinded to the experiment assignment. After finishing the experimental protocol, the animals were killed by infusing KCl. Transthoracic echocardiography (TTE) All animals before VF were examined by TTE (PHILIPS 7500 with a 2.5–3.5 MHz transducer) to measure the left atrium dimension (LAD), left ventricular diastolic dimensions (LVDd), and left ventricular ejection fraction (LVEF), respectively. All of the above examinations were performed in a parasternal long-axis and M-mode view. Hemodynamic measurement
Materials and methods Animal preparation All experimental protocols were approved by the Animal Care and Use Committee of Qilu Hospital, Shandong University, China. Thirty-two mongreal dogs (obtained from the Center for Experimental Animals of Qilu Hospital of Shandong University) of either sex (weight 11.5–17 kg) were randomly divided into four groups: sham control group (n = 8), 8-min VF group (n = 8), 12-min VF group (n = 8), and 30-min VF group (n = 8). The animals were anesthetized via intravenous injection of pentobarbital-Na (30 mg/kg), then placed in the supine position on the table and restrained at the four extremities. A 5.0 cuffed tracheal tube was inserted into the trachea for intubation. The tube was attached to a ventilator (Newport E-100 M, Newport Medical Instruments, Costa Mesa, CA, USA). Ventilation began at the tidal volume of 10–15 ml/kg, ventilator rate of 16–20 breaths/min, and inspiration to expiration ratio of 1:1.5–1:2.0. Three surface electrodes were placed under three limbs separately for standard lead II electrocardiogram (ECG). The right femoral artery and bilateral femoral veins were cannulated. 6-F catheters (Cordis Corp., Miami, FL, USA) were positioned in the intrathoracic ascending aorta and the right atrium under fluoroscopic guidance. The remaining cannulated femoral vein was used for drug infusion. Heparin was administered at 100 U/kg for anticoagulation, and added when necessary. Creation of VF model VF was induced by delivering a 5-s alternating current at 80 V across the thorax through two needles. Successful VF
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Catheter positions were confirmed by X-ray fluoroscopy. Both catheters in the intrathoracic ascending aorta and the right atrium were connected to the pressure transducers attached to a PRO EP recording system (PowerLab/16sp, AD Instruments, Australia), by which aortic systolic pressure (AOSP), diastolic aortic pressure (AODP), and right atrial pressure (RAP) could be recorded. Coronary perfusion pressure (CPP) was calculated as AODP minus RAP and mean aortic pressure (MAP) was calculated as 1/3 AOSP plus 2/3AODP. Tissue collection Tissue samples of the left ventricular myocardium were collected within 15 s following the death. Then, the samples were immediately snap-frozen in liquid nitrogen (stored at -80 °C) to minimize time-dependent effects of myocardial ischemia on the changes of connexins, MMPs and TIMPs. Western blot analysis Tissue samples (20 mg) were homogenized in 200 ll of icecold lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 15,000 r/min for 10 min at 4 °C. Supernatants were collected. Equal amounts of protein from each sample were subjected to SDS-PAGE (10 % polyacrylamide) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked for 2 h with 1 % bovine serum albumin, then incubated overnight at 4 °C with the antibodies, including rabbit polyclonal anti-Cx43, anti-pCx43 (Uscnlife, Wuhan, China), anti-MMP-2, anti-TIMP-2
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(Santa Cruz CA, USA), and b-actin (Sigma, USA). After being washed, the membrane was incubated with the corresponding secondary antibody (Santa Cruz, CA, USA) conjugated to horseradish peroxidase (HRP). Immunoreactive bands were visualized with the SuperSignal West Pico enhanced chemiluminescence kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. b-actin was used as loading control. Band intensities were quantified with Quantity One (Bio-Rad Laboratories, Inc).
overnight with primary antibody for MMP-2 or TIMP-2 (Santa Cruz, CA, USA) followed by incubation with HRPconjugated secondary antibody (1:500 dilution). Nuclei were counterstained with hematoxylin. For negative controls, PBS solution was used instead of primary antibody. The images were analyzed by Image-Pro plus 6.0 software (Media Cybernetics, Inc).
Immunofluorescence and confocal laser scanning microscopy
Data were presented as mean ± SEM and analyzed by SPSS13 software. All variables had normal distribution and were analyzed by ANOVA analysis. P \ 0.05 was considered significant.
Left ventricular preparations were fixed in 4 % paraformaldehyde for 24 h. Tissue samples were embedded with paraffin, cut into 4 lm sections, mounted on gelatin-coated slides, then dewaxed and rehydrated with graded alcohols. The slides were microwaved in boiling 0.01 M sodium citrate buffer for 15 min, then blocked in goat serum/PBS (1:20) solution for 20 min at room temperature. Next, the slides were incubated with primary antibody for Cx43 and p-Cx43 (1:100 dilutions in PBS) overnight at 4 °C. Finally, the slides were incubated with isothiocyanate (FITC)-conjugated secondary antibody (1:500 dilution) for 30 min at 37 °C. For negative controls, PBS solution was used instead of primary antibody. Samples were examined by laser scanning confocal microscope (TCS SP2, Leica, Germany). High-intensity signal was measured and analyzed with Image-Pro plus 6.0 software (Media Cybernetics, Inc).
Statistical analysis
Results Baseline characteristics of different groups Among 32 dogs employed in this study, one dog in the sham control group was excluded because of death during catheterization, and one dog in 30-min VF group died during VF. There were no significant differences among the animals in each group in body weight, heart rate, LAD, LVDd, LVEF, and hemodynamic variables at baseline (Table 1).
Immunohistochemistry staining
The expression of Cx43 and p-Cx43 in hearts of different groups
Five micrometer ventricular tissue sections were rehydrated and blocked with 3 % hydrogen peroxide followed by incubation in 20 % (v/v) normal goat serum (Santa Cruz, CA, USA). Tissue sections were incubated at 4 °C
Confocal microscopy showed that with the duration of VF, Cx43, and p-Cx43 signals became weaker and distributed in heterogeneity. The percentage area and the integral optical density (IOD) of Cx43 and p-Cx43 in VF groups
Table 1 Baseline characteristics of experimental groups
Contents
Sham group
8-minVF group
12-minVF group
30-minVF group
Subjects
8
8
8
8
Weight (kg)
13.36 ± 1.69
13.43 ± 1.72
13.01 ± 1.75
13.26 ± 1.67
Heart (bpm)
156 ± 9
154 ± 13
157 ± 11
150 ± 10
Echocardiography bpm beat per minute, LAD left atrial diameter, LVDd left ventricle diastolic diameter, LVEF left ventricular ejection fraction, AOSP systolic aortic pressure, AODP diastolic aortic pressure, MAP mean aortic pressure, RAP right atrial pressure, CPP coronary perfusion pressure Values were expressed as mean ± SEM
LAD (mm)
16.73 ± 0.83
15.92 ± 1.47
17.28 ± 1.09
16.83 ± 1.26
LVDd (mm)
24.83 ± 1.90
25.27 ± 1.75
24.36 ± 1.43
25.32 ± 1.69
0.67 ± 0.08
0.68 ± 0.06
0.69 ± 0.09
0.69 ± 0.07
LVEF Hemodynamics AOSP (mmHg)
137.67 ± 17.34
133.73 ± 16.96
135.12 ± 19.52
136.31 ± 18.43
AODP (mmHg)
109.47 ± 15.37
102.39 ± 24.16
114.15 ± 17.84
116.24 ± 16.27
MAP (mmHg)
118.87 ± 16.28
112.84 ± 19.62
121.14 ± 18.73
122.93 ± 17.53
RAP (mmHg)
8.26 ± 3.67
7.72 ± 2.68
7.46 ± 3.25
6.95 ± 2.37
CPP (mmHg)
101.21 ± 11.71
94.67 ± 21.48
106.69 ± 14.59
109.29 ± 14.25
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262 Fig. 1 Immunofluorescence analysis of Cx43 and p-Cx43 distribution in heart samples. With the duration of VF, Cx43, and p-Cx43 signals became weaker and distributed in heterogeneity. (Color figure online)
Mol Cell Biochem (2014) 391:259–266
Sham
8-min VF
12-min VF
30-min VF
Cx43
p-Cx43
were significantly decreased compared to the sham control group (Fig. 1). Western blot analysis further confirmed that compared to the sham control group, Cx43 level in the heart was significantly decreased in three VF groups and Cx43 level declined with the duration of VF (0.83 ± 0.04 vs. 0.71 ± 0.06 vs. 0.66 ± 0.05 vs. 0.52 ± 0.07, P \ 0.05) (Fig. 2a). p-Cx43 levels in 12-min VF group and 30-min VF group were significantly reduced (0.76 ± 0.07 vs. 0.56 ± 0.05 vs. 0.41 ± 0.03, P \ 0.05), while there was no significant difference between the sham control group and 8-min VF group (0.76 ± 0.07 vs. 0.69 ± 0.05, P [ 0.05) (Fig. 2b). In addition, the ratio of p-Cx43/Cx43 was decreased with the duration of VF (0.87 ± 0.03 vs. 0.85 ± 0.04 vs. 0.76 ± 0.06 vs. 0.64 ± 0.04) (Fig. 2c). The expression of MMP-2 and TIMP-2 in hearts of different groups Compared with sham controls, dogs under VF showed significantly decreased levels of TIMP-2 in the hearts, and the decline of TIMP-2 level was correlated to the duration of VF (0.86 ± 0.08 vs. 0.75 ± 0.06 vs. 0.68 ± 0.04 vs. 0.51 ± 0.06, P \ 0.05) (Figs. 3, 4). MMP-2 levels showed no significant difference between the sham control group and the 8-min VF group (0.51 ± 0.03 vs. 0.54 ± 0.04, P [ 0.05) (Fig. 4a), however, MMP-2 levels were increased in other two longer-duration VF groups (0.59 ± 0.07 and 0.71 ± 0.05, P \ 0.05 compared to sham control group) (Fig. 4b). The ratios of MMP-2/TIMP-2 were higher in VF groups, and increased gradually with the duration of VF (0.64 ± 0.08, 0.85 ± 0.09, 1.12 ± 0.07 and 1.39 ± 0.09 in four groups, respectively) (Fig. 4c).
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Correlation between p-Cx43/Cx43 and MMP-2/TIMP-2 To investigate the correlation between p-Cx43/Cx43 and MMP-2/TIMP-2, we performed correlation analysis. The results showed a remarkable correlation between the ratios of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P \ 0.01; Fig. 5).
Discussion Since the mechanisms implicated in the initiation and progression of VF are still controversial, fundamental knowledge about cellular basis of arrhythmia is crucial for the development of novel and effective approaches for the prevention and therapy of heart diseases. Cx43 is the predominant ventricular gap junction channel protein [4, 10]. Numerous studies have demonstrated that gating properties of Cx43-formed channels (hemichannels, gap junctions) play an essential role in normal rapid electrical signal propagation and cardiac muscle synchronization [10–12]. In turn, alterations of Cx43 expression are implicated in the development of ventricular tachyarrhythmias [13, 14]. Previous studies have shown that Cx43-deficient mice exhibited markedly slow myocardial conduction that facilitates re-entrant arrhythmias and sudden arrhythmic death [15, 16]. Down-regulation and/or abnormal distribution of myocardial Cx43 linked with increased susceptibility to hypokalemia induced VF has been reported in diabetic rats [17]. A peptide, ZP123 has been shown to increase gap junction conductance and prevent re-entrant ventricular tachycardia in a myocardial ischemia dog model [18]. In addition, ZP123 decreased defibrillation
Mol Cell Biochem (2014) 391:259–266 Sham
A
8-minVF 12-minVF 30-minVF
Sham
B
8-minVF 12-minVF 30-minVF p-Cx43
Cx43
β-actin
β-actin 1
p-Cx43/Cx43
#
*
*
0.6
**
0.4 0.2 0
C
#
0.8
Sham
8-minVF
12-minVF
Δ #
1
30-minVF
Relative value of p-Cx43
1
Relative value of Cx43
Fig. 2 Western blot analysis of Cx43 and p-Cx43 levels in the heart samples. a Cx43 levels and statistical analysis. b pCx43 levels and statistical analysis. c Analysis of p-Cx43/ Cx43 comparison. Data were shown as mean ± SEM. *P \ 0.05, **P \ 0.01 versus sham control; #P \ 0.05 versus 30-min VF; DP \ 0.05 versus 12-min VF. b-actin was loading control
263
Δ #
0.8
#
*
0.6
**
0.4 0.2 0
Sham
8-minVF
12-minVF
30-minVF
#
*
0.8
* 0.6 0.4 0.2 0
Fig. 3 Immunohistochemistry staining of MMP-2 and TIMP-2 in heart samples. Yellow staining indicated positive expression of MMP-2 and TIMP-2. They were mainly in the interstitium of the cardiomyocytes. Amplification: 940. (Color figure online)
Sham
Sham
8-minVF
12-minVF
8-min VF
30-minVF
12-min VF
30-min VF
MMP-2
TIMP-2
threshold in a VF rabbit model [19]. Furthermore, the change of phosphorylation status of Cx43 has been implicated in the regulation of the function of channel gating that determines channel conductance [20, 21]. Dephosphorylation could regulate Cx43 turnover, change channel properties and result in gap junction uncoupling [22, 23]. In present study, we first performed echocardiographic and hemodynamic measurements to ensure that every group had similar basic characteristics. VF was induced by delivering an alternating current on healthy animals. Thus, the potential confounders such as various chronic diseases
have been eliminated. Dynamic alterations in total amount, distribution, and phosphorylation status of Cx43 were observed within 30 min duration of VF. The results showed that the amount of Cx43 decreased significantly in VF groups compared with the sham control group and the decrease was correlated with the duration of VF. In addition, the distribution of Cx43 became confused and disordered in VF groups. These results were consistent with our previous findings [24]. Moreover, we observed that the level of p-Cx43 was decreased in VF groups, but it was not significant different between the 8-min VF group and the
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264 Sham
0.8
C
β-actin
β-actin
*
#
0.4
0.2
0
Sham
8-minVF
12-minVF
1.6
8-minVF 12-minVF 30-minVF TIMP-2
*
0.6
Sham
MMP-2
#
30-minVF
1
Δ #
*
0.8
#
*
0.6
**
0.4 0.2 0
Sham
8-minVF
12-minVF
30-minVF
**
1.4
MMP-2/TIMP-2
B
8-minVF 12-minVF 30-minVF
Relative value of TIMP-2
A
Relative value of MMP-2
Fig. 4 Western blot analysis of MMP-2 and TIMP-2 in heart samples. a MMP-2 expression and statistical analysis. b TIMP2 expression and statistical analysis. c Analysis of MMP-2/ TIMP-2 comparison. Data were shown as mean ± SEM. *P \ 0.05, ** P \ 0.01 versus sham control; # P \ 0.01 versus 30-min VF; DP \ 0.05 versus 12-min VF. b-actin was loading control
Mol Cell Biochem (2014) 391:259–266
1.2 1
#
Δ #
*
8-minVF
12-minVF
*
0.8 0.6 0.4 0.2 0
Sham
Fig. 5 Correlation between p-Cx43/Cx43 and MMP-2/TIMP-2
sham control group. This may imply that the phosphorylation effect is more powerful than dephosphorylation effect at the primary stage of VF. Although it had been proposed that homogeneous conduction of electrical activity can be affected by ECM [6], the role of ECM remodeling in the initiation and/or persistence of VF is also unclear. MMPs primarily degrade the components of ECM and are inhibited by TIMPs [25, 26]. MMPs and TIMPs play important roles in a variety of diseases, such as ventricular remodeling of acute myocardial infarction, chronic heart failure, hypertension, occurrence or maintenance of atrial fibrillation, and dilated cardiomyopathy [27, 28]. The imbalance between MMPs and TIMPs is involved in acute pathological or
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30-minVF
physiological processes, such as platelet aggregation, the regulation of vascular tone, inflammation, and ischemia– reperfusion injury [29–33]. In this study, we found that the level of TIMP-2 in heart samples decreased significantly in VF groups compared with sham control group, while the level of MMP-2 increased in VF groups. Consequently, the ratios of MMP2/TIMP-2 were higher in VF groups than in control. Furthermore, a remarkable correlation was observed between the ratio of p-Cx43/Cx43 and the ratio of MMP-2/TIMP-2. These results are consistent with previous study [34]. There are several limitations in the present study. The data were collected from dogs with physiological VF, which may have different properties from VF that occurs in human. In this study, we only examined the changes of the markers during VF, further investigations are needed to employ pharmacological intervention. In addition, we only studied Cx43, MMP-2, and TIMP-2, and did not investigate other isoforms of Cx, MMPs, and TIMPs. In conclusion, the alteration of Cx43 and/or p-Cx43 levels and the imbalance between MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Therefore, maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate the prognosis of VF. Acknowledgments This study was sponsored by the Natural Science Foundation of China (81270238) and supported by the Scientific Development Plan of Shandong Province of China (2012G0021850).
Mol Cell Biochem (2014) 391:259–266 Conflict of interest of interest.
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The authors declare that they have no conflict 17.
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