Calcineurin B subunit acts as a potential agent for ... - Springer Link

Mol Cell Biochem (2012) 370:163–171 DOI 10.1007/s11010-012-1407-7

Calcineurin B subunit acts as a potential agent for preventing cardiac ischemia/reperfusion injury Junxia Guo • Shengquan Mi • Jing Li Wei Liu • Yanxia Yin • Qun Wei

•

Received: 18 March 2012 / Accepted: 25 July 2012 / Published online: 28 August 2012 Ó Springer Science+Business Media, LLC. 2012

Abstract Calcineurin B subunit (CnB) is the regulatory subunit of calcineurin (Cn), a Ca2?/calmodulin-dependent serine/threonine protein phosphatase. It has been reported that mice deleting the CnB gene lose nearly all Cn activity and show poor tolerance to cardiac stress; CnB gene expression is downregulated in the hearts of rats that have suffered ischemia/reperfusion (I/R) injury. Therefore, we wonder whether injection of exogenous CnB protein can prevent the rats from suffering I/R injury. In cardiomyocytes, fluorogenic labeling shows that exogenous CnB quickly enters the cell. Pretreatment of cardiomyocytes with CnB reduces apoptosis in response to hypoxia/ reoxygenation injury (an in vitro model mimicking ischemia/reperfusion injury), and CsA reverses this effect by inhibiting Cn activity. Furthermore, CnB upregulates Bcl-2 and Bcl-XL expression in the process of hypoxia/reoxygenation injury, which may contribute to protecting cardiomyocytes against apoptosis. In vivo experiments shows that pretreatment with CnB improves cardiac contractile function and reduces the frequency of arrhythmias induced by global I/R injury. These findings reveal a novel function for CnB protein in cardiac stress response and suggest a possible application of CnB in coronary disease therapy.

J. Guo J. Li W. Liu Y. Yin Q. Wei (&) Department of Biochemistry and Molecular Biology, Beijing Key Laboratory of Genetic Engineering Medicine and Biotechnology, Beijing Normal University, Beijing 100875, China e-mail: [email protected] J. Guo S. Mi Beijing Key Laboratory of Bioactive Substances and Functional Foods, College of Arts and Science of Beijing Union University, Beijing 100191, China

Keywords Calcineurin CnB Cardiomyocyte Ischemia/reperfusion injury

Introduction Despite recent advances in medical science, coronary heart disease remains a major health problem worldwide, and therapeutic interventions aimed at ameliorating the myocardial consequences of ischemia–reperfusion (I/R) are being intensively explored. There is increasing evidence that cardiomyocyte injury and death following I/R injury, which can result from apoptosis, is an important cause of morbidity and mortality [1]. Ischemic preconditioning is a well-established phenomenon that brief ischemia/reperfusion applied prior to a longer coronary artery occlusion effectively reduces myocardial infarct size [2, 3]. Preconditioning mimetic agents that stimulate the biochemical pathways of ischemic preconditioning and protect the heart without inducing ischemia have been developed in recent years. These agents include lycopene [4], wild garlic [5], adenosine, adenosine agonists, the KATP channel/opener and nitrate-like agent nicorandil, delta opioids, volatile anesthetics, and nitroglycerin [6]. Calcineurin (Cn) is a calcium–calmodulin activated serine–threonine protein phosphatase. It is essential for a number of signal transduction pathways [7], including those involved in the development of T lymphocytes and the nervous system, fiber-type switching in skeletal muscle, development of vascular and heart valves and of bone, and the control of cardiovascular disease [8–11]. It consists of a 61 kDa catalytic subunit (CnA) and a 19 kDa regulatory subunit (CnB). CnB functions in signal transduction pathways by up-regulating the phosphatase activity of CnA [12], and knock-out of the CnB gene in mice results in loss

123

164

of nearly all calcineurin activity [13]. Moreover, in recent years, it has been reported that CnB has some new functions such as activation of innate immunity [14–16], antiplatelet aggregation [17], regulation of proteasome pathway [18]. Recent studies have indicated that mice lacking the CnB1 gene lose nearly all Cn activity and show poor tolerance to cardiac stress, which increases cardiomyocyte apoptosis and can be lethal [13]. The data of microarrays have shown that CnB expression is downregulated in the hearts of old rats that have suffered I/R injury [19]. These findings suggest that CnB may play an important role in sustaining cardiac functions in cardiac stress. In this study, we investigated whether exogenous CnB protein shared a protective effect on hypoxia/reoxygenation (H/R) injury in vitro and ischemia/reperfusion injury in vivo and further explored the probable molecular mechanism of the process. The construction of CnB vector and the expression and the purification of CnB have been described previously [20, 21].

Materials and methods Cells culture Primary neonatal rat cardiomyocytes were prepared from hearts of 1- to 2-day-old Sprague–Dawley rat pups. The hearts were collected, atria were removed, and the ventricles were cut into pieces and subjected to enzymatic digestion with 0.08 % pancreatin (GIBCO, Sigma). The cells were differentially plated for 1.5 h to remove contaminating nonmyocytes and plated on gelatinized cell culture dishes in DMEM medium supplemented with 10 % fetal bovine serum, penicillin/streptomycin (100 U/ml), and L-glutamine (2 mmol/l) for 48 h. Then they were transferred to DMEM medium with 5 % serum to synchronize cell beat and cultured overnight in DMEM without serum before being exposed to H/R injury. H9C2 rat heart myoblasts were cultured in DMEM medium supplemented with 10 % fetal bovine serum. They were then grown in serum-free DMEM medium overnight and exposed to H/R injury. H/R injury Cells were switched to serum/glucose-free DMEM and exposed to hypoxic conditions (H) for 2 h using an AnaeroPack pouch (Mitsubishi Gas Chemical), in which the oxygen concentration was\2 % at the end of the exposure. Reoxygenation (R) was achieved by returning cells to normal DMEM containing 10 % serum, followed by incubation at 37 °C in a humidified atmosphere containing

123

Mol Cell Biochem (2012) 370:163–171

95 % air and 5 % CO2 for different periods, as indicated in the text. Assessment of apoptosis in cultured cardiomyocytes Apoptosis-positive cells were detected by Annexin V-EGFP/PI (Propidium Iodide) and Hoechst/PI staining according to the manufacturer’s instructions. In brief, cells were incubated for 15–30 min at 37 °C in medium containing appropriate concentrations of Annexin V/PI and Hoechst/PI, respectively, followed by analysis in a Becton– Dickinson FACScalibur cytofluorometer and by epifluorescence microscopy. Calcineurin phosphatase activity analysis Cellular calcineurin phosphatase activity was measured in cell extracts. In brief, cell extracts were obtained in lysis buffer (50 mM Tris, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.2 % NP-40, 50 lg/ml soybean trypsin inhibitor, 10 lg/ml leupeptin, and 10 lg/ml antipain). Total phosphatase activity due to calcineurin was determined as phosphate released from RII in the presence of okadaic acid minus phosphate released in the presence of okadaic acid plus EGTA, based on the malachite green dye reaction. Colorimetric measurements were performed at 620 nm on a microplate reader (BIO-RAD Model 860). Protein tracing Fluorescently labeled protein was traced in live cells by confocal microscopy (Olympus FV300). Cardiomyocytes were maintained in DMEM with *1 lM fluorescently labeled CnB-RBITC (Fig. 3b) or RBITC (Fig. 3a) at 37 °C for 20 min and fixed with formaldehyde. Quantitative RT-PCR Total RNA was isolated from cultured cells by the TRIzol method (BioTeke), and cDNA was obtained from purified RNA using oligo(dT) priming and M-MLV reverse transcriptase (Takara).Transcription of selected genes was quantified on an ABI PRISM 7500 (Applied Biosystems Inc.) Expression levels were determined from threshold cycle numbers (CT) using the DD CT method, and normalized against GAPDH. Western blotting Extracts were prepared in ice-cold RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 0.1 mM EDTA, 0.1 mM sodium orthovanadate, 10 mM sodium fluoride). 60 lg


samples of protein were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked for 1 h at room temperature in buffer containing 5 % skim milk and incubated overnight at 4 °C with appropriate primary antibody. The blots were then incubated with HRP-conjugated secondary antibody and washed again. Primary antibodies for blotting included caspase-3 from Cell Signaling Technology, Bcl-2 from BD Pharmingen, and b-actin from Biosynthesis Biotechnology. Preparation of isolated Langendorff-perfused rat hearts and I/R injury analysis Male Sprague–Dawley rats, body weight 300–350 g (Vital River Laboratory Animal Technology Co. Ltd), were acclimated in a quarantine room and maintained on a standard diet for a week before experiments. Rats were injected once with 1.0 mg/kg CnB protein (dissolved in normal saline, i.p.) or with normal saline (1.0 ml i.p.). Two days after injection, hearts were isolated and subjected to normothermic global I/R. The rats were anesthetized with pentobarbital Na (45 mg/kg, i.p.) and beating hearts were rapidly excised into oxygenated Krebs–Henseleit solution containing (in mmol/l) 5.5 glucose, 1.2 CaCl2, 4.7 KCl, 25 NaHCO3, 119 NaCl, 1.17 MgSO4, and 1.18 KH2PO4 (pH 7.4). Normothermic retrograde perfusion was performed with the Langendorff technique and perfusion pressure was maintained at 70 mmHg. A latex balloon was inserted through the left atrium into the left ventricle, and the balloon was filled with water (0.18–0.28 ml) to achieve an LVEDP (left ventricular end-diastolic pressure) of 5–10 mmHg. LVDP (left ventricular developed pressure) and LVEDP were continuously recorded with a computerized pressure amplifier-digitizer and rates of LVP rise (?dP/dt) and fall (-dP/dt) were recorded. Electrocardiograms (ECG) were monitored by means of three silver electrodes, two being inserted into the heart chamber and connected to an amplifier, and the third clipped to the cannula. Data were transferred to a Data Acquisition System (BIOPAC, MP150WS, USA). All parameters were recorded continuously during experiments. A three-way stopcock was mounted above the aortic cannula to create global ischemia. After 15 min of perfusion (equilibration), hearts were subjected to 20 min of normothermic global ischemia followed by 40 min of reperfusion. Statistical analysis Data are expressed as mean ± SE. Statistical differences between groups were calculated by two-tailed t test (in vitro experiments) and the Bonferroni/Dunnett test (animal

165

experiments). significant.

P \ 0.05

was

considered

statistically

Results Exogenous CnB enters cardiomyocytes quickly To investigate whether exogenous CnB enters cardiomyocytes so as to affect cellular signaling pathways, CnB protein conjugated with Rhodamine B isothiocyanate (CnB-RBITC) was used as a probe and added directly to the H9C2 cells or isolated neonatal rat cardiomyocytes culture medium for 20 min. Confocal imaging showed that the cytoplasm was efficiently stained by the CnB-RBITC protein probe (Fig. 1b, c). This result demonstrates that CnB can cross the plasma membrane into cardiomyocytes and hence is able to interact with biomolecules in the cytoplasm. Pretreatment with CnB markedly reduces H/R-induced apoptosis in cultured cardiomyocytes Here, we used the response to H/R in vitro to simulate I/R injury in vivo. Usually, apoptosis is more pronounced during reperfusion or reoxygenation than during ischemia or hypoxia [1, 22]. To determine the effects of CnB on H/Rinduced apoptosis, H9C2 cells or neonatal rat cardiomyocytes were treated with CnB (2.6 lM) alone or CnB plus CsA (1.5 lM) in serum-free medium for 18 h followed by H/R treatment. Under our experimental conditions, H/R markedly increased the number of apoptotic cells, as reflected in condensed Hoechst/PI stained nuclei (Fig. 2a) and percent of Annexin V-positive cells (Fig. 2b, c). Pretreatment with CnB significantly reduced H/R-induced apoptosis while inhibition of Cn with CsA partially reversed the effects of CnB. To confirm the inhibitory effect of CnB on cardiomyocytes apoptosis, we analyzed caspase-3 precursor and cleaved caspase-3 by Western blotting to assess whether CnB inhibited activation of the caspase cascade. Caspase-3 is expressed as an inactive precursor from which the p17 and p12 subunits of the active caspase-3 are proteolytically generated during apoptosis [23]. As illustrated in Fig. 2d, H/R treatment decreased the level of caspase-3 precursor as well as the level of cleaved caspase-3 in H9C2 cells; CnB inhibited this decrease and CsA reversed the effect of CnB. Taken together, these findings demonstrate that CnB has an antiapoptotic effect on H/Rinduced apoptosis in cardiomyocytes. CnB blocks the decrease of calcineurin activity after H/ R in cardiomyocytes We further investigated if the CnB-mediated antiapoptotic effect is due to its ability to activate calcineurin. As

123

166


Fig. 1 Exogenous CnB enters cardiomyocytes quickly. Confocal microscopy images show that CnB-RBITC (b) but not RBITC (a) enters the H9C2 cardiomyocytes after incubation for 20 min at

37 °C. CnB-RBITC, 1 lM. c CnB-RBITC enters neonatal rat cardiomyocytes primary culture cells after incubation for 20 min at 37 °C

illustrated in Fig. 3, CnB blocked the decrease of calcineurin activity induced by H/R treatment and CsA reversed this inhibition. This suggests that CnB exerts an antiapoptotic effect on cardiomyocytes exposed to H/R injury by activating calcineurin activity.

Pretreatment with CnB promotes the recovery of cardiac contractile function and reduces arrhythmias after I/R injury

CnB upregulates the expression of Bcl-2 family proteins after H/R The Bcl-2 family of proteins is crucial for apoptosis and is induced by various stimuli [24, 25]. To determine whether stimulation of cardiomyocytes with CnB induces the expression of Bcl-2 family proteins, and, if so, whether the Cn pathway is involved in this process, cardiomyocytes were treated with CnB in the presence or the absence of CsA prior to H/R injury. As shown in Fig. 4a, b, d, CnB induced a higher level of expression of Bcl-2 and Bcl-XL in cardiomyocytes than H/R alone, and blockade of Cn activation by CsA inhibited this induction. Bax expression was not obviously affected by CnB (Fig. 4c). These results indicate that Cn activation by CnB is required for induction of the expression of the antiapoptotic molecules, Bcl-2 and Bcl-XL.

123

We first examined the direct effects of CnB on I/R hearts. Rats were injected with CnB (1.0 mg/kg) or normal saline for 2 days before I/R treatment. After I/R, cardiac function was markedly decreased and LVEDP, LVDP, ?dP/dtmax, and -dP/dtmax was significantly improved in the hearts of rats pretreated with CnB (Fig. 5a–d). In particular, the left ventricular diastolic end pressure (LVEDP) of the heart during reperfusion was reduced to 24.3 ± 8.2 mmHg in the CnB group, compared with 53.9 ± 15.1 mmHg in the control group (P \ 0.05) (Fig. 5a). These findings suggest that pretreatment with CnB before ischemia increases the recovery of cardiac contractile function after reperfusion. To evaluate the influence of CnB pretreatment on subsequent I/R-induced arrhythmias, we monitored the arrhythmias on ECG during periods of ischemia and reperfusion. ECGs were weak during ischemia, and 5 of 10 hearts stopped beating in different phases after reperfusion in the control group, whereas only one of six hearts stopped beating in the CnB group. The arrhythmias manifested as


A

a

167

b

c

B Apoptosis cell %

**

***

100 80 60 40 20 0 H/R injury

Apoptosis cell %

80

CnB+CsA

*

**

60

40

Caspase-3 precuror / -actin 1.5

Relative Intensity

C

CnB

20

0 H/R injury

CnB

CnB+CsA

D

1.0

0.5

0.0 H/R injury CnB CsA

-

Relative Intensity

20

+

+

+

-

+

+

-

+

Cleaved caspase-3 /

-actin

15 10 5 0

H/R injury CnB CsA

Fig. 2 Pretreatment with CnB markedly reduces H/R-induced apoptosis in cultured neonatal rat cardiomyocytes or H9C2 cells. Neonatal rat cardiomyocytes or H9C2 cells were exposed to H/R injury in the presence or the absence of CnB (2.6 lM) or CsA (1.5 lM) and subjected to apoptosis detections. a Neonatal rat cardiomyocytes were staining with Hoechst 33342 (blue) and PI (red). Normal cells (a), H/R control cells (b) and CnB treated cells (c) are illustrated in Fig. 2a. The insets show cells with evidence of apoptosis, including Hoechst-positive staining and chromatin condensation. b Neonatal rat cardiomyocytes apoptosis was assayed by Annexin V and PI staining.

-

+

+

+

-

+

+

-

+

The x axis is Annexin V fluorescence and the y axis PI fluorescence. The right graph is the quantitative analysis of the left graph. Annexin V-positive cells were counted and are expressed as percentages of total cells. c H9C2 cells apoptosis was assayed by Annexin V and PI staining. d Western blot analysis of caspase-3 precursor and cleaved caspase-3. Band intensities were quantified with Bandscan software (right two graphs). *P \ 0.05, **P \ 0.01, ***P \ 0.001. This figure is one representative result of three independent experiments. (Color figure online)

123

168


**

g protein) (PO 4 nmol/

CnA activity

1.0

**

0.8

**

0.6 0.4 0.2 0.0

H/R injury CnB CsA

-

+

+

+

-

+

+

-

+

Fig. 3 CnB blocks the decrease of calcineurin activity after H/R in H9C2 cardiomyocytes. H9C2 cardiomyocytes were incubated with or without exogenous CnB (2.6 lM) for 18 h followed by H/R treatment, and then calcineurin activity was analyzed. *P \ 0.05, **P \ 0.01, ***P \ 0.001. This figure is one representative result of three independent experiments

premature ventricular contraction (VPC), ventricular tachycardia, and ventricular fibrillation during reperfusion, and are described in Table 1. The results demonstrate that arrhythmia was reduced in the hearts of the CnB pretreatment group.

Discussion Accumulating evidence suggests that cardiomyocyte apoptosis is the central mechanism of cell death in hearts undergoing I/R in vivo and in vitro [26]. Because adult cardiac myocytes have lost the capacity to proliferate, survival of cardiac cells is critical for recovery of cardiac function. Whether calcineurin activation is potentially protective or an essentially deleterious factor in cardiovascular disease has long been disputed. In the present study, we have demonstrated that pretreatment with CnB, the native regulatory subunit of calcineurin, improves cardiac function and reduces apoptosis by upregulating calcineurin activity after ischemia/reperfusion injury. In isolated perfused rat hearts, CnB improved cardiac contractive function and reduced the frequency of arrhythmias induced by global ischemia/reperfusion injury. Consistent with our proposal, CnB-deleted mice have impaired cardiac function and are highly sensitive to cardiac stress, which results in complete lethality after pressure overload. Arrhythmias with few periods of stable rhythm were also detected [13], and calcineurin Ab knockout mice also exhibit similar effects [27]. These findings appear to support our proposal that CnB

123

supplementation protects cardiomyocytes from I/R-induced injury. We also found that: first, exogenous CnB entered cultured cardiomyocytes rapidly; second, pretreatment with CnB clearly prevented apoptosis in response to H/R injury and CsA reversed this effect; third, calcineurin activity increased after CnB pretreatment and this effect was reversed by CsA. These results suggest that exogenous CnB activates cellular calcineurin and protects cardiomyocytes from apoptosis. Other studies have also shown that calcineurin activation exerts a potent inhibitory effect on apoptosis in cardiomyocytes [27–30]. For example, in cultured cardiomyocytes, calcineurin activation conferred protection against H2O2-, staurosporine- and 2-deoxyglucose-induced apoptosis [29, 30]. Calcineurin inhibition has also been reported to have adverse effects on cardiomyocyte apoptosis [28]. Thus, CnB-deleted mice lost calcineurin activity and displayed cardiac dysfunction, and this was associated with increased apoptosis [13]. The behavior of these mice was very similar to that of calcineurin Ab knockout mice [27]. These findings are consistent with the idea that CnB has a potent protective effect on cardiomyocyte apoptosis. A likely interpretation is that CnB, as the native regulatory subunit, upregulates calcineurin activity. We speculate that exogenous CnB enters the cells and activates calcineurin, which then prevents the progression of apoptosis. On the other hand, a proapoptotic effect of calcineurin has also been reported in cardiomyocytes [31, 32]. For example, Saito et al. [31] reported that isoproterenol stimulation promoted cardiomyocyte apoptosis, in part by stimulating calcineurin activity. Cyclosporin A and FK506 blocked the increase in cardiomyocyte apoptosis induced by isoproterenol stimulation and, more significantly, transgenic mice expressing a dominant-negative calcineurin in the heart were refractory to isoproterenol-induced apoptosis in vivo [31]. These results suggest that calcineurin activation is associated with enhanced apoptosis in cardiomyocytes. However, Saito et al. [31] also reported that transgenic mice expressing the dominant-negative calcineurin in the heart underwent increased apoptosis in response to ischemia/reperfusion injury, a result that supports the opposite conclusion to the hypothesis favored by their previous results, namely that calcineurin activation is cardioprotective. Similarly, calcineurin signaling has been shown to both inhibit and induce apoptosis after stress stimulation in the same cells [33]. These seemingly contradictory data underscore the complexity of intracellular signaling networks within mammalian cells, such that related stress stimuli can elicit fundamentally opposite responses. Molkentin [34] proposed that the most probable explanation for such divergent findings is that calcineurin


B

15

** 12

Bcl-XL Relative mRNA levels (fold)

Bcl-2 Relative mRNA levels (fold)

A

169

*

**

9 6 3 0

-

H/R injury CnB CsA

2.5

* 1.5 1.0 0.5 0.0

+

+

H/R injury

-

+

+

CnB

-

+

+

+

+

-

+

+

-

+

+

*

2.0

-

CsA

+

+

+

-

+

+

-

+

Bax Relative mRNA levels (fold)

C 1.5

1.0

0.5

0.0

H/R injury CnB CsA

-

1.2

Relative Intensity

D

CsA

-actin

0.9 0.6 0.3

0.0 H/R injury CnB

Bcl-2 /

-

+

+

+

-

+

+

-

+

Fig. 4 CnB upregulates the expression of Bcl-2 family proteins after H/R in H9C2 cardiomyocytes. H9C2 cardiomyocytes were pretreated with CnB (2.6 lM) or CsA (1.5 lM) prior to H/R treatment. Normoxic treatment was used as negative control. At the end of indicated time, total RNA and protein were extracted and subjected to

quantitative RT-PCR (a–c) and western blot analysis (d), respectively. Band intensities of the western blot data were quantified with Bandscan software (Fig. 4d, right graph). *P \ 0.05, **P \ 0.01, ***P \ 0.001. This figure is one representative result of three independent experiments

signaling is interpreted within the context of other signaling pathways. The Bcl-2 family proteins are essential for the program of apoptosis and consist of proapoptotic and antiapoptotic factors. Some of the proteins within this family, including Bcl-2 and Bcl-XL, inhibit apoptosis; others, such as Bax and Bak, promote apoptosis and in some instances are sufficient to cause apoptosis independent of additional signals [25, 35, 36]. Direct interactions between Bcl-2 and

calcineurin have been reported in BHK-21 cells transfected with these two proteins. Bcl-2 forms a tight complex with calcineurin, suggesting a functional role for this interaction [37]. The present study demonstrates that stimulation of cardiomyocytes with CnB increases the expression of Bcl-2 and Bcl-XL, and CsA reverses these effects. The protective effects of calcineurin signaling are functionally linked to Akt phosphorylation or p38 MAPK activation [30, 33]. Calcineurin activation affects multiple signaling pathways,

123

170


A

Normal saline i.p. + I/R

100

B 100

CnB i.p. + I/R

**

80

LVDP (mmHg)

LVEDP (mmHg)

80 60

* **

40

**

**

*

20

60 40 Normal saline i.p. + I/R CnB i.p. + I/R

20

0

0

5

10

20

30

0

40

Untouched group

0

5

Reperfusion (min)

D **

** **

1000 Normal saline i.p. + I/R CnB i.p. + I/R

500

Untouched group

0

20

30

40

Reperfusion (min)

**

0

-dp/dt (mmHg/s)

2000

1500

10

Reperfusion (min)

C

dp/dt (mmHg/s)

**

*

Untouched group

0

5

10

20

30

40

**

*

-500

*

-1000

-1500 Normal saline i.p. + I/R

0

5

10

20

30

40

CnB i.p. + I/R

Reperfusion (min)

Untouched group

Fig. 5 Pretreatment with exogenous CnB promotes the recovery of cardiac contractile function after ischemia/reperfusion injury. Rats were injected once with CnB protein (1.0 mg/kg i.p.) or normal saline (0.2 ml i.p.). Two days after injection, hearts were isolated and subjected to global ischemia/reperfusion. Untouched group was used as a negative control. LVEDP (a), LVDP (b) and ± dp/dt (c and

d) were assessed during ischemia and reperfusion. Data are expressed as mean ± SE. Statistical differences at different time points between groups were calculated by the Bonferroni/Dunnett test. Compared with normal saline group, *P \ 0.05, **P \ 0.01, ***P \ 0.001. This figure is one representative result of three independent experiments

Table 1 Incidence and duration of ventricular arrhythmia induced by I/R injury Group

Ventricular tachycardia

Ventricular fibrillation

Incidence (%)

Duration (s)

Incidence (%)

Duration (s)

Ventricular premature contraction (VPC, beats)

Normal saline i.p. ?I/R (n = 10)

50

54.5 ± 25.5

50.0

60.5 ± 23.6

97.8 ± 17.4

CnB i.p. ? I/R (n = 6)

20

5.5 ± 2.5

16.7

10.9 ± 8.6

63.6 ± 19.7

0

0

0

Untouched group (n = 6)

0

and upregulation of Bcl-2 expression may not be the only mechanism involved in the antiapoptotic effects of CnB. However, the potent ability of Bcl-2 to prevent apoptosis in cardiomyocytes suggests that this upregulation is, at least in part, involved in the protective effects of CnB. In summary, pretreatment with CnB protects against I/R injury in vivo and against apoptosis induced by H/R treatment in vitro. The discovery of this novel role of CnB raises the possibility that it may be a potent survival factor against apoptosis and a potential agent in the clinical

123

3.4 ± 1.7

treatment of coronary disease. Acute toxicity experiment indicates that mice can tolerate at least 50-fold normal dose of CnB [38]. The very low toxicity and good efficacy makes CnB an excellent candidate for coronary disease agent. Acknowledgments This work was partially supported by the National Natural Science Foundation of China, the International Cooperation Project, the National Important Novel Medicine Research Project, and the Fundamental Research Funds for the Central Universities.


References 1. Machado NG, Alves MG, Carvalho RA, Oliveira PJ (2009) Mitochondrial involvement in cardiac apoptosis during ischemia and reperfusion: can we close the box? Cardiovasc Toxicol 9:211–227 2. Ferdinandy P, Schulz R, Baxter GF (2007) Interaction of cardiovascular risk factors with myocardial ischemia/reperfusion injury, preconditioning, and postconditioning. Pharmacol Rev 59: 418–458 3. Maulik N, Yoshida T, Engelman RM, Deaton D, Flack JE (1998) Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion. Mol Cell Biochem 186:139–145 4. Bansal P, Gupta SK, Ojha SK, Nandave M, Mittal R, Kumari S, Arya DS (2006) Cardioprotective effect of lycopene in the experimental model of myocardial ischemia–reperfusion injury. Mol Cell Biochem 289:1–9 5. Rietz B, Isensee H, Strobach H, Makdessi S, Jacob R (1993) Cardioprotective actions of wild garlic (Allium ursinum) in ischemia and reperfusion. Mol Cell Biochem 119:143–150 6. Kloner RA, Rezkalla SH (2006) Preconditioning, postconditioning and their application to clinical cardiology. Cardiovasc Res 70:297–307 7. Kurji K, Sharma RK (2010) Potential role of calcineurin in pathogenic conditions. Mol Cell Biochem 338:133–141 8. Wu H, Peisley A, Graef IA, Crabtree GR (2007) NFAT signaling and the invention of vertebrates. Trends Cell Biol 17:251–260 9. Hogan PG, Chen L, Nardone J, Rao A (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17: 2205–2232 10. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228 11. Vega RB, Bassel-Duby R, Olson EN (2003) Control of cardiac growth and function by calcineurin signaling. J Biol Chem 278: 36981–36984 12. Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiol Rev 80:1483–1521 13. Maillet M, Davis J, Auger-Messier M, York A, Osinska H, Piquereau J, Lorenz JN, Robbins J, Ventura-Clapier R, Molkentin JD (2010) Heart-specific deletion of CnB1 reveals multiple mechanisms whereby calcineurin regulates cardiac growth and function. J Biol Chem 285:6716–6724 14. Liu L, Su Z, Xin S, Cheng J, Li J, Xu L, Wei Q (2012) The calcineurin B subunit (CnB) is a new ligand of integrin aM that mediates CnB-induced Apo2L/TRAIL expression in macrophages. J Immunol 188:238–247 15. Su Z, Xin S, Xu L, Cheng J, Guo J, Li L, Wei Q (2012) The calcineurin B subunit induces TNF-related apoptosis-inducing ligand (TRAIL) expression via CD11b-NF-jB pathway in RAW264.7 macrophages. Biochem Biophys Res Commun 417:777–783 16. Li J, Guo J, Su Z, Hu M, Liu W, Wei Q (2011) Calcineurin subunit B activates dendritic cells and acts as a cancer vaccine adjuvant. Int Immunol 23:327–334 17. Su Z, Xin S, Li J, Guo J, Long X, Cheng J, Wei Q (2011) A new function for the calcineurin b subunit: antiplatelet aggregation and anticoagulation. IUBMB Life 63:1037–1044 18. Li N, Zhang Z, Zhang W, Wei Q (2011) Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1a activity via the proteasome pathway. Biochem Biophys Res Commun 405:468–472 19. Simkhovich BZ, Marjoram P, Poizat C, Kedes L, Kloner RA (2003) Age-related changes of cardiac gene expression following myocardial ischemia/reperfusion. Arch Biochem Biophys 420: 268–278

171 20. Wu W, Jia Z, Liu P, Xie Z, Wei Q (2005) A novel PCR strategy for high-efficiency, automated site-directed mutagenesis. Nucleic Acids Res 33:e110 21. Jiang G, Wei Q (2003) Function and structure of N-terminal and C-terminal domains of calcineurin B subunit. Biol Chem 384: 1299–1303 22. Kang PM, Haunstetter A, Aoki H, Usheva A, Izumo S (2000) Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation. Circ Res 87:118–125 23. Han Z, Hendrickson EA, Bremner TA, Wyche JH (1997) A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. J Biol Chem 272:13432–13436 24. Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334–336 25. Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59 26. Whelan RS, Kaplinskiy V, Kitsis RN (2010) Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu Rev Physiol 72:19–44 27. Bueno OF, Lips DJ, Kaiser RA, Wilkins BJ, Dai YS, Glascock BJ, Klevitsky R, Hewett TE, Kimball TR, Aronow BJ, Doevendans PA, Molkentin JD (2004) Calcineurin Abeta gene targeting predisposes the myocardium to acute ischemia-induced apoptosis and dysfunction. Circ Res 94:91–99 28. Pu WT, Ma Q, Izumo S (2003) NFAT transcription factors are critical survival factors that inhibit cardiomyocyte apoptosis during phenylephrine stimulation in vitro. Circ Res 92:725–731 29. Kakita T, Hasegawa K, Iwai-Kanai E, Adachi S, Morimoto T, Wada H, Kawamura T, Yanazume T, Sasayama S (2001) Calcineurin pathway is required for endothelin-1-mediated protection against oxidant stress-induced apoptosis in cardiac myocytes. Circ Res 88:1239–1246 30. De Windt LJ, Lim HW, Taigen T, Wencker D, Condorelli G, Dorn GW 2nd, Kitsis RN, Molkentin JD (2000) Calcineurinmediated hypertrophy protects cardiomyocytes from apoptosis in vitro and in vivo: an apoptosis-independent model of dilated heart failure. Circ Res 86:255–263 31. Saito S, Hiroi Y, Zou Y, Aikawa R, Toko H, Shibasaki F, Yazaki Y, Nagai R, Komuro I (2000) Beta-adrenergic pathway induces apoptosis through calcineurin activation in cardiac myocytes. J Biol Chem 275:34528–34533 32. Chu CH, Tzang BS, Chen LM, Liu CJ, Tsai FJ, Tsai CH, Lin JA, Kuo WW, Bau DT, Yao CH, Huang CY (2009) Activation of insulin-like growth factor II receptor induces mitochondrial-dependent apoptosis through G(alpha)q and downstream calcineurin signaling in myocardial cells. Endocrinology 150:2723–2731 33. Lotem J, Kama R, Sachs L (1999) Suppression or induction of apoptosis by opposing pathways downstream from calcium-activated calcineurin. Proc Natl Acad Sci USA 96:12016–12020 34. Molkentin JD (2001) Calcineurin, mitochondrial membrane potential, and cardiomyocyte apoptosis. Circ Res 88:1220–1222 35. Antonsson B (2004) Mitochondria and the Bcl-2 family proteins in apoptosis signaling pathways. Mol Cell Biochem 256(257): 141–155 36. Maulik N, Engelman RM, Rousou JA, Flack JE 3rd, Deaton D, Das DK (1999) Ischemic preconditioning reduces apoptosis by upregulating anti-death gene Bcl-2. Circulation 100:II369–II375 37. Shibasaki F, Kondo E, Akagi T, McKeon F (1997) Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386:728–731 38. Wei Q, Lian ML, Jing FZ, Zhang N, Yan MS, Chen Y, Gao QS (2002) Studies of calcineurin B subunit from genetic engineering for use in medicine. Drug Dev Res 56:40–43

123