At baseline, G-CSF at 50 ng/ml and CDP alone did not cause any changes in cardiac mitochondria. However, CsA alone caused a slight decrease in ΔΨm (mitochondrial depolarization) (Figure 10). When H2O2 was added to cardiac mitochondria, ΔΨm was significantly altered, compared to the control group. However, applying H2O2 to mitochondria pretreated with G-CSF or CsA or CDP did not alter the ΔΨm. When H2O2 was added to mitochondria pretreated with (G-CSF+CsA) or (G-CSF+CDP) or (CsA+CDP) or (G-CSF+CsA+CDP), the ΔΨm was also not altered, compared to the H2O2 group. No difference was found for ΔΨm among these combined treatment groups. Furthermore, the ΔΨm in these groups did not differ from that in the control group (Figure 10).
3.7 Effects of G-CSF on the complex I and III of the electron transport chain
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At baseline, rotenone (2 µM) and antimycin A (2 µM) did not cause any change in the ROS level, whereas H2O2 caused an increase in the ROS level in cardiac mitochondria (Figure 11). G-CSF could significantly decrease the ROS level in cardiac mitochondria when it was applied to the mitochondria before the addition of rotenone, antimycin, and H2O2 (i.e. ROS level in MG, MGR, and MGA groups was lower than that in the H2O2, MRH and MAH groups in Figure 11). However, the effectiveness of G-CSF in reducing the ROS level was decreased when G-CSF was added to the mitochondria after rotenone and antimycin A application since the ROS levels in MRGH and MAGH were significantly higher than those in the MG, MGRH and MGAH groups.
Mitochondria have been shown to play a key role in cardiac dysfunction caused by myocardial ischemia (Xiao et al., 2010). A previous study demonstrated that oxidative stress occurring in ischemic myocardium caused an increased ROS production in cardiac mitochondria (Tompkins et al., 2006). The increased ROS level in one or a few mitochondria has been shown to trigger the release of ROS from neighboring mitochondria, a mechanism known as ROS-induced ROS release (Zorov et al., 2006), resulting in a large amount of ROS accumulated in the ischemic myocardium. The increased ROS level could lead to the disruption of the electron transport chain in cardiac mitochondria, causing cardiac mitochondrial dysfunction and eventually leading to myocardial cell death (Tompkins et al., 2006). Pharmacological interventions to attenuate mitochondrial dysfunction have been shown to have cardioprotective effects, including prevention of arrhythmia and reduction of infarct size (Matejikova et al., 2009; Moncada, 2010).
Granulocyte-colony stimulating factor (G-CSF) has recently been shown to improve cardiac function, increase myocardial blood supply, and reduce mortality after cardiac injury under several conditions including ischemic heart (Cheng et al., 2008; Brunner et al., 2008; Okada et al., 2008). However, its effect on cardiac mitochondria undergo oxidative stress induced by H2O2 has never been investigated. In the present study, we found that G-CSF could prevent mitochondrial swelling, decrease ROS level, and attenuate mitochondrial membrane depolarization in cardiac mitochondria under oxidative stress condition.
In the present study, the effective dose of G-CSF (50 ng/ml) could successfully prevent mitochondrial damage. G-CSF at higher concentrations (up to 200 ng/ml) shared equally similar effects to G-CSF at 50 ng/ml. Although G-CSF could completely prevent mitochondrial swelling and ΔΨm changes after oxidative stress induced by H2O2, it could only decrease ROS to a certain level, which was still higher than that in the control group. These findings indicate that G-CSF may act more effectively in preventing mitochondrial swelling and mitochondrial depolarization, but less effective in preventing ROS production.
Mitochondrial swelling is described as an increase of mitochondrial volume. The cause of mitochondrial swelling is due to the prolonged opening of mPTP, which causes an increase of permeability to solutes with molecular masses up to 1500 Da. Since mPTP opening can be triggered by the increased ROS level (Hausenloy et al., 2003), the prolonged mPTP opening can lead to free bi-directional movement of low molecular weight molecules across the inner membrane, while proteins remain in the matrix. Consequently, colloidal osmotic pressure increases and causes mitochondrial swelling (the inner membrane cristae unfolded), the rupture of outer mitochondrial membrane, and the release of cytochrome c (Azzone and Azzi, 1965; Crompton, 1999). In this study, G-CSF effectively prevented mitochondrial swelling caused by H2O2, and this protection is as effective as the effect of the blocker of mPTP opening (CsA). These findings suggested that G-CSF might act on mPTP, thus inhibiting the opening of this pore and leading to the prevention of mitochondrial swelling.
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Mitochondria is known as a major organelle for producing ROS in cells. ROS is normally produced at sites of Complex I and III in the electron transport chain (Chen et al., 2008). Under physiological conditions, O2- is transported to the electron transport chain for oxidative phosphorylation in the mitochondria and converted into a small amount of H2O2 (Chen et al., 2008). However, these ROS are generally degraded by catalase and glutathione peroxidase in the mitochondria. In some pathological conditions such as oxidative stress, ROS production is markedly increased and the ROS degradation process is not sufficiently maintained, resulting in an excess of ROS including O2-.
Under physiological conditions, O2- can be released across the inner mitochondrial membrane via IMAC, causing mitochondrial membrane depolarization (Brady et al., 2006). The release of ROS via IMAC opening could activate the IMAC of its neighboring mitochondria, resulting in a great increase of ROS (Vanden Hoek et al., 1998). This process is called ROS-induced ROS release mechanism, leading to severe oxidative stress in cells (Zorov et al., 2000). IMAC opening is normally terminated by a reduction of ROS level (i.e. via decreasing ROS production and efflux from the mitochondrial matrix) and ROS scavenging by the antioxidant enzymes (Cortassa et al., 2004). Aon et al. (2003) clearly demonstrated in their excellent study that a certain amount of ROS produced from the electron transport chain is required to accumulate in the mitochondrial matrix up to a critical levels in order to trigger the opening of IMAC (Aon et al., 2003). Once the ROS accumulation reaches a point of mitochondrial critical threshold, mitochondrial depolarization is observed and the mitochondrial membrane potential becomes unstable in almost the entire population of mitochondria in the mitochondrial network (Aon et al., 2003).
In the present study, our finding indicated that H2O2 used in this study could cause a markedly increased ROS level in cardiac mitochondria, which must be sufficiently high, resulting in the opening of IMAC and eventually leading to a decrease in ΔΨm. Through this mechanism, our findings that G-CSF, CsA, and CDP shared a similar efficacy in the prevention of the mitochondrial depolarization, despite the fact that they had different efficacy in the prevention of ROS production, indicated that these pharmacological interventions can effectively attenuate the ROS level in cardiac mitochondria to a level below the critical threshold required for triggering the IMAC opening. Our findings that low concentrations of H2O2 (0.5 and 1 mM) could increase the ROS level in cardiac mitochondria but did not cause mitochondrial membrane potential changes, whereas high-concentration (2 mM) H2O2 could cause much increase in ROS level and produced mitochondrial membrane depolarization, thus supporting the critical threshold hypothesis.
In the present study, the ROS level was lowest in mitochondria pretreated with CsA, followed by higher levels in the CDP and G-CSF groups. Since the efficacy of G-CSF in preventing mitochondrial depolarization was similar to that of CsA, our findings suggest that the decreased ROS level caused by G-CSF was sufficient to bring the ROS level below the critical threshold. As a result, the trigger of IMAC opening was inhibited, resulting in no change in ΔΨm after H2O2 application. All these findings suggest that the accumulation of ROS in cardiac mitochondria up to a critical threshold level could be a key determinant for the cardiac mitochondrial protection.
Without H2O2 application, ROS levels in the G-CSF and CsA groups were modestly increased. This could be due to the fact that G-CSF itself can increase ROS production by stimulating the angiogenic factors production (Carrao et al., 2009). In cardiomyocytes, it has been shown that G-CSF can directly stimulate ROS production, which plays a pivotal role in triggering adaptations of the heart to ischemia including growth of the coronary collaterals (Carrao et al., 2009). However, the ROS level caused by G-CSF alone was not sufficient to cause the opening of the IMAC.
Our results also showed that applying CsA alone could modestly raise the ROS level in cardiac mitochondria. CsA is known to effectively block the mPTP opening, therefore the release of ROS from mitochondria can be blocked inside the matrix at some degree. Our study also showed that CsA alone can cause a modest decrease in ΔΨm, compared to the control group. This evidence suggests that under physiological condition, mPTP flickering (i.e. opening and closing) is essential for the exchange of metabolites between cardiac mitochondria and cytosol, thus maintaining cardiac mitochondrial membrane potential (Kroemer et al., 2007). Therefore, blocking the opening of mPTP by CsA could prevent this exchange process, resulting in a slight change in ΔΨm.
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In the present study, CsA had the highest efficacy in preventing ROS production in cardiac mitochondria. The lower efficacy of CDP than CsA in preventing ROS production could be due to the fact that blocking IMAC only prevents the release of ROS from the mitochondrial matrix, while the remaining ROS could still be released from the intermembrane space and out of the mitochondria via mPTP. Thus, ROS-induced ROS release mechanism could still occur within the mitochondrial network, resulting in a higher level of ROS in the CDP group. Since combined treatment with G-CSF and CDP could effectively decrease ROS level to a similar degree as that in CsA, these findings suggested that G-CSF may act as a blocker of mPTP opening, but with less efficacy than CsA in preventing ROS production.
In the present study, our results indicated that the protective effect of G-CSF on mitochondria could involve complex I and III on the electron transport chain. Complexes I and III are known as the main sources of ROS production in the mitochondria (Andrukhiv et al., 2006). In a previous study, Chen et al. reported that rotenone did not alter net ROS generation in intact mitochondria (Chen et al., 2003). However, under the pathological condition such as myocardial ischemia, administration of rotenone could decrease the production of ROS in mitochondria (Becker et al., 1999). Our results were consistent with these previous reports. In the present study, although rotenone has a tendency to decrease ROS production in cardiac mitochondria pretreated with rotenone prior to H2O2 application, compared to mitochondria treated with H2O2, it did not reach statistical significance. Antimycin A is previously reported that it can increase ROS production (Chen et al., 2008). In present study, antimycin A-treated cardiac mitochondria under the H2O2-induced oxidative stress condition can increase ROS production compared to control group. Similar to rotenone, antimycin A did not alter ROS level in normal cardiac mitochondria, and did not change ROS level when H2O2 was applied. However, the ROS level measured in the groups in which G-CSF was administered prior to the application of rotenone and antimycin A was significantly lower than that in the groups in which G-CSF was administered after rotenone and antimycin A. This finding indicated that the effect of G-CSF on mitochondria could involve complex I and III activity in the electron transport chain. It is also important to note that although these findings indicate that G-CSF could affect complex I and III activity, future studies are needed to elucidate the definite mechanism of G-CSF as well as its involvement on complex IV and V in the electron transport chain.
Under oxidative stress, G-CSF can effectively prevent mitochondrial swelling, mitochondrial membrane potential changes, and ROS production in cardiac mitochondria. Its mechanism could be due to the inhibition of mPTP opening and could involve the complex I and III activity on the electron transport chain. These beneficial effects of G-CSF may be used to prevent cardiac mitochondrial damage under oxidative stress conditions.