In this study, we showed that tf-LC3 can be used to evaluate the level of autophagic flux in cardiac myocytes both in vitro and in vivo. By using Tg-tf-LC3, we demonstrated that I/R increases autophagic flux in cardiac myocytes through an oxidative stress–dependent mechanism in vivo.
tf-LC3 is a useful tool for evaluating the formation of both autophagosomes and autolysosomes simultaneously in cardiac myocytes. The results of our experiments with tf-LC3 showed that AAD increases both autophagosomes and autolysosomes, an observation also supported by conventional indexes of autophagy, including increases in LC3-II and decreases in p62 accumulation in cardiac myocytes. Although autophagosomes accumulate when fusion between autophagosomes and lysosomes is inhibited, a concurrent increase in both autophagosomes and autolysosomes indicates that autophagic flux is stimulated (14
). Although autophagic flux can be evaluated by comparing the extent of autophagosome accumulation in the presence or absence of inhibitors of lysosomal fusion or degradation or both, such interventions are usually toxic, which would confound analyses of the role of autophagy in mediating biologic functions in vivo
). Tg-tf-LC3 mice are useful not only in that the extent of autophagic flux can be monitored in vivo
, but also in that one can investigate the effect of various interventions on formation of autophagosomes and autolysosomes independently.
Terada and colleagues (23
) previously reported double-transgenic mice generated by cross-breeding cardiac-specific mCherry-LC3 mice with systemic GFP-LC3 mice. Compared with those mice, our transgenic mice with cardiac-specific expression of a single transgene, tf-LC3, have several advantages. First, because either green or red color can be emitted from the same LC3 molecule in Tg-tf-LC3, more-accurate quantification of autophagosomes and autolysosomes can be made than in the cross between cardiac-specific mCherry-LC3 and systemic GFP-LC3 mice, in which the number of mCherry-LC3 and GFP-LC3 molecules are not identical and GFP-LC3 is not cardiac myocyte specific. Second, the mouse model reported by Terada requires cross-breeding between mCherry-LC3 mice and GFP-LC3 mice, whereas our model does not, because both GFP and mRFP are expressed in a single transgene. Finally, because tf-LC3 is expressed in a cardiac myocyte–specific manner in Tg-tf-LC3, our model allows one to quantitate the extent of autophagic flux specifically in cardiac myocytes.
With our unique mouse model, we here provide evidence that oxidative stress plays an important role in mediating autophagy during reperfusion (). Reperfusion causes various cellular responses relevant to autophagy, including oxidative stress, mitochondrial permeability transition pore (mPTP) opening, endoplasmic reticulum stress, Ca2+
overloading, and mitochondrial damage (7
). However, to our knowledge, the role of oxidative stress in mediating increases in autophagic flux during I/R has not been clearly demonstrated. Our results suggest that increases in oxidative stress are both necessary and sufficient for inducing autophagy during I/R in cardiac myocytes.
FIG. 6. Schematic representation of the role of oxidative stress in mediating autophagy and myocardial injury in response to I/R. Myocardial I/R stimulates autophagy through increases in oxidative stress. Stimulation of autophagy plays an important role in mediating (more ...)
Oxidative stress affects autophagy through multiple mechanisms. During starvation, H2
directly oxidizes HsAtg4 and inhibits its cysteine protease activity, thereby causing increased LC3 lipidation and autophagy in cancer cell lines (27
). Oxidative stress affects lysosomal membrane permeability through disulfide bond formation in the lysosomal membrane proteins (6
). Autophagy is also activated as a compensatory response to damages in intracellular organelles (12
). For example, increases in oxidative stress in mitochondria induce opening of the mPTP, leading to depolarization of the mitochondrial membrane potential, swelling, and damage to mitochondrial proteins (3
). Cyclophilin D, a component of the mPTP, is involved in starvation-induced mitophagy in cardiac myocytes (4
). BH3-only proteins, such as Bnip3, are upregulated during mPTP opening and mediate autophagy during I/R (10
). Oxidative stress also triggers ER stress, which, in turn, induces autophagy (17
Although the molecular mechanism by which oxidative stress induces autophagy during myocardial reperfusion remains to be elucidated, we showed previously that Beclin-1 is dramatically upregulated during the reperfusion phase in the mouse heart, which, in turn, plays an essential role in mediating increases in autophagy (18
). Because MPG treatment significantly inhibited upregulation of Beclin-1 during the reperfusion phase, it is possible that oxidative stress induces autophagy through upregulation of Beclin-1. Whether the dramatic upregulation of Beclin-1 during the reperfusion phase is mediated through a transcriptional mechanism, and, if so, which transcription factor is involved in this process, remains to be elucidated.
Our results showed that MPG treatment inhibited formation of both autophagosomes and autolysosomes during reperfusion. Interestingly, we noted that the formation of the latter is more strongly affected. Although the precise mechanism by which this regulation takes place is yet to be identified, it is possible that mechanisms mediating autolysosome formation, such as the fusion of autophagosomes and lysosomes, may be sensitive to oxidative stress. Interestingly, a study by Li et al.
) showed that MPG neutralizes 3-aminopropanol, a neurotoxin formed during cerebral ischemia that damages the lysosome.
Although oxidative stress activates various mechanisms leading to an increase in death of cardiac myocytes (19
), we propose that oxidative stress induces reperfusion injury in part through stimulation of autophagy. Several lines of evidence support our hypothesis. First, suppression of oxidative stress inhibits autophagy during the reperfusion phase, and a similar extent of autophagy suppression by Beclin-1 downregulation was sufficient to reduce the level of reperfusion injury. Second, although MPG significantly reduced the size of MI/AAR in control mice, it did not further reduce the size of MI/AAR when autophagy is suppressed in beclin1+/−
mice, consistent with the notion that oxidative stress regulates cell survival/death through regulation of autophagy. Whether stimulation of autophagy can be detrimental during reperfusion is controversial (8
). Although our results indicating that MPG suppresses both autophagy and myocardial injury support the notion that autophagy during reperfusion is detrimental, further studies are needed to elucidate the functional significance of autophagy during I/R.
At present, the contribution of autophagic cell death to overall myocardial injury is unknown. Because I/R-induced increases in apoptosis are attenuated in beclin1+/− mice, it is possible that autophagy and apoptosis may be linked. Although mPTP opening may initially induce both autophagy and apoptosis, severe depletion of ATP caused by mPTP opening may lead to necrosis as well. Whether strong activation of lysosomal degradation by autophagy contributes to necrotic cell death during I/R remains to be elucidated.
In summary, our results suggest that tf-LC3 is a useful tool for investigating the level of autophagic flux in the heart and the cardiac myocytes therein. With Tg-tf-LC3 mice, we showed that oxidative stress plays an important role in stimulating autophagic flux, which contributes to myocardial injury during I/R in vivo. We propose that suppression of oxidative stress during the reperfusion phase prevents myocardial injury in part through suppression of excessive autophagy.