Parkin has been shown to play a central role in mitochondrial quality control in the brain, but its role in heart has not been examined to date, although both organs consist of long-lived cells with a high mitochondrial content. Because we have previously shown a role for autophagy in cardioprotection by IPC, we considered whether mitochondria were an important target of autophagy; in particular, we considered whether Parkin might play an important role in mitophagy in the setting of IPC. The results presented herein reveal an important role for Parkin and p62 in mediating mitophagy in response to ischemic stress in HL-1 cells, and in mediating IPC-dependent cardioprotection in vivo.
Studies dating back to the 1970s have described the occurrence of mitochondria in autophagosomes in heart tissue 
. More recently, the mitochondrial permeability transition pore has been implicated in signaling autophagy 
, but the mechanism by which cardiac mitochondria might be targeted for selective removal has not been elucidated. We have shown that HL-1 cells undergo cyclophilin D-dependent mitophagy in response to starvation 
, but this is the first study to examine the processes mediating mitophagy in response to ischemic stress in the heart.
Why would removal of mitochondria be beneficial? Selective removal of the mitochondria with the lowest threshold for opening of the mitochondrial permeability transition pore (MPTP) would leave behind a population of robust mitochondria better-equipped to resist ischemic stress. Since depolarized mitochondria will hydrolyze ATP in a futile effort to restore membrane potential, elimination of these mitochondria would actually result in increased ATP content after I/R despite the reduced numbers of mitochondria. Selective removal of damaged mitochondria might also reduce the production of reactive oxygen species (ROS). Although we did not measure ROS production in this study, we previously reported that sulfaphenazole attenuates ROS production and protects the heart via autophagy, and that stimulation of autophagy attenuates ROS production by HL-1 cells exposed to lipopolysaccharide 
The limitations of this study are that we have not measured PINK1, the mitochondrial outer membrane kinase that is stabilized as membrane potential decreases, nor have we measured mitochondrial membrane potential concurrently with Parkin or p62 translocation. We saw a consistent reduction in both mitochondrial antigens in Parkin-transfected cells. Although the magnitude of reduction was greater for Tom70 than COX4, the immunostaining for COX4 was done on a different set of cells, non-concurrently. Further work will be needed to determine if outer membrane and inner membrane antigens disappear concurrently—consistent with organellar destruction—or if outer membrane proteins are selectively degraded as suggested by Chan et al. 
. We expect that mitophagy is selective and self-limited since the process is restricted to depolarized mitochondria 
. Global depolarization of mitochondria with an uncoupler such as FCCP is known to trigger generalized mitophagy and extensive loss of mitochondria 
. Cells that eliminate mitochondria will need to replace them in order to fully restore ATP production capacity. Thus it will be important to assess mitochondrial biogenesis in future studies. Since biogenesis likely counterbalances mitophagy, it will be important to develop better methods to measure mitochondrial turnover in vivo.
The evidence presented in this study indicates that Parkin triggers mitophagy and mediates cytoprotection in HL-1 cells subjected to ischemic stress. These findings, coupled with the fact that IPC is abolished in Parkin knockout mice, lead us to conclude that Parkin mediates IPC through ubiquitination of mitochondrial membrane proteins and recruitment of p62 to facilitate autophagic engulfment of the decorated mitochondria.
The autophagic removal of depolarized mitochondria mediated by Parkin and p62 may represent an important element of mitochondrial quality control. Loss-of-function mutations of Parkin are linked to some cases of Parkinson Disease and are associated with mitochondrial dysfunction 
. While the role of Parkin in the heart is as yet unknown, it is interesting to note that the incidence of heart failure and coronary artery disease is doubled in elderly patients with Parkinson Disease compared to age-matched peers