Preconditioning Involves Selective Mitophagy Mediated by Parkin and p62/SQSTM1

Abstract

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

“…7A and C), paralleling the observed impairment in autophagic flux with accumulation of abnormal appearing mitochondria in these models. Taken together, these observations suggest that intermittent fasting preconditions the myocardium via enhanced autophagy of damaged mitochondria, 39 with consequent reduction in oxidative stress.…”

“…7), suggesting that intermittent fasting stimulates lysosome function to remove damaged organelles that are the source for deleterious ROS generation, 38,64 as a mechanism for preconditioning the myocardium to ischemia-reperfusion injury. 39 Additional upstream signaling events that link fasting to stimulation of lysosomal pathways, including autophagy, may also participate in preconditioning responses. Fastinginduced activation of AMPK and suppression of MTOR signaling, are 2 candidate pathways that have been previously demonstrated to have no effect (as with AMPK 65 ) or facilitate ischemic preconditioining (as with rapamycin-mediated inhibiton of MTOR 66 ) in the myocardium.…”

Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart

“…Mitochondria can divide asymmetrically into functional progeny, which can reintegrate the mitochondrial network by fusion, and dysfunctional organelles with low membrane potential, which are specifically destined for mitophagy 9 . In addition to neurodegenerative diseases, Parkin-mediated mitophagy was also shown to contribute to cardiac pathophysiology 42 . Previous studies investigated whether Parkin mutations associated with familial AR-JP were defective in supporting mitophagy.…”

The Dynamics of the Mitochondrial Organelle as a Potential Therapeutic Target

“…In neurons in particular, where mitochondrial alterations occur over a wide spatiotemporal plane, capturing and quantifying mitophagy remains challenging. A variety of techniques have been used to attempt to quantify mitophagy (reviewed in Zhu et al 41 ) Some studies examine loss of a specific mitochondrial protein (e.g., Tom70, see Huang et al 42 ) as evidence for mitochondrial clearance, but reliance on one protein alone does not indicate either a specialized mitochondrial -targeted process or a loss of the entire organelle, as it is confounded by the possible proteasomal clearance of that particular protein rather than mitophagic degradation. The current standard for observing autophagic mitochondrial engulfment remains by observation of EM images of mitochondria surrounded by an autophagic membrane.…”

“…42 Overexpression of Parkin led to decreased mitochondrial loss (measured by Tom70 immunofluorescence), and Parkin knockout resulted in a loss of preconditioning-afforded ischemic cardioprotection.…”