Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation.
However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32-ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis.
Zhu Y, etal., Protein Cell. 2013 Sep;4(9):711-21. doi: 10.1007/s13238-013-3910-3. Epub 2013 Sep 10.
Mutations in LR RK2 (Leucine rich repeat kinase 2) are a major cause of Parkinson's disease (PD). We and others reported recently that expression of the pathogenic gainof-function mutant form of LRRK2, LRRK2 G2019S, induces mitochondrial fission in neurons through DLP1. Here we provide evidence that
expression of LRRK2 G2019S stimulates mitochondria loss or mitophagy. We have characterized several LRRK2 interacting proteins and found that LRRK2 interacts with ULK1 which plays an essential role in autophagy. Knockdown of either ULK1 or DLP1 expression with shRNAs suppresses LRRK2 G2019S expression-induced mitochondrial clearance, suggesting that LRRK2 G2019S expression induces mitochondrial fission through DLP1 followed by mitophagy via an ULK1 dependent pathway. In addition to ULK1, we found that LRRK2 interacts with the endogenous MKK4/7, JIP3 and coordinates with them in the activation of JNK signaling. Interestingly, LRRK2 G2019S-induced loss of mitochondria can also be suppressed by 3 different JNK inhibitors, implying the involvement of the JNK pathway in the pathogenic mechanism of mutated LRRK2. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies.
Joshi A, etal., Cell Death Differ. 2016 Feb;23(2):216-30. doi: 10.1038/cdd.2015.88. Epub 2015 Jul 3.
Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1
;'>ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H2O2). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H2O2-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.
Di Nardo A, etal., Hum Mol Genet. 2014 Jul 15;23(14):3865-74. doi: 10.1093/hmg/ddu101. Epub 2014 Mar 5.
Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mam
malian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.
Young AR, etal., J Cell Sci. 2006 Sep 15;119(Pt 18):3888-900. Epub 2006 Aug 29.
Autophagy, fundamentally a lysosomal degradation pathway, functions in cells during normal growth and certain pathological conditions, including starvation, to maintain homeostasis. Autophagosomes are formed through a mechanism that is not well understood, despite the identification of many genes re
quired for autophagy. We have studied the mammalian homologue of Atg9p, a multi-spanning transmembrane protein essential in yeast for autophagy, to gain a better understanding of the function of this ubiquitious protein. We show that both the N- and C-termini of mammalian Atg9 (mAtg9) are cytosolic, and predict that mAtg9 spans the membrane six times. We find that mAtg9 is located in the trans-Golgi network and late endosomes and colocalizes with TGN46, the cation-independent mannose-6-phosphate receptor, Rab7 and Rab9. Amino acid starvation or rapamycin treatment, which upregulates autophagy, causes a redistribution of mAtg9 from the TGN to peripheral, endosomal membranes, which are positive for the autophagosomal marker GFP-LC3. siRNA-mediated depletion of the putative mammalian homologue of Atg1p, ULK1, inhibits this starvation-induced redistribution. The redistribution of mAtg9 also requires PI 3-kinase activity, and is reversed after restoration of amino acids. We speculate that starvation-induced autophagy, which requires mAtg9, may rely on an alteration of the steady-state trafficking of mAtg9, in a Atg1-dependent manner.
Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the seri
ne/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.
To explore the potential roles of miRNAs in controlling the survival of mycobacteria in macrophages, miR-17-5p in the regulation of Bacillus Calmette-Guerin(BCG)growth in the macrophage RAW264.7 cells was interrogated. Our results reveal that an infection of BCG shows a time-dependent up-regulation
of miR-17-5p in RAW264.7 cells in early phase; importantly, excessive expression of miR-17-5p in these cells exhibits an increased propagation of intracellular BCG. Mechanistically, the Unc-51 like autophagy activating kinase 1 (ULK1), an initial molecular of autophagy are identified as novel target of miR-17-5p, the miR-17-5p is capable of targeting down-regulating the expression of ULK1 protein. In addition, an overexpression of miR-17-5p in RAW264.7 cells is correlated with repression of ULK1 and the autophagosome related proteins LC3I/II. These results imply that miR-17-5p may be able to arrest the maturation of mycobacterial phagosomes in part by targeting ULK1, subsequently reduces the ability of host cells to kill intracellular BCG.
Yan J, etal., Biochem Biophys Res Commun 1998 May 8;246(1):222-7.
A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conse
rvation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.
Birt-Hogg-Dube (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, cur
rently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.
Qi S, etal., Structure. 2015 Oct 6;23(10):1848-57. doi: 10.1016/j.str.2015.07.011. Epub 2015 Aug 20.
The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad
2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners.
Li R, etal., J Immunol. 2015 Oct 15;195(8):3901-11. doi: 10.4049/jimmunol.1500967. Epub 2015 Sep 14.
Earlier studies reported that a cell membrane protein, Annexin A2 (AnxA2), plays multiple roles in the development, invasion, and metastasis of cancer. Recent studies demonstrated that AnxA2 also functions in immunity against infection, but the underlying mechanism remains largely elusive. Using a m
ouse infection model, we reveal a crucial role for AnxA2 in host defense against Pseudomonas aeruginosa, as anxa2(-/-) mice manifested severe lung injury, systemic dissemination, and increased mortality compared with wild-type littermates. In addition, anxa2(-/-) mice exhibited elevated inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ), decreased bacterial clearance by macrophages, and increased superoxide release in the lung. We further identified an unexpected molecular interaction between AnxA2 and Fam13A, which activated Rho GTPase. P. aeruginosa infection induced autophagosome formation by inhibiting Akt1 and mTOR. Our results indicate that AnxA2 regulates autophagy, thereby contributing to host immunity against bacteria through the Akt1-mTOR-ULK1/2 signaling pathway.
Liu CC, etal., Mol Cell. 2016 Jan 7;61(1):84-97. doi: 10.1016/j.molcel.2015.11.001. Epub 2015 Dec 10.
Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ... (more)
n style='font-weight:700;'>ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism.
Saturated fatty acid (SFA)-induced lipotoxicity is caused by the accumulation of reactive oxygen species (ROS), which is associated with damaged mitochondria. Moreover, lipotoxicity is crucial for the progression of nonalcoholic steatohepatitis (NASH). Autophagy is required for the clearance of prot
ein aggregates or damaged mitochondria to maintain cellular metabolic homeostasis. The NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2)-KEAP1 (kelch like ECH associated protein 1) pathway is essential for the elimination of ROS. ULK1 (unc-51 like autophagy activating kinase 1; yeast Atg1) is involved in the initiation of autophagy; however, its role in lipotoxicity-induced cell death in hepatocytes and mouse liver has not been elucidated. We now show that ULK1 potentiates the interaction between KEAP1 and the autophagy adaptor protein SQSTM1/p62, thereby mediating NFE2L2 activation in a manner requiring SQSTM1-dependent autophagic KEAP1 degradation. Furthermore, ULK1 is required for the autophagic removal of damaged mitochondria and to enhance binding between SQSTM1 and PINK1 (PTEN induced kinase 1). This study demonstrates the molecular mechanisms underlying the cytoprotective role of ULK1 against lipotoxicity. Thus, ULK1 could represent a potential therapeutic target for the treatment of NASH.Abbreviations: ACTB: actin beta; CM-H2DCFDA:5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; GSTA1: glutathione S-transferase A1; HA: hemagglutinin; Hepa1c1c7: mouse hepatoma cells; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch like ECH associated protein 1; LPS: lipopolysaccharides; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK8/JNK: mitogen-activated protein kinase 8; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NASH: nonalcoholic steatohepatitis; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PA: palmitic acid; PARP: poly (ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PRKAA1/2: protein kinase AMP-activated catalytic subunits alpha1/2; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; PRKC/PKC: protein kinase C; RBX1: ring-box 1; ROS: reactive oxygen species; SFA: saturated fatty acid; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUBA: tubulin alpha; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; ULK1: unc-51 like autophagy activating kinase 1.
Liu W, etal., Int J Mol Med. 2016 Feb;37(2):309-18. doi: 10.3892/ijmm.2015.2425. Epub 2015 Dec 7.
In a previous study by our group, we demonstrated that electroacupuncture (EA) activates the class I phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. There is considerable evidence that the downstream mammalian target of rapamycin complex 1 (mTORC1) plays an important role in autophagy follo
wing ischemic stroke. The aim of the present study was to determine whether EA exerts a neuroprotective effect through mTORC1-mediated autophagy following ischemia/reperfusion injury. Our results revealed that EA at the LI11 and ST36 acupoints attenuated motor dysfunction, improved neurological deficit outcomes and decreased the infarct volumes. The number of autophagosomes, autolysosomes and lysosomes was decreased following treatment with EA. Simultaneously, the levels of the autophagosome membrane maker, microtubule-associated protein 1 light chain 3 beta (LC3B)/, Unc-51-like kinase 1 (ULK1), autophagy related gene 13 Atg13) and Beclin1 (ser14) were decreased, whereas mTORC1 expression was increased in the peri-infarct cortex. These results suggest that EA protects against ischemic stroke through the inhibition of autophagosome formation and autophagy, which is mediated through the mTORC1-ULK complex-Beclin1 pathway.
Hieke N, etal., Autophagy. 2015;11(9):1471-83. doi: 10.1080/15548627.2015.1068488.
Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 ha
s been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.
Chen Y, etal., Sci Rep. 2015 Jul 17;5:11035. doi: 10.1038/srep11035.
Autophagy, referring to an evolutionarily conserved, multi-step lysosomal degradation process, has been well-known to be initiated by Unc-51 like kinase 1 (ULK1) with some links to Parkinson's disease (PD). MicroRNAs (miRNAs), small and non-coding endogenous RN
As 22 ~ 24 nucleotides (nt) in length, have been demonstrated to play an essential role for modulating autophagy. Recently, the relationships between miRNAs and autophagy have been widely reported in PD; however, how microRNAs regulate autophagy still remains in its infancy. Thus, in this study, we computationally constructed the ULK1-regulated autophagic kinase subnetwork in PD and further identified ULK1 able to negatively regulate p70(S6K) in starvation-induced autophagy of neuroblastoma SH-SY5Y cells. Combination of in silico prediction and microarray analyses, we identified that miR-4487 and miR-595 could target ULK1 and experimentally verified they could negatively or positively regulate ULK1-mediated autophagy. In conclusion, these results may uncover the novel ULK1-p70(S6K) autophagic pathway, as well as miR-4487 and miR-595 as new ULK1 target miRNAs. Thus, these findings would provide a clue to explore ULK1 and its target miRNAs as potential biomarkers in the future PD therapy.
Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins i
ncluding Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.
Li J, etal., Autophagy. 2015;11(8):1216-29. doi: 10.1080/15548627.2015.1017180.
Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. Accumulating evidence has also shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination throu
gh mitochondrial E3 ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics, and mitophagy. Here we showed that MUL1, a mitochondria-localized E3 ligase, regulates selenite-induced mitophagy in an ATG5 and ULK1-dependent manner. ULK1 partially translocated to mitochondria after selenite treatment and interacted with MUL1. We also demonstrated that ULK1 is a novel substrate of MUL1. These results suggest the association of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent.
Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T3) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity
and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T3 is coupled to oxidative phosphorylation and ROS production. We show that T3 induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, beta) mediated phosphorylation of PRKAA1/AMPK (5' AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T3-treated cells impairs both mitophagy as well as OXPHOS without affecting T3 induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T3-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.
This study was designed to evaluate the role of ULK1 in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge. Wild-type and AMPK knockout mice were challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial functi
on was evaluated using echocardiography and edge-detection. Western blot analysis was employed to evaluate the levels of AMPK, Raptor, mTOR, the AMPK downstream signal ULK1 and autophagy markers Beclin-1 and LC3-II. siRNA was used to knockdown ULK1 in H9C2 myoblasts. GFP-LC3 puncta was used to evaluate autophagosome formation. Alcohol challenge compromised cardiac function as evidenced by decreased fractional shortening, peak shortening and intracellular Ca²¿ rise, prolonged relengthening and intracellular Ca²¿ decay in WT mice, the effects of which were mitigated by AMPK knockout. Ethanol exposure facilitated myocardial autophagy as evidenced by enhanced LC3-II level, as well as phosphorylation of AMPK, Raptor, and dephosphorylation of mTOR and ULKI in WT hearts, which were alleviated by AMPK knockout. Pharmacological inhibition of AMPK using compound C attenuated ethanol-induced autophagosome formation, AMPK phosphorylation, ULK1 dephosphorylation and apoptosis. Ethanol exposure-induced cardiomyocyte contractile defects and autophagosome accumulation were reversed by the autophagy inhibitor 3-MA. Similarly, knockdown of ULK1 using siRNA in H9C2 cells ablated ethanol-induced autophagosome accumulation, LC3-II expression and cell death. Lysosomal inhibition using bafilomycin, E64-D and pepstatin A potentiated ethanol-induced increase in autophagosome formation. Taken together, our results suggest that ULK1 may play a critical role in AMPK-mediated myocardial autophagy, apoptosis and contractile dysfunction following acute alcohol challenge.
