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• W2014943313 abstract "Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell. Autophagy involves processes for the turnover of long lived macromolecules and organelles via the lysosomal degradative pathway (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar, 2Cuervo A.M. Mol. Cell. Biochem. 2004; 263: 55-72Crossref PubMed Scopus (395) Google Scholar). Macroautophagy (referred to hereafter as autophagy) is a specific mode of autophagy in which isolation membranes envelop a portion of the cytosol, containing nonspecific cytosolic components, selectively targeted toxic protein aggregates (3Ravikumar B. Vacher C. Berger Z. Davies J.E. Luo S. Oroz L.G. Scaravilli F. Easton D.F. Duden R. O'Kane C.J. Rubinsztein D.C. Nat. Genet. 2004; 36: 585-595Crossref PubMed Scopus (1990) Google Scholar), intracellular pathogens (4Gutierrez M.G. Master S.S. Singh S.B. Taylor G.A. Colombo M.I. Deretic V. Cell. 2004; 119: 753-766Abstract Full Text Full Text PDF PubMed Scopus (1769) Google Scholar), or organelles such as mitochondria (5Xue L. Fletcher G.C. Tolkovsky A.M. Curr. Biol. 2001; 11: 361-365Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 6Priault M. Salin B. Schaeffer J. Vallette F.M. di Rago J.P. Martinou J.C. Cell Death Differ. 2005; 12: 1613-1621Crossref PubMed Scopus (242) Google Scholar). The autophagosomes are then delivered to the lysosome, forming the autophagolysosome, for subsequent degradation of their contents by lysosomal hydrolases (Fig. 10). Interest in autophagy has increased recently, because of the recognition of its involvement in caspase-independent programmed cell death (PCD 2The abbreviations used are: PCD, programmed cell death; AVs, autophagic vacuoles; GFP, green fluorescent protein; KH, Krebs-Henseleit; 3-MA, 3-methyladenine; PI3K, phosphatidylinositol 3-kinase; I/R, ischemia/reperfusion; sI/R, simulated ischemia/reperfusion; RNAi, RNA interference; PI3P, phosphatidylinositol 3-phosphate; mTOR, mammalian target of rapamycin; ER, endoplasmic reticulum. type II) and its regulation by components of the apoptotic death pathway (PCD type I) (7Saeki K. Yuo A. Okuma E. Yazaki Y. Susin S.A. Kroemer G. Takaku F. Cell Death Differ. 2000; 7: 1263-1269Crossref PubMed Scopus (172) Google Scholar, 8Yanagisawa H. Miyashita T. Nakano Y. Yamamoto D. Cell Death Differ. 2003; 10: 798-807Crossref PubMed Scopus (91) Google Scholar, 9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1192) Google Scholar). Anti-apoptotic Bcl-2 and Bcl-xL have been linked to the autophagic pathway via an interaction with Beclin1, a key mediator of autophagic activity (9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1192) Google Scholar, 10Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar). Autophagy is a vital process in the heart, presumably participating in the removal of dysfunctional cytosolic components and serving as a catabolic energy source during times of starvation. For example, autophagy in cardiac myocytes has been suggested to provide a necessary source of energy between birth and suckling (11Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2405) Google Scholar), and in a GFP-LC3 transgenic mouse, cardiac myocytes from starved animals displayed high numbers of autophagosomes, some of which contained mitochondria (12Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1942) Google Scholar). On the other hand, impaired autophagy may play a causative role in cardiac disease. Incomplete autophagic removal of mitochondria may be the source of lipofuscin, a toxic waste product that builds up during the life span (13Brunk U.T. Terman A. Eur. J. Biochem. 2002; 269: 1996-2002Crossref PubMed Scopus (608) Google Scholar), and chronic impairment of the lysosome results in reduced myocardial function (14Saftig P. Tanaka Y. Lullmann-Rauch R. von Figura K. Trends Mol. Med. 2001; 7: 37-39Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Furthermore, disruption of the autophagic pathway may contribute to cardiac cell death under conditions where lysosomal integrity is lost and lysosomal proteases are released into the cytosol (15Decker R.S. Wildenthal K. Am. J. Pathol. 1980; 98: 425-444PubMed Google Scholar). In the study presented here, we investigated the role and regulation of autophagy during ischemia/reperfusion (I/R) injury. Following a bout of ischemia (a reduction of blood flow resulting in oxygen and nutrient starvation), reperfusion must be achieved in order to rescue affected tissue. However, reperfusion can activate pathways that either preserve cell viability (preconditioning) or lead to cell death (I/R injury). Autophagy may be a protective response to I/R injury, as increased prevalence of autophagosomes has been documented in response to sub-lethal ischemia in the perfused heart (15Decker R.S. Wildenthal K. Am. J. Pathol. 1980; 98: 425-444PubMed Google Scholar). Moreover, it was recently reported that increased Beclin1 expression in the heart correlated with the onset of protection in an in vivo model of myocardial stunning (16Yan L. Vatner D.E. Kim S.J. Ge H. Masurekar M. Massover W.H. Yang G. Matsui Y. Sadoshima J. Vatner S.F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13807-13812Crossref PubMed Scopus (455) Google Scholar). The cardiac HL-1 cell line was subjected to simulated I/R (sI/R) as an in vitro model of I/R injury to the heart. Using three-dimensional high resolution fluorescence imaging, we analyzed the autophagic response to sI/R. Our results indicate that in HL-1 cardiac myocytes subjected to sI/R, autophagic flux is impaired at the level of both induction and degradation, yet remains a vital underlying protective response against sI/R injury. Moreover, increasing autophagic capacity of the cardiac myocyte is protective against sI/R injury. Reagents—3-Methyladenine, wortmannin, rapamycin, pepstatin A methyl ester, E64D, and bafilomycin A1 were purchased from EMD Biosciences. Cell Culture and Transfections—Cells of the atrially derived cardiac cell line HL-1 (17Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2979-2984Crossref PubMed Scopus (1243) Google Scholar) were plated in gelatin/fibronectin-coated culture vessels and maintained in Claycomb medium (JRH Biosciences) supplemented with 10% fetal bovine serum, 0.1 mm norepinephrine, 2 mm l-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 μg/ml amphotericin B. Cells were transfected with the indicated vectors using the transfection reagents Effectene (Qiagen) or Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions, achieving at least 40 and 60% transfection efficiency, respectively. RNA Interference—Sequences with 100% homology to regions within the open reading frame of mouse Beclin1 (gi: 27764874) were generated using the BLOCK-iT™ RNAi Designer, a construct that embeds a small hairpin RNA within a micro RNA fold, which is then processed by the endogenous RNAi machinery (18Amarzguioui M. Rossi J.J. Kim D. FEBS Lett. 2005; 579: 5974-5981Crossref PubMed Scopus (170) Google Scholar). The obtained target sequences, 5′-tgaaacttcagacccatctta-3′ (a) and 5′-taatggagctgtgagttcctg-3′ (b), showed no significant homology to other mouse proteins as determined by Blast analysis. The sequence was used to generate oligonucleotide pairs, which were inserted into the pcDNA™6.2-GW/EmGFP-miR, which has co-cistronic expression of EmGFP, allowing for determination of transfection efficiency by fluorescence microscopy. The vectors, pcDNA™6.2-GW/EmGFP-miR-Beclin1 (a) and -Beclin1 (b), were sequence-verified, and cells were co-transfected with both vectors to achieve maximal knockdown. To control for nonspecific RNAi effects, the construct pcDNA™6.2-GW/EmGFP-miR-LacZ (targeting β-galactosidase) was used as a control. Simulated Ischemia/Reperfusion (sI/R)—Cells were plated in 14-mm diameter glass bottom microwell dishes (MatTek), and ischemia was introduced by a buffer exchange to ischemia-mimetic solution (in mm: 125 NaCl, 8 KCl, 1.2 KH2PO4, 1.25 MgSO4, 1.2 CaCl2, 6.25 NaHCO3, 5 sodium lactate, 20 HEPES, pH 6.6) and placing the dishes in hypoxic pouches (GasPak™ EZ, BD Biosciences) equilibrated with 95% N2, 5% CO2. After 2 h of ischemia, reperfusion was initiated by a buffer exchange to normoxic Krebs-Henseleit solution (KH, in mm: 110 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.25 MgSO4, 1.2 CaCl2, 25 NaHCO3, 15 glucose, 20 HEPES, pH 7.4) and incubation at 95% room air, 5% CO2. Controls incubated in normoxic KH solution were run in parallel for each condition for periods of time that corresponded with those of the experimental groups. Under control conditions cell viability was not compromised. Wide Field Fluorescence Microscopy—Cells were observed through a Nikon TE300 fluorescence microscope (Nikon) equipped with a ×10 lens (0.3 N.A., Nikon), a ×40 Plan Fluor, and a ×60 Plan Apo objective (1.4 N.A. and 1.3 N.A. oil immersion lenses; Nikon), a Z-motor (ProScanII, Prior Scientific), a cooled CCD camera (Orca-ER, Hamamatsu), and automated excitation and emission filter wheels controlled by a LAMBDA 10–2 (Sutter Instrument) operated by MetaMorph 6.2r4 (Universal Imaging). Fluorescence was excited through an excitation filter for fluorescein isothiocyanate (HQ480/×40) and Texas Red (D560/×40). Fluorescent light was collected via a polychroic beam splitter (61002bs) and an emission filter for fluorescein isothiocyanate (HQ535/50m) and Texas Red (D630/60m). All filters were from Chroma. Acquired wide field Z-stacks were routinely deconvolved using 10 iterations of a three-dimensional blind deconvolution algorithm (AutoQuant) to maximize spatial resolution. Unless stated otherwise, representative images shown are maximum projections of Z-stacks taken with 0.3- μm increments capturing total cellular volume. Quantification of Cellular Autophagosome Content—LC3 forms I and II are known to be differentially recognized by the LC3 antibodies (19Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1117) Google Scholar). Furthermore, in our hands immunodetection of endogenous LC3 in HL-1 cells was inconclusive (data not shown). Therefore, cellular contents of autophagosomal structures were quantified via fluorescence imaging of GFP-LC3 (20Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar) or mCherry-LC3. To generate pmCherry-LC3, mCherry was amplified from the pRSET-mCherry vector (21Shaner N.C. Campbell R.E. Steinbach P.A. Giepmans B.N. Palmer A.E. Tsien R.Y. Nat. Biotechnol. 2004; 22: 1567-1572Crossref PubMed Scopus (3507) Google Scholar) and swapped with enhanced GFP of the vector pEGFP-LC3 (20Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar); HL-1 cells were transfected with (mCherry-/)GFP-LC3, and 48 h after transfection, cells were subjected to sI/R as indicated. Cells were fixed with 4% formaldehyde in phosphate-buffered saline, pH 7.4, for 15 min. To quantify the autophagic response in a population of cells, cells were inspected at ×60 magnification and classified as either having predominantly diffuse (mCherry-/)GFP-LC3 fluorescence or as having numerous punctate (mCherry-/)GFP-LC3 structures, representing autophagic vacuoles, AVs. At least 150 cells were scored in each of three or more independent experiments. For quantification of the autophagic response of single cells, Z-stacks of (mCherry-/)GFP-LC3 fluorescence of 7–10 representative cells per condition in three separate experiments were acquired through the ×60 oil immersion lens with 0.3-μm increments through the entire volume of the cell. Z-stacks were thresholded, and total number and volume of the autophagosome per cell were determined (AutoQuant). Determination of LC3-II Degradation—To analyze autophagic flux, (mCherry-/)GFP-LC3-expressing cells were subjected to the indicated experimental conditions with and without a mixture of the cell-permeable lysosomal inhibitors bafilomycin A1 (100 nm, vacuolar H+-ATPase inhibitor) to inhibit autophagosome-lysosome fusion (22Yamamoto A. Tagawa Y. Yoshimori T. Moriyama Y. Masaki R. Tashiro Y. Cell Struct. Funct. 1998; 23: 33-42Crossref PubMed Scopus (1077) Google Scholar), E64D (5 μg/ml, inhibitor of cysteine proteases, including cathepsin B), and pepstatin A methyl ester (5 μg/ml, cathepsin D inhibitor) to inhibit lysosomal protease activity. Fluorescence microscopy of GFP-LC3 was used to determine cellular autophagosomal content as described above. Activity of the Lysosomal Compartment—LysoTracker Red is a cell-permeable acidotropic probe that selectively labels vacuoles with low internal pH and thus can be used to label functional lysosomes (23Bucci C. Thomsen P. Nicoziani P. McCarthy J. van Deurs B. Mol. Biol. Cell. 2000; 11: 467-480Crossref PubMed Scopus (804) Google Scholar). Following sI/R and control experiments, cells were loaded with 50 nm LysoTracker Red for 5 min in KH solution; the medium was then exchanged with dye-free KH solution, and cells were analyzed by fluorescence microscopy. Activity and intracellular distribution of cathepsin B, a predominant lysosomal protease, were assessed using (z-RR)2-MagicRed-Cathepsin B substrate (B-Bridge). MagicRed cathepsin B substrate was added to the cells during the last 30 min of an experiment according to the manufacturer's instructions. Quantification of Cellular Injury—GFP-Bax (24Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1577) Google Scholar) or mCherry-Bax (25Brady N.R. Hamacher-Brady A. Gottlieb R.A. Biochim. Biophys. Acta. 2006; 1757: 667-678Crossref PubMed Scopus (96) Google Scholar) distribution was used as a parameter to quantify irreversible cellular injury. Cells were cotransfected with (mCherry-/)GFP-Bax and the indicated vectors and allowed to express for 48 h. Cells were then subjected to 2 h of ischemia in hypoxic pouches followed by 5 h of reperfusion, and live cells were analyzed by fluorescence microscopy. Cells were classified as cells with either diffuse or punctate mitochondrial (mCherry-/)GFP-Bax fluorescence. Approximately 300 transfected cells per condition were scored at ×60 magnification in each of three independent experiments. Immunoblotting—Cells were harvested by scraping and centrifugation at 550 × g for 5 min at 4 °C and washed once with cold phosphate-buffered saline, pH 7.4. To prepare whole cell lysates, cell pellets were suspended in cold RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, and 1× complete protease inhibitor mixture (Roche Applied Science)) and left on ice for 20 min. The cell extracts were centrifuged at 20,000 × g for 5 min to remove cellular debris. After addition of sample buffer and reducing agent (Bio-Rad), samples were incubated at 95 °C for 5 min, electrophoresed on SDS-polyacrylamide gels, and transferred to nitrocellulose membranes (Bio-Rad). Immunodetection was performed using antibodies against actin (clone AC-40; Sigma), Bcl-2 (C-2, Santa Cruz Biotechnology), Bcl-xL (H-5, Santa Cruz Biotechnology), Beclin1 (D-18; Santa Cruz Biotechnology), and fluorescent protein (BD Biosciences). Attempts to detect endogenous LC3-I and -II using F-14 and H-50 (Santa Cruz Biotechnology) and A0973 (Biosignatures) antibodies were unsatisfactory because of inconsistencies in immunoreactivity and nonspecificity, perhaps due to the fact that these are mouse antibodies being used against mouse proteins. Blots shown are representative of at least three independent experiments. Statistics—The probability of statistical differences between experimental groups was determined by the Student's t test. Values are expressed as mean ± S.E. of at least three independent experiments unless stated otherwise. sI/R Induces Programmed Cell Death in HL-1 Cardiac Myocytes—The HL-1 cell line is an excellent model for studying many aspects of cardiac cell physiology (26White S.M. Constantin P.E. Claycomb W.C. Am. J. Physiol. 2004; 286: H823-H829Crossref PubMed Scopus (333) Google Scholar). In our hands HL-1 cells reproducibly underwent PCD in response to simulated I/R via pathways resembling in vivo cardiac I/R injury (25Brady N.R. Hamacher-Brady A. Gottlieb R.A. Biochim. Biophys. Acta. 2006; 1757: 667-678Crossref PubMed Scopus (96) Google Scholar). One key feature of sI/R-induced cell death is the participation of the pro-apoptotic Bcl-2 protein Bax in the mitochondrial death pathway. Bax activation, a point-of-noreturn in the PCD pathway, is reflected by a redistribution from the cytosol to punctate clusters at the mitochondria and can be quantified via fluorescent imaging of a GFP-Bax fusion protein (Fig. 1A) (24Wolter K.G. Hsu Y.T. Smith C.L. Nechushtan A. Xi X.G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1577) Google Scholar). Using GFP-Bax redistribution as an index to monitor activation of PCD, we found that sI/R induced PCD in a hypoxia/reoxygenation-dependent manner (Fig. 1B). Overexpression of both Bcl-2 and Bcl-xL, known protectors against cardiac I/R injury (27Brocheriou V. Hagege A.A. Oubenaissa A. Lambert M. Mallet V.O. Duriez M. Wassef M. Kahn A. Menasche P. Gilgenkrantz H. J. Gene Med. 2000; 2: 326-333Crossref PubMed Scopus (155) Google Scholar, 28Huang J. Nakamura K. Ito Y. Uzuka T. Morikawa M. Hirai S. Tomihara K. Tanaka T. Masuta Y. Ishii K. Kato K. Hamada H. Circulation. 2005; 112: 76-83Crossref PubMed Scopus (49) Google Scholar, 29Imahashi K. Schneider M.D. Steenbergen C. Murphy E. Circ. Res. 2004; 95: 734-741Crossref PubMed Scopus (168) Google Scholar), significantly reduced sI/R-induced GFP-Bax redistribution, further demonstrating suitability of the model (Fig. 1, C and D). Cellular Autophagosomal Content Is Increased during the Early Phase of sI/R Injury—We used HL-1 cells to explore the role of autophagy during sI/R injury. To determine whether autophagic activity is modulated in response to sI/R, we first characterized changes in cellular autophagosomal content using high resolution three-dimensional imaging of GFP-LC3. During the initiation of autophagy, cytosolic LC3 (LC3-I) is cleaved and lipidated to form LC3-II (19Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1117) Google Scholar, 30Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (940) Google Scholar). LC3-II is then recruited to the autophagosomal membrane (31Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). Thus, punctate GFP-LC3-labeled structures represent autophagosomes, also referred to as autophagic vacuoles (AVs). Importantly, overexpression of (GFP-)LC3 does not affect autophagic activity, and transgenic mice expressing GFP-LC3 display no detectable abnormalities (12Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1942) Google Scholar, 32Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (714) Google Scholar). We transfected HL-1 cardiac myocytes with GFP-LC3 and compared the abundance of AVs in cells subjected to sI/R to normoxic control cells. Under normoxic conditions in KH solution, GFP-LC3 was diffusely distributed throughout the cell, with very few detectable AVs (Fig. 2A, upper panel). Cells subjected to sI/R, however, displayed increased numbers of AVs (Fig. 2A, bottom panel). In addition, in control cells the few pre-existing AVs were randomly distributed, whereas AVs in cells subjected to sI/R were typically more clustered at the center of the cell. This distinctive distribution contrasts with the autophagic response to starvation in hepatocytes, where no such clustering was observed (33Kochl R. Hu X.W. Chan E.Y. Tooze S.A. Traffic. 2006; 7: 129-145Crossref PubMed Scopus (345) Google Scholar). To quantify the increase in GFP-LC3-labeled AVs, the percentage of cells displaying numerous punctate GFP-LC3 structures was determined. Only a small fraction of cells displayed punctate GFP-LC3 fluorescence when incubated in fully supplemented medium or KH solution (Fig. 2B). In cells subjected to sI/R, however, the number of cells with numerous AVs was significantly increased (Fig. 2B). Quantitative analysis performed on Z-stacks of GFP-LC3 fluorescence revealed that sI/R significantly increased the number of AVs per cell and, likewise, the total autophagosomal volume (Fig. 2C). Changes in Autophagic Activity during Ischemia and Reperfusion—Our results demonstrate that cellular AV content was increased early in the reperfusion period. We subsequently addressed the effect of sI/R on actual autophagic activity. Autophagy involves the delivery of the autophagosomes and their contents to lysosomes that contain the degradative enzymes needed to complete the catabolic processes of autophagy (1Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar). Therefore, the increased presence of AVs may reflect enhanced formation of AVs, impaired fusion of AVs with lysosomes to generate autophagolysosomes, or a combination of the two. Moreover, LC3-II may be removed by lysosomal degradation at a rate that exceeds our imaging capabilities, i.e. the transit is so rapid and/or the AVs so small that only a few AVs can be detected at any given time. Accordingly, a low number of GFP-LC3-labeled AVs may be due to either low or high autophagic activity. To characterize autophagic activity, we therefore determined two relevant parameters of autophagosome-lysosome fusion, the index of LC3-II degradation and downstream lysosomal activity. Flux of LC3-II Degradation during sI/R—Using an approach based on the inhibition of downstream lysosomal degradation of AVs and their cargo, we determined whether the increase in cellular AVs during sI/R was indicative of increased or impaired autophagy. Cells were subjected to various experimental conditions and treated with a mixture of lysosomal inhibitors to inhibit autophagolysosome formation (with bafilomycin A1) and lysosomal protease activity (with E64D and pepstatin A). By analyzing the lysosomal inhibitor-mediated increase in GFP-LC3-II (AV) accumulation within a cell population, we were able to obtain a quantitative index of the flux of AV formation and degradation. Bar graphs with offset superimposed bars depict the percentage of cells exhibiting high AV levels in the absence and presence of lysosomal inhibitors, per condition. The difference between the two bars (see values in graphs) is a measure of the percentage of cells demonstrating high autophagic activity, or flux. We found that in KH solution AV content was dramatically increased in the presence of inhibitors (Fig. 3, A and B). Thus, under control conditions in KH solution, which lacks the serum and amino acid component of full medium, autophagy was strongly active. Notably, this response is only revealed through the use of inhibitors; based on GFP-LC3 imaging alone (Fig. 2), low autophagic activity in KH solution would be incorrectly assumed. sI/R augmented the number of cells with increased numbers of AVs (Fig. 3B). Under lysosomal inhibition the number of cells with high AV content was increased only slightly more, indicating that the previously described increase in cellular AV content in sI/R (Fig. 2 and 3B) is a reflection of an accumulation of AVs, presumably due to impairment in the autophagic pathway at a point(s) following AV formation and before AV degradation. As the level of AV accumulation was substantially smaller than the inhibitor-mediated response seen in KH solution for the same period of time, it can be concluded that autophagy is also impaired at the level of AV formation. Most AVs were formed during the reperfusion period, as cells fixed immediately after the ischemic period were essentially devoid of AVs, either with or without lysosomal inhibitors, indicating a complete blockage of autophagy during the ischemic period (Fig. 3C). Interestingly, hypoxia was a necessary component of the insult, as cells incubated in ischemia/mimetic solution alone, under normoxic conditions, exhibited only a minor reduction of autophagic flux, which recovered completely upon reperfusion (Fig. 3B). Lysosomal Activity during sI/R—One possible explanation for the observed accumulation of AVs during sI/R was a nonfunctional lysosomal compartment. To investigate down-stream lysosomal activity, HL-1 cells were incubated in LysoTracker Red, which labels the highly acidic lysosomal vacuoles and thus reports activity of the vacuolar H+-ATPase (v-ATPase). Before and after sI/R, we observed similar patterns of LysoTracker Red fluorescence, indicating that, consistent with its importance in cell survival during I/R (34Karwatowska-Prokopczuk E. Nordberg J.A. Li H.L. Engler R.L. Gottlieb R.A. Circ. Res. 1998; 82: 1139-1144Crossref PubMed Scopus (75) Google Scholar), activity of the v-ATPase is maintained during the reperfusion period (Fig. 4A). Furthermore, we determined the activity and subcellular localization of cathepsin B, a predominant lysosomal protease, using a MagicRed substrate that fluoresces when cleaved by cathepsin B (35Lamparska-Przybysz M. Gajkowska B. Motyl T. J. Physiol. Pharmacol. 2005; 56: 159-179PubMed Google Scholar). We did not detect a decrease in cathepsin B activity following sI/R, as MagicRed fluorescence was still punctate (lysosomal) and displayed an intensity comparable with the normoxic control (Fig. 4B). Moreover, we found that cathepsin B activity was not detected in the cytosol, indicating that cathepsin B is not released from the lysosomes following sI/R. Together, these results indicate a functional lysosomal compartment durin" @default.
• W2014943313 created "2016-06-24" @default.
• W2014943313 creator A5017792849 @default.
• W2014943313 creator A5031285183 @default.
• W2014943313 creator A5072292859 @default.
• W2014943313 date "2006-10-01" @default.
• W2014943313 modified "2023-09-30" @default.
• W2014943313 title "Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes" @default.
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