This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/6/914    most recent 01.RES.0000261924.76669.36v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsui, Y. Articles by Sadoshima, J. Search for Related Content PubMed PubMed Citation Articles by Matsui, Y. Articles by Sadoshima, J. Related Collections Cardio-renal physiology/pathophysiology Animal models of human disease Apoptosis Cell signalling/signal transduction Heart failure - basic studies

(Circulation Research. 2007;100:914.)
© 2007 American Heart Association, Inc.

Integrative Physiology

Distinct Roles of Autophagy in the Heart During Ischemia and Reperfusion

Roles of AMP-Activated Protein Kinase and Beclin 1 in Mediating Autophagy

Yutaka Matsui, Hiromitsu Takagi, Xueping Qu, Maha Abdellatif, Hideyuki Sakoda, Tomoichiro Asano, Beth Levine, Junichi Sadoshima

From the Cardiovascular Research Institute (Y.M., H.T., M.A., J.S.), Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark; Departments of Internal Medicine (X.Q., B.L.) and Microbiology (B.L.), University of Texas Southwestern Medical Center, Dallas; Department of Internal Medicine (H.S.), Graduate School of Medicine, University of Tokyo, Japan; and Division of Molecular Medical Science (T.A.), Programs for Biomedical Research, Hiroshima University, Japan.

Correspondence to Junichi Sadoshima, Cardiovascular Research Institute, UMDNJ, 185 S Orange Ave, MSB G-609, Newark, NJ 07103. E-mail Sadoshju{at}umdnj.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autophagy is an intracellular bulk degradation process for proteins and organelles. In the heart, autophagy is stimulated by myocardial ischemia. However, the causative role of autophagy in the survival of cardiac myocytes and the underlying signaling mechanisms are poorly understood. Glucose deprivation (GD), which mimics myocardial ischemia, induces autophagy in cultured cardiac myocytes. Survival of cardiac myocytes was decreased by 3-methyladenine, an inhibitor of autophagy, suggesting that autophagy is protective against GD in cardiac myocytes. GD-induced autophagy coincided with activation of AMP-activated protein kinase (AMPK) and inactivation of mTOR (mammalian target of rapamycin). Inhibition of AMPK by adenine 9-ß-D-arabinofuranoside or dominant negative AMPK significantly reduced GD-induced autophagy, whereas stimulation of autophagy by rapamycin failed to cause an additive effect on GD-induced autophagy, suggesting that activation of AMPK and inhibition of mTOR mediate GD-induced autophagy. Autophagy was also induced by ischemia and further enhanced by reperfusion in the mouse heart, in vivo. Autophagy resulting from ischemia was accompanied by activation of AMPK and was inhibited by dominant negative AMPK. In contrast, autophagy during reperfusion was accompanied by upregulation of Beclin 1 but not by activation of AMPK. Induction of autophagy and cardiac injury during the reperfusion phase was significantly attenuated in beclin 1^+/– mice. These results suggest that, in the heart, ischemia stimulates autophagy through an AMPK-dependent mechanism, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1–dependent but AMPK-independent mechanism. Furthermore, autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion.

Key Words: autophagy • AMP-activated protein kinase (AMPK) • beclin 1 • ischemia/reperfusion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autophagy is an intracellular bulk degradation process whereby cytosolic, long-lived proteins and organelles are degraded and recycled.¹ Autophagy occurs at basal levels but can be further induced by stresses, such as nutrient depletion.² Autolysosomal degradation of membrane lipids and proteins generates free fatty acids and amino acids, which can be reused to maintain mitochondrial ATP production and protein synthesis, and promote cell survival. Disruption of this pathway prevents cell survival in diverse organisms.² Interestingly, autophagy also promotes programmed cell death in some circumstances.^3,4 Thus, autophagy has a dual role in cell survival, although, in cardiac myocytes, it remains to be elucidated whether autophagy is required for survival, and is thereby salutary, or whether it mediates cell death, and is detrimental during pathologically relevant stresses, such as ischemia and reperfusion.

The mTOR (mammalian target of rapamycin) pathway is a key regulator of cell growth and proliferation and integrates signals regarding nutrients and growth factors to regulate many cellular processes, including ribosome biogenesis and metabolism.⁵ The class I phosphatidylinositol 3-kinase/TOR pathway has been identified as a negative regulator of autophagy in mammalian cells.^6,7 In addition, cellular ATP depletion also markedly inhibits mTOR without affecting phosphatidylinositol 3-kinase activation or intracellular amino acid levels.⁸ In this case, in response to changes in the intracellular ATP/AMP ratio, 5'-AMP-activated protein kinase (AMPK) is activated and phosphorylates TSC2, thereby inhibiting mTOR.⁹ Thus, the AMPK–mTOR pathway is thought to be an important regulator of autophagy in response to starvation,^10,11 although there has been little direct evidence to support the critical involvement of AMPK in regulating autophagy of cardiac myocytes.

Autophagy is commonly observed in the heart with acute and chronic ischemia, heart failure, and aging.¹² We have recently shown that repetitive myocardial stunning induces autophagy in the pig heart.¹³ In this model, although autophagy was observed in an area of the myocardium with less apoptosis, heart function was fully recovered after normalization of the coronary flow,¹³ suggesting that autophagy may promote survival of hibernating myocardium. Induction of autophagy is also essential for survival of cardiac myocytes during neonatal starvation in vivo.¹⁴ On the other hand, inhibition of autophagy, by 3-methyladenine (3-MA), an inhibitor of class III phosphatidylinositol 3-kinase, prevents death of H9c2 cells in vitro.¹⁵ Thus, it remains unclear whether autophagy is required for survival or whether it mediates cell death and is detrimental for cardiac myocytes under pathological conditions. Elucidation of the function of autophagy in cardiac myocytes under stress may lead to the development of novel strategies to attain cardioprotection.

Thus, the goals of this study were (1) to establish both in vitro and in vivo models of autophagy in the heart and cardiac myocytes therein; (2) to determine the molecular mechanism mediating autophagy, including the AMPK–mTOR pathway and Beclin 1–dependent mechanisms, in cardiac myocytes; and (3) to examine the role of autophagy in mediating either survival or death of cardiac myocytes in response to ischemia and reperfusion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

For starvation experiments, cardiac myocytes were washed 3 times with PBS and incubated in complete cardiac myocyte medium (CM), glucose-free medium, or amino acid–free medium. Glucose-free medium consisted of glucose-free and serum-free DMEM (11966-025; Invitrogen). Amino acid–free medium consisted of serum-free Hank’s balanced salt solution (24020-117, Invitrogen). Heterozygous green fluorescent protein/light chain 3 (GFP-LC3) transgenic mice (strain GFP-LC3 no. 53, RIKEN BioResource Center, C57BL/6J background) containing a rat LC3/enhanced GFP fusion under the control of the chicken ß-actin promoter,¹⁶ heterozygous beclin 1 knockout mice (C57BL/6J background),¹⁷ and transgenic mice with cardiac specific expression of a dominant negative ₂ subunit of AMPK (D157A) (FVB background)¹⁸ were bred in house.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glucose Deprivation Strongly Induces Cardiac Myocyte Autophagy
We searched for starvation conditions in which autophagy is induced in cardiac myocytes in vitro. Serum starvation, for up to 24 hours, did not increase the LC3-II to LC3-I ratio (LC3-II/LC3-I) (Figure 1A), which is an established indicator of autophagy,¹⁹ suggesting that serum starvation alone is not sufficient to induce autophagy in cardiac myocytes. Under glucose deprivation (GD), however, LC3-II/LC3-I was significantly elevated compared with under CM (Figure 1B). Amino acid deprivation (AAD) also increased LC3-II/LC3-I, but to a lesser extent than GD (Figure 1B). Punctate fluorescence staining, produced by exogenously introduced GFP-LC3, is used as a reliable indicator of autophagy.¹⁹ Either GFP-LC3 or GFP alone was expressed in cardiac myocytes through adenovirus-mediated transduction. When GFP-LC3 was overexpressed in control myocytes, diffuse cytoplasmic staining was observed (Figure 1C). After GD, however, punctate staining of GFP-LC3 was extensively induced. AAD also increased GFP-LC3 dots, but to a lesser extent. When GFP alone was expressed, the punctate staining pattern of GFP was not observed (data not shown). Induction of autophagy was also confirmed using electron microscopy. Under normal conditions, autophagosomes and autolysosomes were rarely detected (Figure 1Da), whereas their levels were increased with AAD (Figure 1Db) and were even greater with GD (Figure 1Dc). These results suggest that cardiac myocytes undergo autophagy in response to AAD or GD, but not to serum starvation alone.

View larger version (44K): [in this window] [in a new window]   Figure 1. GD strongly induces autophagy in cardiac myocytes. A and B, Immunoblot analyses of LC3-I and LC3-II. A, Cardiac myocytes were incubated with CM or serum-free medium for 3 hours. Similar results were obtained after 24 hours of treatment with serum-free medium. B, Cardiac myocytes were treated with CM or glucose-free [Glu (–)] or amino acid–free [a.a.(–)] medium for 3 hours. In A and B, results represent data from 4 independent experiments. C and D, Cardiac myocytes were treated with CM (a), amino acid–free medium (b), or glucose-free medium (c) for 3 hours. In C, cardiac myocytes were transduced with Ad-GFP-LC3 (30 multiplicities of infection) 24 hours before GD or AAD. Representative images of GFP-LC3 staining (C) and electron microscopy (D) are shown. In C, light microscopic quantitation of GFP-LC3 dots is shown. Results are from 3 independent experiments. In D, autophagosomes and autolysosomes are indicated by arrows.

Inhibition of Autophagy by 3-MA Enhances Cardiac Myocyte Death in Response to GD
Cardiac myocytes were treated with 3-MA for 24 hours under GD. Under these conditions, the previously observed increases in LC3-II/LC3-I and GFP-LC3 dots were not observed, indicating that starvation-induced autophagy was inhibited by 3-MA in cardiac myocytes (Figure 2A and 2B). GD reduced cell viability to 60% at 72 hours (Figure 2C). GD-induced cell death was partially but significantly attenuated by either expression of Bcl-xL or treatment with a caspase inhibitor, suggesting that apoptosis plays an important role in mediating GD-induced cell death. Inhibition of autophagy by 3-MA significantly reduced cell viability and increased both caspase-dependent and -independent cell death in the presence of GD at 72 hours, suggesting that autophagy is protective against GD (Figure 2D; Figures I and II in the online data supplement). GD significantly reduced the cellular ATP content in cardiac myocytes and 3-MA further reduced it, consistent with the notion that autophagy preserves cellular ATP content during GD (supplemental Figure III).

View larger version (31K): [in this window] [in a new window]   Figure 2. Inhibition of autophagy and enhanced GD-induced cell death by 3-MA. Myocytes were treated with CM, glucose-free [Glu (–)] or amino acid–free [a.a.(–)] medium with or without 3-MA (10 mmol/L) for 24 hours. A, Immunoblot analyses of LC3-I and LC3-II. Results are from 3 independent experiments. B, Myocytes were transduced with Ad-GFP-LC3. After 24 hours, myocytes were subjected to GD with (b) or without (a) 3-MA (10 mmol/L). Representative images of GFP-LC3 staining are shown. C and D, Time course of the cell viability after GD estimated using the Cell Titer Blue assay. Some myocytes were transduced with adenovirus harboring LacZ or Bcl-xL (30 multiplicities of infection) 24 hours before GD (C) or treated with a pan-caspase inhibitor (ApoBlock, 100 µmol/L) (C) or 3-MA (10 mmol/L) (D) before GD. Results shown are from 3 independent experiments.

GD Activates AMPK and Inhibits mTOR and p70^S6K
Unique among many signaling molecules, the activity of mTOR is regulated not only by cellular levels of amino acids but also by those of ATP.⁸ mTOR activity is negatively regulated by AMPK, a sensitive nutrient sensor.⁹ When the cellular level of ATP/AMP is decreased, AMPK is activated and phosphorylates TSC2, thereby inhibiting mTOR.⁹ Previous studies have suggested that mTOR plays an important role in mediating starvation-induced autophagy.² Although the involvement of AMPK in starvation-induced autophagy has been shown recently in hepatocytes,¹⁰ this notion has not been clearly demonstrated in cardiac myocytes. Thus, we examined whether activation of AMPK and/or inhibition of mTOR coincided with the occurrence of autophagy in response to GD. GD resulted in a significant increase in the LC3-II/LC3-I and Thr172 AMPK phosphorylation, both of which occurred within 2 hours of GD and persisted for more than 24 hours (Figure 3A and 3B). Activation of autophagy and AMPK by GD also coincided with decreases in Ser2481 phosphorylation of mTOR (Figure 3A and 3B). In addition, GD induced increases in Thr56 phosphorylation of eEF2, a downstream target of AMPK, and decreases in Thr389 phosphorylation of p70^S6K, a downstream target of mTOR (Figure 3A and 3B). GD did not significantly affect protein expression of Beclin 1, another critical mediator of autophagy (supplemental Figure IV). These results suggest that GD activates AMPK and inhibits mTOR, accompanied by stimulation of autophagy in cardiac myocytes.

View larger version (35K): [in this window] [in a new window]   Figure 3. GD increases AMPK and eEF2 phosphorylation and decreases p70S6K and mTOR phosphorylation. Cardiac myocytes were treated with CM or glucose-free medium. Cell lysates were subjected to immunoblots for detection of LC3-I, LC3-II, p-AMPK (Thr172), AMPK, p-eEF2 (Thr56), eEF2, p-p70S6K (Thr389), p70S6K, p-mTOR (Ser2481), mTOR, and -actin, where p indicates phosphorylated forms. A, Representative immunoblots. B, Quantitative analysis of protein expression. Data are mean of 3 culture preparations.

Inhibition of AMPK Suppresses Autophagy While Increasing Cell Death in Response to GD
To examine whether AMPK is a critical mediator of autophagy, we treated cardiac myocytes with adenine 9-ß-D-arabinofuranoside (Ara-A) (1 mmol/L), an AMPK inhibitor.²⁰ Ara-A decreased phosphorylation of AMPK in response to GD, accompanied by a nearly complete lack of increase in LC3-II/LC3-I (Figure 4A) and reduced cell viability (Figure 4B) under GD. Specific inhibition of AMPK, together with reduced phosphorylation of acetyl–coenzyme A carboxylase, an endogenous substrate of AMPK, and activation of mTOR, was also attained by adenovirus transduction of dominant negative AMPK (DN-AMPK), which was accompanied by attenuation of both the increase in LC3-II/LC3-I and cell survival after GD (supplemental Figure V). These results suggest that activation of AMPK is required for induction of autophagy and protection of cardiac myocytes from cell death resulting from GD. Although treatment of cardiac myocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a stimulator of AMPK¹¹ increased Thr172 phosphorylation of AMPK more than GD, it failed to increase LC3-II/LC3-I within 24 hours (Figure 4C), suggesting that activation of AMPK is required, but not sufficient, for induction of autophagy by GD.

View larger version (47K): [in this window] [in a new window]   Figure 4. The role of AMPK and mTOR in mediating autophagy. A (left), Cardiac myocytes were incubated with CM or glucose-free [Glu (–)] medium for 24 hours in the presence or absence of Ara-A (1 mmol/L). Cell lysates were subjected to immunoblot analyses for detection of LC3-I, LC3-II, phospho-AMPK (Thr172), AMPK, and -actin. A (right), Quantitative analysis of LC3-II/LC3-I. Data are mean±SEM from 3 culture preparations. B, Augmentation of GD-induced myocyte death by Ara-A (1 mmol/L). Cell viability was determined by the Cell Titer Blue assay after 48 hours of treatment with CM or GD. The data are from 3 independent experiments. C (left), Cardiac myocytes were treated with CM, glucose-free medium, or serum free medium with AICAR (0.5 mmol/L). Cell lysates were subjected to immunoblotting for detection of LC3-I, LC3-II, phospho-AMPK (Thr172), and -actin. C (right), Quantitative analysis of LC3-II/LC3-I at the indicated time points of AICAR treatment. Data are from three culture preparations. D (left), Cardiac myocytes were treated with CM, GD, or serum-free medium with rapamycin (10 ng/mL) or GD with rapamycin. Cell lysates were subjected to immunoblotting for detection of LC3-I, LC3-II, phospho-p70S6K, and -actin. D (right), Quantitative analysis of LC3-II/LC3-I at the indicated time points of rapamycin treatment. Data are means from 3 culture preparations.

To examine whether inactivation of mTOR is sufficient to induce autophagy, myocytes were treated with rapamycin. Rapamycin induced time-dependent inhibition of Thr389 phosphorylation of p70^S6K, suggesting that mTOR was inhibited. Under these conditions, LC3-II/LC3-I was significantly increased, suggesting that inhibition of mTOR is sufficient to induce autophagy. Furthermore, in the presence of GD, rapamycin treatment failed to show an additive effect on the increase in LC3-II/LC3-I (Figure 4D). Thus, GD and rapamycin use a common mechanism to induce autophagy, namely the inhibition of mTOR.

Ischemia or Ischemia/Reperfusion Injury Induces Autophagy in the Heart
To examine the role of autophagy in regulating cardiac myocyte survival/death in vivo, we searched for conditions in which autophagy is induced in the heart. The effect of GD on intracellular ATP content in cardiac myocytes in vitro is mimicked by myocardial ischemia in vivo. Thus, we examined whether autophagy was induced in mouse hearts with either ischemia (20 minutes) or ischemia (20 minutes)/reperfusion (20 minutes). Both ischemia and ischemia/reperfusion (I/R) increased LC3-II/LC3-I (Figure 5A and 5B). To further confirm induction of autophagosomes by myocardial ischemia and I/R, we used GFP-LC3 transgenic mice,¹⁶ in which the punctate staining pattern of GFP-LC3 can be observed as a sensitive indicator of autophagy. The number of GFP-LC3 dots was significantly increased by ischemia alone and was further increased after I/R (Figure 5C and 5D). Parallel immunoblot analyses were conducted to detect the activity of the signaling molecules known as energy sensors. Although Thr172 phosphorylation of AMPK was increased by ischemia, phosphorylation of AMPK went back to normal after I/R (Figure 5A and 5B). These results suggest that AMPK is activated by ischemia but not by I/R. In contrast, Thr389 phosphorylation of p70^S6K, a downstream target of mTOR, was induced by I/R but not by ischemia alone (Figure 5A), suggesting that mTOR is activated during I/R. These results suggest that autophagy is induced by ischemia and persists during the reperfusion phase. Induction of autophagy in the ischemic phase was accompanied by activation of AMPK, mimicking the condition of GD. In contrast, autophagosome formation in the I/R phase was accompanied by inactivation of AMPK and activation of mTOR.

View larger version (46K): [in this window] [in a new window]   Figure 5. Autophagy is increased in mouse hearts with ischemia or I/R in vivo. A and B, Control male C57BL/6J mice were subjected to 20 minutes of ischemia alone or 20 minutes of ischemia plus 20 minutes of reperfusion (I/R). A, Immunoblot analyses of heart homogenates with anti-LC3, phospho-AMPK, phospho-p70S6K, and -actin antibodies. B, Quantitative analysis of LC3-I, LC3-II, LC3-II/LC3-I ratio, phospho-p7056K and phospho-AMPK. Data are from 3 independent experiments. C and D, GFP-LC3 transgenic mice were subjected to 20 minutes of ischemia and 20 minutes to 2 hours of reperfusion or sham operation. C, Representative images of GFP-LC3 in LV tissue sections in GFP-LC3 transgenic mice after ischemia or I/R injury. D, Quantitative analysis of GFP-LC3 dots. Data are from 3 independent experiments.

Autophagy Induced by Myocardial Ischemia Was Attenuated in Transgenic Mice With Cardiac-Specific Expression of DN-AMPK
To examine the role of AMPK in mediating autophagy during the ischemic phase in vivo, we used transgenic mice with cardiac specific expression of DN-AMPK (Tg-DN-AMPK).¹⁸ Tg-DN-AMPK mice have a normal cardiac phenotype, and the level of apoptosis and autophagy was not significantly different from control nontransgenic mice at baseline (supplemental Tables I and II and supplemental Figures VI and VII). Tg-DN-AMPK mice have been shown to have enhanced impairment of left ventricular (LV) function and a greater decrease in glucose uptake during no-flow ischemia.¹⁸ Moreover, it has been shown that Tg-DN-AMPK mice have enhanced apoptosis after ischemia and reperfusion.²¹ Because AMPK is activated during ischemia, but not I/R, we examined how inhibition of AMPK affects autophagy during ischemia. We applied 30 minutes of ischemia in Tg-DN-AMPK or control nontransgenic mice. Increases in both Thr172 AMPK phosphorylation and LC3-II/LC3-I after 30 minutes of ischemia were significantly suppressed in Tg-DN-AMPK compared with nontransgenic mice (Figure 6), suggesting that both activation of AMPK and induction of autophagy are suppressed in Tg-DN-AMPK. These results suggest that AMPK is a critical regulator of autophagy in vivo during myocardial ischemia.

View larger version (56K): [in this window] [in a new window]   Figure 6. Induction of autophagy by ischemia is inhibited in Tg-DN-AMPK. A and B, Tg-DN-AMPK or nontransgenic (NTg) mice were subjected to 30 minutes of ischemia or sham operation. A, Immunoblot analyses of heart homogenates after 30 minutes of ischemia with anti-LC3, anti–phospho-AMPK, and anti–-actin antibodies. Note that both activation of AMPK and increases in LC3-II/LC3-I by ischemia were inhibited in Tg-DN-AMPK. B, Quantitative analysis of LC3-II/LC3-I and phospho-AMPK. Data are mean±SEM from 3 independent experiments.

Both Autophagy and Myocardial Injury During I/R Are Attenuated in Beclin 1^+/– Mice
We further examined the mechanism by which autophagosome formation is further increased during I/R, as well as its functional significance. We have shown previously that expression of Beclin 1 is increased in hibernating myocardium in pigs.¹³ In the mouse heart, expression of Beclin 1 in the area at risk was only slightly upregulated by ischemia alone, but its expression was dramatically enhanced after I/R (Figure 7A). To examine the role of Beclin 1 in mediating autophagy and cell survival during I/R, we used beclin 1 heterozygous knockout (beclin 1^+/–) mice. Heterozygous knock out of beclin 1 reduced the amount of Beclin 1 protein expression (Figure 7B) but failed to affect increases in LC3-II/LC3-I after ischemia (20 minutes) or I/R (20 minutes/20 minutes) (data not shown). To evaluate the extent of autophagosome formation, we quantitated the number of GFP-LC3 dots after ischemia and I/R in beclin 1^+/– mice crossed with GFP-LC3 mice (Figure 7C and 7D). The number of GFP-LC3 dots was significantly increased during ischemia and I/R in control GFP-LC3 mice. However, the increase in the number of GFP-LC3 dots during ischemia and reperfusion was significantly attenuated in GFP-LC3/beclin 1^+/– cross mice. Thus, beclin 1 plays an essential role in mediating autophagy, especially at the step between LC3-II and autophagosome formation during both ischemia and reperfusion. Interestingly, the size of myocardial infarction/area at risk after I/R was significantly smaller in beclin 1^+/– mice than in wild-type (WT) mice (Figure 8A and 8B). Furthermore, the number of TUNEL-positive cells in the ischemic area was also smaller in beclin 1^+/– mice than in WT mice (Figure 8C and 8D). The reduced level of cell death in beclin 1^+/– was not accompanied by enhanced activation of Akt (supplemental Figure VIII). The fact that inhibition of autophagy during I/R was accompanied by decreases in apoptosis and myocardial infarction suggests that the functional significance of autophagy may differ between the ischemic and reperfusion phases, behaving in a manner that is protective in the former but possibly detrimental in the latter.

View larger version (28K): [in this window] [in a new window]   Figure 7. I/R-induced autophagy is attenuated in beclin 1+/– mice. A, Wild-type mice were subjected to 20 minutes of ischemia and reperfusion for the indicated time. Immunoblot analyses of heart homogenates with anti–beclin 1 and anti–-actin antibodies. B, Beclin 1 heterozygous (beclin 1+/–) or control WT mice were subjected to 20 minutes of ischemia and 2 hours of reperfusion. Expression of Beclin 1 was determined by immunoblot analyses. The level of Beclin 1 in WT after I/R was set as 1. C and D, GFP-LC3 mice and GFP-LC3/beclin 1+/– mice were subjected to ischemia (20 minutes) alone or ischemia (20 minutes)/reperfusion (2 hours). C, GFP-LC3 dots in WT and beclin 1+/– hearts subjected to I/R. D, GFP-LC3 dots were quantitated.

View larger version (37K): [in this window] [in a new window]   Figure 8. I/R injury is attenuated in beclin 1+/– mice. A, Beclin 1+/– or control WT mice were subjected to 20 minutes of ischemia and 24 hours of reperfusion. Gross appearance of LV tissue sections after Alcian blue and triphenyltetrazolium chloride staining in beclin 1+/– and WT mice subjected to I/R. B, Effect of I/R on the extent of LV myocardial infarction in beclin 1+/– and WT mice. The area at risk/LV size ratio (left) and the myocardial infarction area/area at risk (percentage of infarct size/area at risk) (right) are shown. C, LV tissue sections were subjected to TUNEL and DAPI (4',6-diamidino-2-phenylindole) staining. D, TUNEL-positive myocytes in the ischemic area. The number of TUNEL-positive myocytes was expressed as a percentage of total nuclei detected by DAPI staining (n=4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autophagy is stimulated by nutrient starvation and growth factor deprivation when cells are unable to take up external nutrients.² Interestingly, in cardiac myocytes, GD induced more prominent autophagy than AAD. Primary cultured cardiac myocytes exhibit remarkable tolerance against AAD, compared with transformed cell lines (our unpublished observation, 2006). The lack of cell proliferation or the availability of autocrine growth factors may, in part, alleviate the necessity of activating autophagy in cardiac myocytes under AAD. Autophagy is also activated by decreases in ATP in order for the cell to maintain energy homeostasis and survival.² In this study, autophagy was observed within 20 minutes of ischemia in the mouse heart in vivo, and the number of autophagosomes further increased during the reperfusion phase. Although the possibility that the accumulation of autophagosomes during either ischemia or I/R could be attributable to lysosomal dysfunction cannot be formally excluded, we believe that it is unlikely because increases in GFP-LC3 dots are blocked by inhibition of Beclin 1, a molecule involved in autophagosome formation. The time course of the appearance of autophagy seems faster than that of apoptosis, which is observed predominantly in the reperfusion phase. We have shown previously that autophagy, together with robust upregulation of cathepsin, is induced only after an initial burst of apoptosis in the pig model of chronic ischemia.¹³ Autophagy may serve primarily to maintain energy production during acute ischemia but switch to clearing up damaged organelles during chronic ischemia or reperfusion.

One of the key findings in this report is that AMPK plays an essential role in mediating GD- or ischemia-induced autophagy in cardiac myocytes. Previous studies have suggested that mTOR, a sensor of the cellular nutrient status, is inhibited during energy starvation and that inhibition of mTOR stimulates autophagy.^6,8 Consistent with these findings, our results suggest that induction of autophagy by GD and by rapamycin are mediated by a common mechanism, most likely inhibition of mTOR. The recent discovery that AMPK inhibits mTOR through phosphorylation of TSC2, together with the fact that AMPK is activated by a small decline in cytosolic ATP or an increase in AMP, has made AMPK an attractive candidate as a key regulator of autophagy.^9,10 Yeast cells contain the AMPK homolog, snf1p, which is, indeed, required for autophagy.²² Although AMPK either positively or negatively regulates autophagy in hepatocytes,^10,11 it has not been clearly demonstrated that AMPK is required for autophagy in cardiac myocytes. In this study, strong activation of AMPK and inactivation of mTOR were induced by GD, which was well correlated with induction of autophagy in cardiac myocytes. Induction of autophagy during acute ischemia was also accompanied by activation of AMPK in the mouse heart in vivo. Activation of AMPK, as well as induction of autophagy by GD or ischemia, was significantly reduced by Ara-A or DN-AMPK. Thus, these results suggest that AMPK plays a critical role in mediating autophagy during GD and myocardial ischemia.

Because AMPK is no longer activated during reperfusion, however, autophagosome formation during reperfusion is unlikely to be mediated by AMPK. Importantly, increases in autophagosome formation in the reperfusion phase were accompanied by dramatic upregulation of Beclin 1 protein expression in the myocardium. Because accumulation of autophagosomes during both the ischemia and reperfusion phases was significantly attenuated in beclin 1^+/– mice, upregulation of Beclin 1 seems likely to play an important role in mediating autophagy during the ischemia and reperfusion phases. It should be noted that LC3-II formation during the I/R phase was not attenuated in beclin 1^+/– mice. In yeast, phosphatidyl ethanolamine conjugation of Atg8p (yeast homolog of LC3), equivalent with LC3-II formation, can take place in a atg6/vsp30 (beclin 1 homolog) mutant, whereas the recruitment of Atg8p to the preautophagosome structure does not take place in the same mutant.²³ Thus, it is likely that Beclin 1 is a rate-limiting factor of autophagosome formation from the available LC3-II in cardiac myocytes (See also supplemental Figure X). Importantly, in the reperfusion phase, mTOR is no longer inactive, as evidenced by activation of p70^S6K, an effector of mTOR. Thus, autophagosome formation during the reperfusion phase could take place even in the absence of mTOR inhibition. It has been suggested recently that such mTOR-independent but Beclin 1–dependent autophagy mediates insulin-mediated clearance of protein aggregates in HeLa cells.²⁴ Thus, we propose that the AMPK-dependent signaling mechanism mediates autophagy during GD or ischemia, whereas in the reperfusion phase, beclin 1 mediates autophagy even after the energy supply and the activity of mTOR are restored (supplemental Figure XI).

Although autophagy during energy starvation is generally protective,²⁵ induction of autophagy by other stimuli can lead to autophagic cell death, and thus can be detrimental. Whether or not autophagy is protective against stresses has been controversial in cardiac myocytes. Both protective²⁶ and detrimental^15,27 effects of autophagy have been reported, using either primary cardiac myocytes or cardiac cell lines in vitro (supplemental Table III). The causative role of autophagy in mediating either survival or programmed cell death has not been tested in the adult heart in vivo. In this study, inhibition of GD-induced autophagy by 3-MA or Ara-A/DN-AMPK was accompanied by decreases in cell survival in cardiac myocytes in vitro. Furthermore, in Tg-DN-AMPK mice, a decrease in ischemia-induced autophagy was accompanied by cardiac dysfunction.¹⁸ Although 3-MA, Ara-A, and DN-AMPK could theoretically affect cell survival though autophagy-independent mechanisms,^28–30 these results collectively suggest that autophagy during GD or ischemia is protective in cardiac myocytes.

Importantly, however, our results also suggest that inhibition of autophagy through beclin 1 downregulation was protective during I/R in vivo. Inhibition of autophagy by beclin 1 knockdown also increases the cell viability in response to H₂O₂ in cultured cardiac myocytes in vitro (supplemental Figure IX). Thus, autophagy could be detrimental to cardiac myocytes both in vivo and in vitro in some circumstances. Autophagy may promote cell death often when other modalities of programmed cell death, such as apoptosis, are not available.^3,4 However, apoptotic cell death mechanisms are competent in cardiac myocytes under I/R or H₂O₂. Thus, autophagy may not be a simple back-up mechanism of cell death during I/R. Interestingly, mTOR is not inactivated, whereas endogenous Beclin 1 is upregulated, during reperfusion. Thus, it is possible that mTOR (inhibition)-independent and Beclin 1–dependent autophagy, when cells are not in a starved condition, could be a detrimental process, in contrast to the energy-recovering process during starvation, which is essential for survival. Alternatively, judging from the remarkable upregulation of Beclin 1 during the reperfusion phase, overactivation of autophagy could simply be toxic (supplemental Figure XII).

Furthermore, although beclin 1, an autophagy gene, is important in mediating the localization of other autophagy proteins to pre-autophagosomal structures, as part of a class III phosphatidylinositol 3-kinase complex,¹⁷ the phenotype seen in beclin 1^+/– mice could result from autophagy-independent functions of beclin 1. For example, heterozygous disruption of beclin 1 in mice results in increased spontaneous tumorigenesis.¹⁷ In addition, Beclin 1 augments cis-diamminedichloroplatinum–induced apoptosis by enhancing caspase-9 activity.³¹ Further investigation is therefore needed to clarify the role of autophagy in mediating cell survival or cell death in cardiac myocytes.

In summary, our results indicate that GD and myocardial ischemia stimulate autophagy through AMPK- and Beclin 1–dependent mechanisms. On the other hand, I/R could stimulate autophagy through Beclin 1–dependent mechanisms. Thus, specific modulation of autophagy, such as stimulation of AMPK or inhibition of Beclin 1, might be a novel strategy to enhance survival under ischemia and protect against cardiac myocyte death from reperfusion injury in vivo.

	Acknowledgments

 
We thank Drs Noboru Mizushima (Tokyo Medical Dental University) and Rong Tian (Brigham and Women’s Hospital) for providing GFP-LC3 mice and DN-AMPK mice, respectively; Dr Tamotsu Yoshimori for providing the anti-LC3 antibody; and Daniela Zablocki for critical reading of the manuscript.

Sources of Funding

This work was supported by US Public Health Service grants HL59139, HL67724, HL67727, HL69020, and HL73048 and by American Heart Association Grant 0340123N. Y.M. is supported by the Banyu Life Science Foundation.

Disclosures

None.

	Footnotes

 
Original received December 1, 2006; revision received January 31, 2007; accepted February 20, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004; 6: 463–477.[CrossRef][Medline] [Order article via Infotrieve]

2. Lum JJ, DeBerardinis RJ, Thompson CB. Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol. 2005; 6: 439–448.[CrossRef][Medline] [Order article via Infotrieve]

3. Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S, Baehrecke EH, Lenardo MJ. Regulation of an ATG7-beclin 1 program of autophagic cell death by caspase-8. Science. 2004; 304: 1500–1502.[Abstract/Free Full Text]

4. Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, Tsujimoto Y. Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol. 2004; 6: 1221–1228.[CrossRef][Medline] [Order article via Infotrieve]

5. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006; 124: 471–484.[CrossRef][Medline] [Order article via Infotrieve]

6. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O’Kane CJ, Rubinsztein DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004; 36: 585–595.[CrossRef][Medline] [Order article via Infotrieve]

7. Mousavi SA, Brech A, Berg T, Kjeken R. Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes. Biochem J. 2003; 372: 861–869.[CrossRef][Medline] [Order article via Infotrieve]

8. Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G. Mammalian TOR: a homeostatic ATP sensor. Science. 2001; 294: 1102–1105.[Abstract/Free Full Text]

9. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell. 2003; 115: 577–590.[CrossRef][Medline] [Order article via Infotrieve]

10. Meley D, Bauvy C, Houben-Weerts JH, Dubbelhuis PF, Helmond MT, Codogno P, Meijer AJ. AMP-activated protein kinase and the regulation of autophagic proteolysis. J Biol Chem. 2006; 281: 34870–34879.[Abstract/Free Full Text]

11. Samari HR, Seglen PO. Inhibition of hepatocytic autophagy by adenosine, aminoimidazole-4-carboxamide riboside, and N6-mercaptopurine riboside. Evidence for involvement of amp-activated protein kinase. J Biol Chem. 1998; 273: 23758–23763.[Abstract/Free Full Text]

12. Hein S, Arnon E, Kostin S, Schonburg M, Elsasser A, Polyakova V, Bauer EP, Klovekorn WP, Schaper J. Progression from compensated hypertrophy to failure in the pressure-overloaded human heart: structural deterioration and compensatory mechanisms. Circulation. 2003; 107: 984–991.[Abstract/Free Full Text]

13. Yan L, Vatner DE, Kim SJ, Ge H, Masurekar M, Massover WH, Yang G, Matsui Y, Sadoshima J, Vatner SF. Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A. 2005; 102: 13807–13812.[Abstract/Free Full Text]

14. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, Ohsumi Y, Tokuhisa T, Mizushima N. The role of autophagy during the early neonatal starvation period. Nature. 2004; 432: 1032–1036.[CrossRef][Medline] [Order article via Infotrieve]

15. Aki T, Yamaguchi K, Fujimiya T, Mizukami Y. Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2. Oncogene. 2003; 22: 8529–8535.[CrossRef][Medline] [Order article via Infotrieve]

16. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 2004; 15: 1101–1111.[Abstract/Free Full Text]

17. Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen EL, Mizushima N, Ohsumi Y, Cattoretti G, Levine B. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003; 112: 1809–1820.[CrossRef][Medline] [Order article via Infotrieve]

18. Xing Y, Musi N, Fujii N, Zou L, Luptak I, Hirshman MF, Goodyear LJ, Tian R. Glucose metabolism and energy homeostasis in mouse hearts overexpressing dominant negative alpha2 subunit of AMP-activated protein kinase. J Biol Chem. 2003; 278: 28372–28377.[Abstract/Free Full Text]

19. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000; 19: 5720–5728.[CrossRef][Medline] [Order article via Infotrieve]

20. Pelletier A, Joly E, Prentki M, Coderre L. Adenosine 5'-monophosphate-activated protein kinase and p38 mitogen-activated protein kinase participate in the stimulation of glucose uptake by dinitrophenol in adult cardiomyocytes. Endocrinology. 2005; 146: 2285–2294.[Abstract/Free Full Text]

21. Russell RR 3rd, Li J, Coven DL, Pypaert M, Zechner C, Palmeri M, Giordano FJ, Mu J, Birnbaum MJ, Young LH. AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury. J Clin Invest. 2004; 114: 495–503.[CrossRef][Medline] [Order article via Infotrieve]

22. Wang Z, Wilson WA, Fujino MA, Roach PJ. Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol Cell Biol. 2001; 21: 5742–5752.[Abstract/Free Full Text]

23. Suzuki K, Kirisako T, Kamada Y, Mizushima N, Noda T, Ohsumi Y. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J. 2001; 20: 5971–5981.[CrossRef][Medline] [Order article via Infotrieve]

24. Yamamoto A, Cremona ML, Rothman JE. Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway. J Cell Biol. 2006; 172: 719–731.[Abstract/Free Full Text]

25. Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest. 2005; 115: 2679–2688.[CrossRef][Medline] [Order article via Infotrieve]

26. Hamacher-Brady A, Brady NR, Gottlieb RA. Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes. J Biol Chem. 2006; 281: 29776–29787.[Abstract/Free Full Text]

27. Valentim L, Laurence KM, Townsend PA, Carroll CJ, Soond S, Scarabelli TM, Knight RA, Latchman DS, Stephanou A. Urocortin inhibits Beclin1-mediated autophagic cell death in cardiac myocytes exposed to ischaemia/reperfusion injury. J Mol Cell Cardiol. 2006; 40: 846–852.[CrossRef][Medline] [Order article via Infotrieve]

28. Xue L, Fletcher GC, Tolkovsky AM. Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution. Mol Cell Neurosci. 1999; 14: 180–198.[CrossRef][Medline] [Order article via Infotrieve]

29. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol. 2004; 36: 2491–2502.[CrossRef][Medline] [Order article via Infotrieve]

30. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005; 1: 15–25.[CrossRef][Medline] [Order article via Infotrieve]

31. Furuya D, Tsuji N, Yagihashi A, Watanabe N. Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity. Exp Cell Res. 2005; 307: 26–40.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				E. R. Porrello, A. D'Amore, C. L. Curl, A. M. Allen, S. B. Harrap, W. G. Thomas, and L. M.D. Delbridge Angiotensin II Type 2 Receptor Antagonizes Angiotensin II Type 1 Receptor-Mediated Cardiomyocyte Autophagy Hypertension, June 1, 2009; 53(6): 1032 - 1040. [Abstract] [Full Text] [PDF]

				H. Kanamori, G. Takemura, R. Maruyama, K. Goto, A. Tsujimoto, A. Ogino, L. Li, I. Kawamura, T. Takeyama, T. Kawaguchi, et al. Functional Significance and Morphological Characterization of Starvation-Induced Autophagy in the Adult Heart Am. J. Pathol., May 1, 2009; 174(5): 1705 - 1714. [Abstract] [Full Text] [PDF]

				P. Weisova, C. G. Concannon, M. Devocelle, J. H. M. Prehn, and M. W. Ward Regulation of Glucose Transporter 3 Surface Expression by the AMP-Activated Protein Kinase Mediates Tolerance to Glutamate Excitation in Neurons J. Neurosci., March 4, 2009; 29(9): 2997 - 3008. [Abstract] [Full Text] [PDF]

				A. B. Gustafsson and R. A. Gottlieb Autophagy in Ischemic Heart Disease Circ. Res., January 30, 2009; 104(2): 150 - 158. [Abstract] [Full Text] [PDF]

				W. Zhu, M. H. Soonpaa, H. Chen, W. Shen, R. M. Payne, E. A. Liechty, R. L. Caldwell, W. Shou, and L. J. Field Acute Doxorubicin Cardiotoxicity Is Associated With p53-Induced Inhibition of the Mammalian Target of Rapamycin Pathway Circulation, January 6, 2009; 119(1): 99 - 106. [Abstract] [Full Text] [PDF]

				B. A. Rothermel and J. A. Hill Autophagy in Load-Induced Heart Disease Circ. Res., December 5, 2008; 103(12): 1363 - 1369. [Abstract] [Full Text] [PDF]

				E. Itakura, C. Kishi, K. Inoue, and N. Mizushima Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG Mol. Biol. Cell, December 1, 2008; 19(12): 5360 - 5372. [Abstract] [Full Text] [PDF]

				T. Hara, A. Takamura, C. Kishi, S.-i. Iemura, T. Natsume, J.-L. Guan, and N. Mizushima FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells J. Cell Biol., October 14, 2008; 181(3): 497 - 510. [Abstract] [Full Text] [PDF]

				W.-L. Yen and D. J. Klionsky How to Live Long and Prosper: Autophagy, Mitochondria, and Aging Physiology, October 1, 2008; 23(5): 248 - 262. [Abstract] [Full Text] [PDF]

				R. Maruyama, K. Goto, G. Takemura, K. Ono, K. Nagao, T. Horie, A. Tsujimoto, H. Kanamori, S. Miyata, H. Ushikoshi, et al. Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1599 - H1607. [Abstract] [Full Text] [PDF]

				K. Nishida, O. Yamaguchi, and K. Otsu Crosstalk Between Autophagy and Apoptosis in Heart Disease Circ. Res., August 15, 2008; 103(4): 343 - 351. [Abstract] [Full Text] [PDF]

				A. Melendez and T. P. Neufeld The cell biology of autophagy in metazoans: a developing story Development, July 15, 2008; 135(14): 2347 - 2360. [Abstract] [Full Text] [PDF]

				P. Tannous, H. Zhu, J. L. Johnstone, J. M. Shelton, N. S. Rajasekaran, I. J. Benjamin, L. Nguyen, R. D. Gerard, B. Levine, B. A. Rothermel, et al. Autophagy is an adaptive response in desmin-related cardiomyopathy PNAS, July 15, 2008; 105(28): 9745 - 9750. [Abstract] [Full Text] [PDF]

				J. A. Hill and E. N. Olson Cardiac Plasticity N. Engl. J. Med., March 27, 2008; 358(13): 1370 - 1380. [Full Text] [PDF]

				L. H. Young AMP-Activated Protein Kinase Conducts the Ischemic Stress Response Orchestra Circulation, February 12, 2008; 117(6): 832 - 840. [Full Text] [PDF]

				A. B. Gustafsson and R. A. Gottlieb Heart mitochondria: gates of life and death Cardiovasc Res, January 15, 2008; 77(2): 334 - 343. [Abstract] [Full Text] [PDF]

				N. Mizushima Autophagy: process and function Genes & Dev., November 15, 2007; 21(22): 2861 - 2873. [Abstract] [Full Text] [PDF]

				J. J. Gills, J. LoPiccolo, J. Tsurutani, R. H. Shoemaker, C. J.M. Best, M. S. Abu-Asab, J. Borojerdi, N. A. Warfel, E. R. Gardner, M. Danish, et al. Nelfinavir, A Lead HIV Protease Inhibitor, Is a Broad-Spectrum, Anticancer Agent that Induces Endoplasmic Reticulum Stress, Autophagy, and Apoptosis In vitro and In vivo Clin. Cancer Res., September 1, 2007; 13(17): 5183 - 5194. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/6/914    most recent 01.RES.0000261924.76669.36v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matsui, Y. Articles by Sadoshima, J. Search for Related Content PubMed PubMed Citation Articles by Matsui, Y. Articles by Sadoshima, J. Related Collections Cardio-renal physiology/pathophysiology Animal models of human disease Apoptosis Cell signalling/signal transduction Heart failure - basic studies