Jianjian Shi1, Michelle Surma1, Lei Wei1,2. 1. Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, USA. 2. Department of Cellular and Integrative Physiology, Indiana University, School of Medicine, Indianapolis, Indiana, USA.
Abstract
Doxorubicin is among the essential medicines with a wide antitumor spectrum, but its clinical application is limited by its cardiotoxicity. We recently discovered that ROCK1 is a key molecule in mediating cardiac remodeling in response to various stresses. To determine the roles of ROCK1 in doxorubicin cardiotoxicity, we gave three doses of doxorubicin injections to wild type (WT) and ROCK1-/- mice with one week intervals between treatments, the cumulative dose being 24 mg/kg. ROCK1-/- mice exhibited preserved cardiac function, reduced apoptosis, autophagy and fibrosis compared to the WT mice. To further determine the cellular mechanisms, we have examined the role of ROCK1 in cardiomyocytes using cardiomyocyte-specific knockout mice, MHC-Cre/ROCK1fl/fl, which partially reproduced the cardioprotective characteristics of ROCK1-/- mice, indicating that ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicin cardiotoxicity. To elucidate the molecular mechanisms, a detailed time course study after a single doxorubicin injection at 10 mg/kg was performed in ROCK1-/- and MHC-Cre/ROCK1fl/fl mice. The molecular analysis revealed that both ROCK1-/- and MHC-Cre/ROCK1fl/fl hearts exhibited significant reduction of doxorubicin-induced early responses including increased apoptotic (Bax) and autophagic (p62/SQSTM1 and LC3-II) markers, associated with reduced Beclin 1 phosphorylation on Thr119, supporting reduced Beclin 1-mediated autophagy initiation due to increased association of Beclin 1 with Bcl 2 or Bcl-XL in these hearts compared to the WT or ROCK1fl/fl mice. These results support that ROCK1 deficiency is cardioprotective against doxorubicin-induced cardiotoxicity at least in part through reducing Beclin 1-mediated autophagy initiation in cardiomyocytes and restoring autophagic flux to ameliorate doxorubicin cardiotoxicity.
Doxorubicin is among the essential medicines with a wide antitumor spectrum, but its clinical application is limited by its cardiotoxicity. We recently discovered that ROCK1 is a key molecule in mediating cardiac remodeling in response to various stresses. To determine the roles of ROCK1 in doxorubicincardiotoxicity, we gave three doses of doxorubicin injections to wild type (WT) and ROCK1-/- mice with one week intervals between treatments, the cumulative dose being 24 mg/kg. ROCK1-/- mice exhibited preserved cardiac function, reduced apoptosis, autophagy and fibrosis compared to the WT mice. To further determine the cellular mechanisms, we have examined the role of ROCK1 in cardiomyocytes using cardiomyocyte-specific knockout mice, MHC-Cre/ROCK1fl/fl, which partially reproduced the cardioprotective characteristics of ROCK1-/- mice, indicating that ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicincardiotoxicity. To elucidate the molecular mechanisms, a detailed time course study after a single doxorubicin injection at 10 mg/kg was performed in ROCK1-/- and MHC-Cre/ROCK1fl/fl mice. The molecular analysis revealed that both ROCK1-/- and MHC-Cre/ROCK1fl/fl hearts exhibited significant reduction of doxorubicin-induced early responses including increased apoptotic (Bax) and autophagic (p62/SQSTM1 and LC3-II) markers, associated with reduced Beclin 1 phosphorylation on Thr119, supporting reduced Beclin 1-mediated autophagy initiation due to increased association of Beclin 1 with Bcl 2 or Bcl-XL in these hearts compared to the WT or ROCK1fl/fl mice. These results support that ROCK1 deficiency is cardioprotective against doxorubicin-induced cardiotoxicity at least in part through reducing Beclin 1-mediated autophagy initiation in cardiomyocytes and restoring autophagic flux to ameliorate doxorubicincardiotoxicity.
The anthracyclines, mainly doxorubicin, are among the most widely used and successful drugs to treat a wide spectrum of hematologic malignancies and solid tumors. But anthracycline-induced dose-dependent and cumulative cardiotoxic side effects cause persistent and progressive damage to the cardiovascular system [1-4]. Therefore, their usefulness in cancer treatment is significantly compromised. Children and adolescents are particularly vulnerable to anthracyclinecardiotoxicity. About half of the young adult survivors of childhood cancer have received anthracyclines in their treatment [4-7]. Regardless of the severe cardiotoxicity, anthracyclines are still irreplaceable in cancer therapeutic schemes.Although intensive investigations have improved our insights on anthracyclinecardiotoxicity, the underlying mechanisms have not yet been completely elucidated; therefore new strategies providing effective prevention of cardiac side effects continue being in great demand. Various mechanisms have been proposed; they include, but are not limited to: free radical-induced oxidative stress, damage to nuclear DNA, dysregulation of calcium handling and cellular contractility, suppression of transcription factors that regulate cell survival and sarcomere protein synthesis, disruption of sarcomere stability, and mitochondrial dysfunction in cardiomyocytes [8-13]. Most of these cellular events eventually contribute to cardiomyocyte death. Indeed, there is accumulating experimental evidence that cardiomyocyte apoptosis [13-15] and impaired autophagic function [16-21] play important roles in doxorubicin-induced cardiomyopathy.Autophagy is a cell survival mechanism aimed at maintaining cell and tissue homeostasis under normal as well as stress conditions, including nutrient starvation, metabolic intervention, energy machinery dysfunction, and oxidative stress. Autophagy, characterized by the formation of autophagosomes, is an orchestrated process involving several steps: initiation, nucleation, elongation, maturation and degradation. Numerous studies have demonstrated that doxorubicin-induced cardiac injury is associated with dysregulation in autophagic function [16-21]. Recent studies support the notion that doxorubicin-induced cardiotoxicity causes an over-activation of autophagy initiation due to increased cellular damage while preventing autophagy completion due to deleterious effects on lysosomes, which results in the accumulation of un-degraded protein aggregates or damaged organelles [16-21]. Attenuating autophagic dysregulation in doxorubicin-treated hearts represents an attractive strategy to prevent or mitigate doxorubicin-induced cardiomyopathy [16-21].ROCKs are central regulators of the actin cytoskeleton downstream of the small GTPase RhoA [22-31]. The two ROCK isoforms, ROCK1 and ROCK2, are highly homologous with an overall amino acid sequence identity of 65% [22-24]. We recently found that ROCK1 is a key molecule in mediating apoptotic signaling in cardiomyocytes under pressure overload and in genetically-induced pathological cardiac hypertrophy [32-36]. Using mouse embryonic fibroblasts as an in vitro system, we observed that ROCK1 deficiency has a unique protective benefit of preserving actin cytoskeleton stability, which acts additively with antioxidant treatment to suppress excessive production of doxorubicin-induced reactive oxygen species and apoptosis [37-40]. The present study is focused on the in vivo role of ROCK1 in mediating doxorubicincardiomyopathy, particularly on doxorubicin-induced autophagy dysregulation. We used both systemic ROCK1 deficient mice (ROCK1−/−) and cardiomyocyte-specific ROCK1 knockout mice using MHC-Cre mice [41] crossed into ROCK1fl/fl in this study. In addition to demonstrating an in vivo role of ROCK1 in mediating doxorubicincardiotoxicity and cardiomyocyte apoptosis, we have uncovered a role for ROCK1 in mediating doxorubicin-induced dysregulation of autophagic flux in cardiomyocytes, possibly through promoting Beclin 1-mediated autophagy initiation, further supporting that ROCK1 represents a potential therapeutic target to prevent chemotherapeutic drug doxorubicin-induced heart failure.
RESULTS
ROCK1 deficient mice are protected from doxorubicin cardiotoxicity associated with attenuation of apoptosis and autophagy dysregulation
Mice 8 to 9 weeks old were injected intraperitoneally with doxorubicin at 8 mg/kg or normal saline (NS) once weekly for 3 consecutive weeks, the cumulative dose totaling 24 mg/kg (Figure 1A). This treatment is based on the approach previously described [15, 42], which causes progressive cardiac dysfunction within three weeks after the initial injection, a scenario that mimics early-onset doxorubicin-induced cardiotoxicity in humans. Echocardiograms revealed that doxorubicin treatment resulted in reproducible and progressive left ventricular (LV) dilation in WT mice as evidenced by increased end systolic dimension (LVESD) and end diastolic dimension (LVEDD; Figure 1B) over 3 weeks after initiation of the treatment. Consistent with cardiac dilation, LV contractile function (as measured by LV fractional shortening, FS) was reduced in doxorubicin-treated WT mice (Figure 1C, 2A, 2B). Doxorubicin had a significant impact on body weight (Figure 1D), heart weight (Figure 2C, 2D), and cardiomyocyte size (Figure 2E). For subsequent studies of phenotype, we focused on day 21 after the initial injection, when no significant animal death was detected in WT mice (Figure 2F).
Figure 1
Time course study of doxorubicin cardiotoxicity
(A). Schematic of serial doxorubicin administration protocol. FVB WT and ROCK1 deficient mice 8 to 9 weeks old received three serial injections weekly of normal saline (NS) or doxorubicin (DOX) (8 mg/kg). Mice were sacrificed on day 21 after the initial injection. (B). Representative short-axis echocardiograms from WT mice before each injection, and in 1 week after the 3rd dose. (C). Cardiac function in WT mice, but not in ROCK1 deficient mice, was significantly impaired on day 14, 1 week after the 2nd dose. FS, fractional shortening. (D). DOX significantly affected body weight on day 21 after the initial injection in both WT and ROCK1 deficient mice. N = 10-15 in each group. *p < 0.05 for DOX-injected vs. NS-injected mice. #p < 0.05 for DOX-treated ROCK1 deficient mice vs. DOX-injected WT mice.
Figure 2
ROCK1 deficient mice are protected from doxorubicin cardiotoxicity
(A-B). Echocardiography analysis of WT and ROCK1 deficient mice on day 21 after the initial injection. Cardiac dimension were preserved in DOX-treated ROCK1 deficient mice compared with WT mice. LVESD, left ventricular end systolic dimension; LVEDD, left ventricular end diastolic dimension. N = 8-10 in each group. (C-D). Quantitative analysis of heart weight (C), heart weight (Heart wt)/tibial length (TL) ratios from WT and ROCK1 deficient mice on day 21 after the initial injection. (E). Quantitative analysis of cardiac myocyte area measured from laminin stained sections. Each column represents results obtained from approximately 200 myocytes from at least four hearts per group. ROCK1 deficiency doesn’t prevent DOX-induced reduction in heart weight and cardiomyocyte size. (F). Kaplan-Meier survival curves (over 6 weeks after the initial DOX injection) indicate that the mortality rate was significantly lower in the ROCK1 deficient mice than that in WT mice. N = 10 in each group. p < 0.01 for DOX-injected ROCK1 deficient vs. DOX-injected WT mice.
Time course study of doxorubicin cardiotoxicity
(A). Schematic of serial doxorubicin administration protocol. FVB WT and ROCK1 deficient mice 8 to 9 weeks old received three serial injections weekly of normal saline (NS) or doxorubicin (DOX) (8 mg/kg). Mice were sacrificed on day 21 after the initial injection. (B). Representative short-axis echocardiograms from WT mice before each injection, and in 1 week after the 3rd dose. (C). Cardiac function in WT mice, but not in ROCK1 deficient mice, was significantly impaired on day 14, 1 week after the 2nd dose. FS, fractional shortening. (D). DOX significantly affected body weight on day 21 after the initial injection in both WT and ROCK1 deficient mice. N = 10-15 in each group. *p < 0.05 for DOX-injected vs. NS-injected mice. #p < 0.05 for DOX-treated ROCK1 deficient mice vs. DOX-injected WT mice.
ROCK1 deficient mice are protected from doxorubicin cardiotoxicity
(A-B). Echocardiography analysis of WT and ROCK1 deficient mice on day 21 after the initial injection. Cardiac dimension were preserved in DOX-treated ROCK1 deficient mice compared with WT mice. LVESD, left ventricular end systolic dimension; LVEDD, left ventricular end diastolic dimension. N = 8-10 in each group. (C-D). Quantitative analysis of heart weight (C), heart weight (Heart wt)/tibial length (TL) ratios from WT and ROCK1 deficient mice on day 21 after the initial injection. (E). Quantitative analysis of cardiac myocyte area measured from laminin stained sections. Each column represents results obtained from approximately 200 myocytes from at least four hearts per group. ROCK1 deficiency doesn’t prevent DOX-induced reduction in heart weight and cardiomyocyte size. (F). Kaplan-Meier survival curves (over 6 weeks after the initial DOX injection) indicate that the mortality rate was significantly lower in the ROCK1 deficient mice than that in WT mice. N = 10 in each group. p < 0.01 for DOX-injected ROCK1 deficient vs. DOX-injected WT mice.To elucidate the role of ROCK1 in doxorubicincardiotoxicity, ROCK1−/− mice were treated with doxorubicin in parallel to the WT mice. In contrast to the WT mice, doxorubicin-induced LV dilation was blocked in ROCK1−/− mice, and cardiac function was preserved (Figure 1C, 2A, 2B) over 3 weeks after initiation of the treatment. Doxorubicin had a similar impact on body weight (Figure 1D), heart weight (Figure 2C, 2D), and cardiomyocyte size (Figure 2E) in ROCK1−/− mice compared to WT mice, indicating that the protective effects of ROCK1 deletion are not mediated by inhibition of weight loss and cardiomyocyte atrophy. Kaplan-Meier survival curves over 6 weeks after the initial doxorubicin injection indicate that the mortality rate was significantly lower in the treated ROCK1−/− group (about 10%) than that in the treated WT group (about 70%) (Figure 2F). In addition, all treated WT mice died within 3 months after starting doxorubicin treatment, whereas the mortality rate of treated ROCK1 deficient mice reached to 55% at 3 months and to 70% at 6 months. These results support that ROCK1 deficiency reduces not only cardiac toxicity but also systemic toxicity caused by doxorubicin.Since doxorubicin treatment induced significant increases in cardiomyocyte and non-cardiomyocyte apoptosis [14], we assessed apoptosis by TUNEL staining. The number of TUNEL positive cardiac cells was significantly increased in doxorubicin-treated WT mouse hearts (Figure 3A), and was associated with increased mitochondrial translocation of Bax (Figure 3B). Cardiac fibrosis was also increased in doxorubicin-treated WT mice (Figure 3C). However, these characteristics of doxorubicincardiotoxicity were effectively blocked in ROCK1−/− mice (Figure 3A–3C). Molecular analysis also revealed increased ROCK1 expression in doxorubicin-treated WT mice, associated with increased Bax levels and decreased focal adhesion kinase (FAK) phosphorylation (Figure 3D–3G), further supporting a role of ROCK1 in doxorubicin-induced cardiotoxicity. During autophagy, the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-II, which is recruited to autophagosomal membranes [43]. Interestingly, the levels of LC3-II were increased by about 3-fold in doxorubicin-treated WT hearts but not in ROCK1−/− hearts (Figure 3D, 3H), suggesting that ROCK1 deletion prevents the dysregulation of autophagy induced by doxorubicin. Consistent with this notion, transmission electron microscopy revealed more accumulation of autophagosomes in doxorubicin-treated WT mouse hearts than in ROCK1−/− hearts (Figure 3I). Moreover, this increased LC3-II accumulation was not accompanied by increased levels of Beclin 1 and AMP-activated protein kinase (AMPK) phosphorylation (Figure 3D), suggesting that the accumulation of autophagosomes was not due to the activation of either AMPK-mediated autophagy or a general up-regulation of the autophagy system.
Figure 3
ROCK1 deletion inhibited doxorubicin-induced apoptosis, fibrosis and autophagy
(A). Quantification of total TUNEL positive nuclei per 105 total nuclei in ventricular myocardium from WT and ROCK1 deficient hearts on day 21 after the initial injection. N = 4-6 in each group. (B). Representative images (top) of Western blot analysis of Bax and Cox IV in mitochondrial fraction of ventricular homogenates from WT and ROCK1 deficient hearts. Quantitative analysis (bottom) of immunoreactive bands of Bax (N = 4-6 in each group) expressed as fold change relative to NS-treated WT group. (C). Representative heart sections (top) stained with picrosirius red/Fast green (scale bar, 50 μm) showing collagen deposition. Quantitative analysis (bottom) of the collagen deposition in WT and ROCK1 deficient hearts, expressed as percentage change relative to NS-treated WT hearts. Four to six hearts in each group. At least 10 randomly chosen high power fields per section and four transverse sections from each heart, sampled from the midpoint between the apex and base, were analyzed. (D). Representative images of Western blot analysis of ROCK1, ROCK2, LC3, Beclin 1, p-AMPK-Thr172, Bax, FAK and p-FAK-Tyr397 in ventricular homogenates of WT and ROCK1 deficient hearts on day 21 after the initial injection. (E-H) Quantitative analysis of immunoreactive bands of ROCK1 (E), Bax (F), p-FAK-Tyr397/FAK (G) and LC3-II (H). N = 4-6 in each group. (I). Representative transmission electron microscopy images of WT and ROCK1 deficient hearts on day 21 after the initial injection. DOX-treated WT heart showed increased numbers of autophagic vacuoles (red arrows). Scale bar, 0.5 μm.
ROCK1 deletion inhibited doxorubicin-induced apoptosis, fibrosis and autophagy
(A). Quantification of total TUNEL positive nuclei per 105 total nuclei in ventricular myocardium from WT and ROCK1 deficient hearts on day 21 after the initial injection. N = 4-6 in each group. (B). Representative images (top) of Western blot analysis of Bax and Cox IV in mitochondrial fraction of ventricular homogenates from WT and ROCK1 deficient hearts. Quantitative analysis (bottom) of immunoreactive bands of Bax (N = 4-6 in each group) expressed as fold change relative to NS-treated WT group. (C). Representative heart sections (top) stained with picrosirius red/Fast green (scale bar, 50 μm) showing collagen deposition. Quantitative analysis (bottom) of the collagen deposition in WT and ROCK1 deficient hearts, expressed as percentage change relative to NS-treated WT hearts. Four to six hearts in each group. At least 10 randomly chosen high power fields per section and four transverse sections from each heart, sampled from the midpoint between the apex and base, were analyzed. (D). Representative images of Western blot analysis of ROCK1, ROCK2, LC3, Beclin 1, p-AMPK-Thr172, Bax, FAK and p-FAK-Tyr397 in ventricular homogenates of WT and ROCK1 deficient hearts on day 21 after the initial injection. (E-H) Quantitative analysis of immunoreactive bands of ROCK1 (E), Bax (F), p-FAK-Tyr397/FAK (G) and LC3-II (H). N = 4-6 in each group. (I). Representative transmission electron microscopy images of WT and ROCK1 deficient hearts on day 21 after the initial injection. DOX-treated WT heart showed increased numbers of autophagic vacuoles (red arrows). Scale bar, 0.5 μm.
Cardiomyocyte-specific ROCK1 knockout mice are also protected from doxorubicin cardiotoxicity
To determine if ROCK1 in cardiomyocytes has an important role in doxorubicincardiotoxicity, we generated cardiomyocyte-specific ROCK1 knockout mice. In myosin heavy chain (MHC)-Cre/ROCK1fl/fl mice, the ROCK1 protein is truncated from residue 137 to the end of the protein (Figure 4A–4C) only in cardiomyocytes. Similar to ROCK1−/− mice in which the ROCK1 protein is also truncated from residue 180 to the end of the protein in all cells [32], the conditional targeting approach results in the removal of a large portion of the kinase domain and amino acids from the coiled-coil and the PH domains (Figure 4A). We have noted that MHC-Cre alone had no significant cardiotoxic effects at baseline as no significant morphological or molecular differences in apoptosis and autophagy markers were observed in MHC-Cre mice compared to WT mice at 8 to 9 weeks old (data not shown). We first examined the cardiac response to doxorubicin in MHC-Cre/ROCK1fl/fl mice 8 to 9 weeks old subjected to the three-injection treatment compared to ROCK1fl/fl mice (Figure 4D–4F). Similar to the finding from WT mice, doxorubicin induced cardiac dysfunction (Figure 4D) and increased TUNEL positivity (Figure 4E) and cardiac fibrosis (Figure 4F) in ROCK1fl/fl hearts. However, these cardiotoxic events were significantly reduced in MHC-Cre/ROCK1fl/fl mice (Figure 4D–4F), supporting that ROCK1 in cardiomyocytes contributes to doxorubicin-induced cardiotoxicity. We have also noted the differences between systemic ROCK1 knockout and MHC-Cre/ROCK1fl/fl mice; in the former there were no significant changes in cardiac function, TUNEL positivity and fibrosis after doxorubicin treatment (Figure 1-3), but these parameters were significantly changed in the latter (Figure 4). Though these increases are at lower levels in comparison with ROCK1fl/fl mice, it indicates that ROCK1 in other non-cardiomyocyte cardiac cells also contributes to the cardiac response to doxorubicin.
Figure 4
Cardiomyocyte-specific ROCK1 deletion also inhibited doxorubicin-induced cardiac dysfunction, apoptosis and fibrosis
(A). Schematic illustration of the gene targeting strategy used for conditional disruption of the ROCK1 gene. Schematic representation of the domain structure of ROCK1 indicates the position of exon 5. A fragment of the ROCK1 allele that includes exons 3-6 is shown. A targeting vector was constructed containing loxP sites (arrows) flanking exon 5 of ROCK1 and Frt sites (diamonds) flanking the PGK-Neo cassette. The diagrams also indicate the positions of genomic probes used for distinguishing WT and the targeted alleles by Southern blot analysis and the positions of the restriction enzymes sites for Bam H I (B), Bgl II (Bg), Xba I (X), and Hind III (H). We first obtained germline transmission of the ROCK1fl-neo allele, which contains the loxP-flanked exon 5 and Neo cassette. The Neo cassette was removed via the Flp-Frt system by crossing ROCK1fl-neo/fl-neo mice with Flp mice, generating ROCK1fl/+ mice. (B). Southern blot analysis of genomic DNA obtained from the tail of WT and ROCK1fl-neo/+ mice. (C). Western blot analysis of ROCK1 levels in the heart and tail of WT, ROCK1fl/fl and MHC-Cre/ROCK1fl/fl mice, showing about 80% reduction of ROCK1 expression in the heart samples of the MHC-Cre/ROCK1fl/fl mice compared with WT or ROCK1fl/fl mice, but not in the tail samples of these mice. Residual ROCK1 expression in the heart is due to the presence of ROCK1 in other cell types in hearts (e.g., fibroblasts, vascular endothelial cells, and inflammatory cells). (D). Cardiomyocyte-specific ROCK1 knockout mice (MHC-Cre/ROCK1fl/fl) and ROCK1fl/fl mice 8 to 9 weeks old received three serial injections weekly of NS or DOX (8 mg/kg). Cardiac function was measured by echocardiography analysis on day 21 after the initial injection. (E-F). Quantitation of total TUNEL positive nuclei per 105 total nuclei (E) and the collagen deposition (F, scale bar, 50 μm) in ventricular myocardium from MHC-Cre/ROCK1fl/fl and ROCK1fl/fl mice hearts on day 21. N = 4-6 in each group.
Cardiomyocyte-specific ROCK1 deletion also inhibited doxorubicin-induced cardiac dysfunction, apoptosis and fibrosis
(A). Schematic illustration of the gene targeting strategy used for conditional disruption of the ROCK1 gene. Schematic representation of the domain structure of ROCK1 indicates the position of exon 5. A fragment of the ROCK1 allele that includes exons 3-6 is shown. A targeting vector was constructed containing loxP sites (arrows) flanking exon 5 of ROCK1 and Frt sites (diamonds) flanking the PGK-Neo cassette. The diagrams also indicate the positions of genomic probes used for distinguishing WT and the targeted alleles by Southern blot analysis and the positions of the restriction enzymes sites for Bam H I (B), Bgl II (Bg), Xba I (X), and Hind III (H). We first obtained germline transmission of the ROCK1fl-neo allele, which contains the loxP-flanked exon 5 and Neo cassette. The Neo cassette was removed via the Flp-Frt system by crossing ROCK1fl-neo/fl-neo mice with Flpmice, generating ROCK1fl/+ mice. (B). Southern blot analysis of genomic DNA obtained from the tail of WT and ROCK1fl-neo/+ mice. (C). Western blot analysis of ROCK1 levels in the heart and tail of WT, ROCK1fl/fl and MHC-Cre/ROCK1fl/fl mice, showing about 80% reduction of ROCK1 expression in the heart samples of the MHC-Cre/ROCK1fl/fl mice compared with WT or ROCK1fl/fl mice, but not in the tail samples of these mice. Residual ROCK1 expression in the heart is due to the presence of ROCK1 in other cell types in hearts (e.g., fibroblasts, vascular endothelial cells, and inflammatory cells). (D). Cardiomyocyte-specific ROCK1 knockout mice (MHC-Cre/ROCK1fl/fl) and ROCK1fl/fl mice 8 to 9 weeks old received three serial injections weekly of NS or DOX (8 mg/kg). Cardiac function was measured by echocardiography analysis on day 21 after the initial injection. (E-F). Quantitation of total TUNEL positive nuclei per 105 total nuclei (E) and the collagen deposition (F, scale bar, 50 μm) in ventricular myocardium from MHC-Cre/ROCK1fl/fl and ROCK1fl/fl mice hearts on day 21. N = 4-6 in each group.
To determine the molecular mechanisms underlying the protective effects of ROCK1 deletion, we evaluated the time course of doxorubicin-induced apoptosis and autophagy dysregulation and performed molecular analysis at day 2, 4 and 7 after a 10 mg/kg single dose injection. The maximal increase in the autophagy marker, LC3-II, occurred on day 4 (Figure 5A, 5B). This increased level of LC3-II could derive from an over-activation of autophagy initiation or from the prevention of autophagy completion due to deleterious effects on lysosomes. To distinguish these two possibilities, we measured the accumulation of p62/sequestosome 1 (SQSTM1), another protein marker of autophagy, which was increased at day 2, preceding maximal LC3-II accumulation (Figure 5A, 5C). Since p62/SQSTM1 is an adaptor protein that is cleared by autophagy [43], the increased accumulation suggests that the degradation process of autophagosomes was impaired in doxorubicin treated hearts. The increased LC3-II and p62/SQSTM1 accumulations were not associated with increases in Beclin 1 levels or in phosphorylation of AMPK (Figure 5A), suggesting that the accumulation of autophagosomes was not due to the activation of AMPK-mediated autophagy or a general up-regulation of the autophagy system. Interestingly, ROCK1 deletion abolished doxorubicin-induced increases in LC3-II and p62/SQSTM1 levels (Figure 5A–5C) at these early time points, indicating that ROCK1 deletion improves autophagic flux.
Figure 5
ROCK1 deletion inhibited the early onset of doxorubicin-induced autophagy dysregulation and apoptosis
(A). Schematic of single DOX administration protocol (top). Mice 8 to 9 weeks old received one injection of NS or DOX (10 mg/kg). Mice were sacrificed on day 2, 4 and 7 after the injection. Representative images (bottom) of Western blot analysis of ROCK1, ROCK2, LC3, p62, Beclin 1, p-Beclin 1-Thr119, p-AMPK-Thr172, Bax, FAK and p-FAK-Tyr397 in ventricular homogenates of WT and ROCK1 deficient hearts on day 2, 4 and 7 after a single DOX injection. (B-G). Quantitative analysis of immunoreactive bands of LC3-II on day 4 (B), p62 on day 2 (C), p-Beclin1-Thr119/Beclin 1 on day 2 (D), ROCK1 on day 2 (E), Bax on day 2 (F), p-FAK-Tyr397/FAK on day 2 (G) after single DOX injection. N = 4-6 in each group. (H). Representative images (top) of Western blot analysis of Bax and Cox IV in the mitochondrial fraction of ventricular homogenates from WT and ROCK1 deficient hearts. Quantitative analysis (bottom) of immunoreactive bands of Bax on day 2 after single DOX injection (N = 4-6 in each group) expressed as fold change relative to NS-treated WT group. (I). Quantification of total TUNEL positive nuclei per 105 total nuclei in ventricular myocardium from WT and ROCK1 deficient hearts on day 2 after single DOX injection. N = 4-6 in each group. (J-K). Mice 8 to 9 weeks old received one injection of saline or DOX (10 mg/kg). On day 4 after the injection, mice received one injection of bafilomycin A1 at 1.5 mg/kg 2 hours before they were sacrificed. Representative images (J) and quantitative analysis (K) of Western blot analysis of LC3-II in ventricular homogenates of WT and ROCK1 deficient hearts. N = 4-6 in each group expressed as fold change relative to NS-treated WT group.
ROCK1 deletion inhibited the early onset of doxorubicin-induced autophagy dysregulation and apoptosis
(A). Schematic of single DOX administration protocol (top). Mice 8 to 9 weeks old received one injection of NS or DOX (10 mg/kg). Mice were sacrificed on day 2, 4 and 7 after the injection. Representative images (bottom) of Western blot analysis of ROCK1, ROCK2, LC3, p62, Beclin 1, p-Beclin 1-Thr119, p-AMPK-Thr172, Bax, FAK and p-FAK-Tyr397 in ventricular homogenates of WT and ROCK1 deficient hearts on day 2, 4 and 7 after a single DOX injection. (B-G). Quantitative analysis of immunoreactive bands of LC3-II on day 4 (B), p62 on day 2 (C), p-Beclin1-Thr119/Beclin 1 on day 2 (D), ROCK1 on day 2 (E), Bax on day 2 (F), p-FAK-Tyr397/FAK on day 2 (G) after single DOX injection. N = 4-6 in each group. (H). Representative images (top) of Western blot analysis of Bax and Cox IV in the mitochondrial fraction of ventricular homogenates from WT and ROCK1 deficient hearts. Quantitative analysis (bottom) of immunoreactive bands of Bax on day 2 after single DOX injection (N = 4-6 in each group) expressed as fold change relative to NS-treated WT group. (I). Quantification of total TUNEL positive nuclei per 105 total nuclei in ventricular myocardium from WT and ROCK1 deficient hearts on day 2 after single DOX injection. N = 4-6 in each group. (J-K). Mice 8 to 9 weeks old received one injection of saline or DOX (10 mg/kg). On day 4 after the injection, mice received one injection of bafilomycin A1 at 1.5 mg/kg 2 hours before they were sacrificed. Representative images (J) and quantitative analysis (K) of Western blot analysis of LC3-II in ventricular homogenates of WT and ROCK1 deficient hearts. N = 4-6 in each group expressed as fold change relative to NS-treated WT group.Regarding the molecular changes implicated in apoptosis, increases in ROCK1 and Bax expression and decreases in phosphorylation of FAK were observed at day 2 (Figure 5A, 5E-5G), coinciding with increased mitochondrial translocation of Bax (Figure 5H) and increased TUNEL positivity (Figure 5I). A trend toward increased Bax, mitochondrial Bax and TUNEL positivity was noticed in doxorubicin-treated ROCK1−/− hearts compared to NS-treated hearts, but the differences were not statistically significant (Figure 5A, 5F, 5H, 5I). These results indicate that both apoptosis and the impairment of autophagic flux occur concurrently at early time points after doxorubicin treatment.To further investigate the mechanisms underlying the beneficial effects of ROCK1 deletion in improving autophagic flux, we examined the phosphorylation levels of Beclin 1 at Thr119 (Figure 5A, 5D). It has been reported that ROCK1-mediated Beclin 1 phosphorylation promotes the dissociation of Beclin 1 from Bcl 2 resulting in increased autophagosome formation induced by nutritional stress [44]. In ROCK1−/− hearts, the levels of p-Beclin 1Thr119 were reduced at all time points tested including the baseline condition, suggesting that ROCK1 deletion inhibits Beclin 1-mediated autophagosome formation. To further demonstrate that ROCK1 deletion can improve autophagic flux, WT and ROCK1−/− mice were treated with either NS or doxorubicin, and mice were euthanized on day 4, 2 hours after an injection of bafilomycin A1 which prevents the fusion of autophagosome with the lysosome and blocks autophagic flux. Bafilomycin A1 injection in control animals resulted in a significant increase in LC3-II levels, reflecting cardiac autophagic flux under basal conditions (Figure 5J, 5K). Bafilomycin A1 injection in doxorubicin-treated WT mice showed no increase in LC3-II levels, indicating a blockage in autophagic flux. Since the absence of added effects of bafilomycin A1 could be the result of saturated activity of LC3-II on day 4 when LC3-II reached maximal levels (Figure 5A), bafilomycin A1 injection was also performed on day 2 in the WT hearts and no added effects were observed (data not shown), further supporting a blockage in autophagic flux in WT hearts. In contrast to WT mice, bafilomycin A1 injection in doxorubicin-treated ROCK1−/− mice on day 4 resulted in a significant increase in LC3-II levels (Figure 5J, 5K), indicating that autophagic flux was maintained in ROCK1−/− mice after doxorubicin treatment. Consistent with a previous report showing that mice with a heterozygous deletion of Beclin 1 are cardioprotective to doxorubicin through reducing the initiation of autophagy, which allows for the preservation of autophagic flux in the presence of impaired lysosome function by doxorubicin [16], our results demonstrate that attenuated autophagy initiation in ROCK1−/− mice results in limiting the accumulation of unprocessed autolysosomes and ameliorating doxorubicincardiotoxicity.
Cardiomyocyte ROCK1 deletion also ameliorates doxorubicin-induced autophagy dysregulation through decreasing Beclin 1-mediated autophagosome formation
Finally, to determine the molecular mechanisms in cardiomyocytes, molecular analysis was performed at day 2, 4 and 7 after a single dose injection in ROCK1fl/fl and MHC-Cre/ROCK1fl/fl mice (Figure 6). Similar to the WT mice, increases in ROCK1 expression were observed from day 2 in ROCK1fl/fl mice (Figure 6A). However, deletion of ROCK1 in cardiomyocytes abolished this molecular change (Figure 6A) indicating that the doxorubicin-induced ROCK1 up-regulation occurs in cardiomyocytes. The time course showing doxorubicin-induced increases in autophagy markers (LC3-II, p62/SQSTM1) and an apoptosis marker (Bax) was also similar in ROCK1fl/fl mice compared to the WT mice (Figure 6A–6CA, 6E), and these marker changes were not associated with increases in Beclin 1 levels or in phosphorylation of AMPK (Figure 6A). ROCK1 deletion in cardiomyocytes significantly reduced the induction of these autophagy and apoptosis markers at the indicated early time points (Figure 6A–6CA, 6E). However, differences existed between systemic ROCK1 knockout and MHC-Cre/ROCK1fl/fl mice: even though they were at lower levels compared to ROCK1fl/fl mice, significant increases of autophagy and apoptosis markers were noted in doxorubicin treated MHC-Cre/ROCK1fl/fl hearts (Figure 6), but not in ROCK1−/− hearts (Figure 5), indicating that ROCK1 in other non-cardiomyocyte cardiac cells also contributes to doxorubicin-induced apoptosis and autophagy dysregulation. Notably, ROCK1-mediated Beclin 1 phosphorylation occurs in cardiomyocytes revealed by reduced levels of p-Beclin 1Thr119 at all tested time points in MHC-Cre/ROCK1fl/fl hearts compared to the ROCK1fl/fl hearts (Figure 6A, 6D). Our results indicate that ROCK1 deletion in cardiomyocytes inhibits Beclin 1-mediated autophagosome formation, resulting in reduced autophagy initiation and subsequently reduced accumulation of unprocessed autolysosomes, consequently contributing to the amelioration of doxorubicin-induced cardiotoxicity.
Figure 6
Cardiomyocyte-specific ROCK1 deletion also inhibited the early onset of doxorubicin-induced autophagy dysregulation
(A). Cardiomyocyte-specific ROCK1 knockout mice (MHC-Cre/ROCK1fl/fl) and ROCK1fl/fl mice 8 to 9 weeks old received one injection of NS or DOX (10 mg/kg). Mice were sacrificed on day 2, 4 and 7 after the injection and Western blot analysis was performed with ventricular homogenates. Representative images of Western blot analysis of ROCK1, ROCK2, LC3, p62, Beclin 1, p-Beclin 1-Thr119, p-AMPK-Thr172 and Bax in ventricular homogenates on day 2, 4 and 7 after single DOX injection. (B-E). Quantitative analysis of immunoreactive bands of LC3-II on day 4 (B), p62 on day 2 (C), p-Beclin1-Thr119/Beclin 1 on day 2 (D) and Bax on day 2 (E) after single DOX injection. N = 4-6 in each group.
Cardiomyocyte-specific ROCK1 deletion also inhibited the early onset of doxorubicin-induced autophagy dysregulation
(A). Cardiomyocyte-specific ROCK1 knockout mice (MHC-Cre/ROCK1fl/fl) and ROCK1fl/fl mice 8 to 9 weeks old received one injection of NS or DOX (10 mg/kg). Mice were sacrificed on day 2, 4 and 7 after the injection and Western blot analysis was performed with ventricular homogenates. Representative images of Western blot analysis of ROCK1, ROCK2, LC3, p62, Beclin 1, p-Beclin 1-Thr119, p-AMPK-Thr172 and Bax in ventricular homogenates on day 2, 4 and 7 after single DOX injection. (B-E). Quantitative analysis of immunoreactive bands of LC3-II on day 4 (B), p62 on day 2 (C), p-Beclin1-Thr119/Beclin 1 on day 2 (D) and Bax on day 2 (E) after single DOX injection. N = 4-6 in each group.
DISCUSSION
The present study has examined the beneficial effects of systemic ROCK1 deficiency and cardiomyocyte-specific ROCK1 deficiency in the context of doxorubicin-induced cardiotoxicity: both systemic and cardiomyocyte-specific ROCK1 deficiency can provide cardioprotection. More specifically, the beneficial effects of ROCK1 deficiency in reducing cell death due to apoptosis and autophagy are consistent with improved cardiac structure and function. Although the favorable effects of ROCK1 deficiency on cardiac remodeling in other pathological cardiac hypertrophy models have been reported, including pressure overload and genetically-induced pathological cardiac hypertrophy [32-36], the current study has further extended the cardioprotective roles of ROCK1 deficiency against doxorubicincardiotoxicity, and discovered a role for ROCK1 in mediating doxorubicin-induced dysregulation of autophagic flux in cardiomyocytes.Doxorubicin-induced increases in autophagosome formation and elevated autophagic markers (p62/SQSTM1 and LC3-II) can be detected within two days after a single dose of doxorubicin, and are also detectable over three weeks with three doses of doxorubicin. ROCK1 deficient mouse hearts exhibited reduced accumulation of autophagosomes and reduced autophagic markers at early time points and during the doxorubicin-induced cardiac remodeling period (Figures 3, 5). Beclin 1 phosphorylation on Thr119 has previously been shown to be involved in the dissociation of Beclin 1 from Bcl 2 [44] or Bcl-XL [45] resulting in Beclin 1-mediated autophagy initiation. We observed in the current study that Beclin 1 phosphorylation was reduced in both systemic and cardiomyocyte-specific ROCK1 knockout mouse hearts at baseline and after doxorubicin treatment (Figures 5, 6), supporting the notion that Beclin 1-mediated autophagy initiation is suppressed by ROCK1 deficiency. This observation is consistent with previously reported protective effects against doxorubicincardiotoxicity in heterozygous Beclin 1 knockout mice [16].Numerous studies have demonstrated that doxorubicin-induced cardiac injury is associated with a dysregulation of autophagic function [16-21]. However, findings have been largely inconsistent regarding how doxorubicindysregulates cardiac autophagy and whether an increase or decrease in autophagy is responsible for doxorubicincardiotoxicity [16-21]. Recent studies support the notion that doxorubicin leads to an over-activation of autophagy initiation while at the same time preventing autophagy completion due to deleterious effects on lysosomes, resulting in the accumulation of un-degraded protein aggregates or damaged organelles which then contribute to doxorubicincardiotoxicity [20, 21]. Our present observations indicate that the increase of p62/SQSTM1 levels preceded the maximal increases in LC3-II levels, and doxorubicin-treated WT mice showed no increase in LC3-II levels associated with bafilomycin A1 treatment (Figure 5), pointing towards the disruptions of autophagic flux by doxorubicin. Similar to a heterozygous deletion of Beclin 1, which decreases autophagy initiation resulting in reduced accumulation of unprocessed autolysosomes to ameliorate doxorubicincardiotoxicity [16], ROCK1 deficiency has a protective role against doxorubicin-induced cardiotoxicity associated with reducing Beclin 1-mediated autophagy initiation and maintaining autophagic flux in cardiomyocytes, thereby reducing the increased demand from an inefficient clearance of autophagosomes by damaged lysosomes.The contribution of doxorubicin-induced apoptosis to cardiotoxicity has been extensively studied [13-15]. Consistent with this view, we observed that doxorubicin induced a 3 to 4-fold increase in TUNEL positivity in both WT and ROCK1fl/fl hearts associated with the increases in Bax expression and Bax translocation to the mitochondria (Figure 3-6). The increase of the apoptotic marker, Bax, occurred concomitantly with the increase of autophagic markers LC3-II and p62/SQSTM1, indicating that both apoptotic upregulation and autophagic dysregulation are triggered by doxorubicin during the early stages of toxicity. Compared to the systemic ROCK1 deficiency, the cardiomyocyte-specific ROCK1 knockout only partially reproduced the beneficial phenotypes, including improvement in cardiac function, attenuation of apoptotic upregulation and autophagic dysregulation, and reduction in cardiac fibrosis; these observations have demonstrated that ROCK1 in both cardiomyocytes and in non-cardiomyocytes contributes to doxorubicin-induced cardiotoxicity. This notion is consistent with our previous finding that the majority of the apoptosis triggered by doxorubicin treatment occurred in non-cardiomyocytes [14]. In addition to suppressing Beclin 1 phosphorylation to inhibit Beclin 1-mediated autophagy initiation in cardiomyocytes, other mechanisms may also contribute to the cardiac effects of ROCK1 deletion, including improved actin cytoskeleton stability and cell adhesion resulting in enhanced survival signaling. In support of improved cell adhesion, FAK phosphorylation was preserved or enhanced in ROCK1 deficient hearts after doxorubicin treatment (Figure 3, 5). This in vivo observation is consistent with previous studies using cultured mouse embryonic fibroblasts in which ROCK1 deficiency, through reducing actin cytoskeleton remodeling, acts additively with antioxidant treatment to suppress excessive production of doxorubicin-induced reactive oxygen species and apoptosis [37-40].Reported by an in vivo study in rats using fasudil, a ROCK inhibitor for both ROCK1 and ROCK2 which also inhibits some other kinases, it was shown that ROCK has a role in mediating doxorubicin-induced cardiotoxicity in rats [46]. Similar to that study, we observed that the doxorubicin treatment increased ROCK1 expression in mouse hearts not only at early time points but also during the doxorubicin-induced cardiac remodeling period (Figures 3, 5, 6). Cardiomyocyte-specific ROCK1 deletion completely suppressed this molecular event, indicating that doxorubicin-induced ROCK1 upregulation occurs largely in cardiomyocytes (Figure 6A). Although fasudil can inhibit doxorubicin-induced apoptosis in rat hearts [46], the effect of the inhibitor on doxorubicin-induced autophagy was not explored. The current study, using a genetic approach, provides novel cellular and mechanistic insights such as promoting Beclin 1-mediated autophagy initiation underlying the detrimental roles of ROCK1 in doxorubicin-induced cardiotoxicity.Numerous studies have demonstrated that RhoA/ROCK signaling contributes to autophagy, however both positive [47, 48] and negative [44, 49] effects of ROCK inhibition on autophagy have been reported. Treatment with the ROCK inhibitor Y27632 increased the degradation of mutant Huntington protein via proteasome degradation and autophagy in mouseneuroblastoma cell lines [48]. In addition, ROCK inhibition has been linked to an increased autophagy response to starvation or rapamycin treatment and associated with the formation of enlarged early autophagosomes and late degradative autolysosomes in humanembryonic kidney293 cells [47]. In contrast, ROCK inhibition impaired the starvation-mediated autophagic response in HeLa cells and CHO cells through its inhibitory effect on actin cytoskeleton formation which participates in the initial membrane remodeling at very early stages of autophagosome formation [49]. Moreover, systemic ROCK1 deletion impaired the starvation-mediated autophagic response in mouse hearts through inhibiting Beclin 1-mediated autophagy initiation [44]. Our study is consistent with this latter report and supports a positive role for ROCK1 in participating in doxorubicin-induced autophagy initiation through phosphorylating Beclin 1. These context dependent roles of ROCK in modulating autophagy are likely due to the complexity of autophagy regulation and also due to the variety of cellular functions controlled by RhoA/ROCK signaling. The roles and precise mechanisms downstream of ROCK1 in regulating autophagy during pathological cardiac remodeling triggered by other stresses require further investigation.In summary, the present study demonstrated an important role for systemic and cardiomyocyte ROCK1 in doxorubicincardiotoxicity, particularly in mediating autophagy dysregulation and apoptosis, which contribute to cardiac remodeling and dysfunction. Altogether, ROCK1 represents a potential therapeutic target in preventing the anti-cancer drug doxorubicin-induced heart failure and possibly other drug-induced cardiotoxicity related heart failure.
MATERIALS AND METHODS
Generation of mouse models
All animal experiments were conducted in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and were approved by the Institutional Animal Care and Use Committee at Indiana University School of Medicine. Generation of ROCK1−/− mice was as previously described [32].Mice bearing a loxP-flanked ROCK1 allele (ROCK1fl/fl) were generated using the same genomic ROCK1 DNA clone containing exons 3-6 isolated from a 129 mouse genomic library as previously used to generate ROCK1−/− mice [32]. In the targeting vector (Figure 4A), a PGK-Neo cassette is flanked by Frt sites and the gene segment containing the exon 5 is flanked by loxP sites. Deletion of exon 5 by Cre recombinase results in a frame-shift mutation in ROCK1, thus removing all residues from residue 137 to the end of the protein. Three independent ROCK1fl-neo/+ embryonic stem cell clones were identified and injected into C57BL/6 blastocysts to generate chimeric mice. The chimeric mice were bred with wild-type C57BL/6 mice for germline transmission of ROCK1fl-neo allele. The genotypes of the offspring were identified by Southern blot analysis (Figure 4B) and PCR on DNA obtained from mouse tails. Mice used in the present study had been backcrossed to C57BL/6 for at least 8 generations. ROCK1fl-neo/+ heterozygous mice were then intercrossed to produce homozygous ROCK1fl-neo/fl-neo mice. The presence of the Neo cassette has no detectable effect on the endogenous ROCK1 expression. ROCK1fl-neo/fl-neo mice were then crossed with Gt (ROSA) 26Sor-Flpmice (Jackson Laboratory) to delete the Frt-flanked Neo cassette from the germ line. Offspring of these mice were heterozygous for the desired ROCK1fl/+ allele. To generate cardiomyocyte-specific ROCK1 knockout mice (MHC-Cre/ROCK1fl/fl), ROCK1fl/fl mice were crossed to the MHC-Cre mice [41] and bred back to ROCK1fl/fl mice (Figure 4C).
In vivo mouse models of doxorubicin cardiomyopathy
Mice 8 to 9 weeks old were injected intraperitoneally with doxorubicin (Sigma) at 8 mg/kg or NS once weekly for 3 consecutive weeks, the cumulative dose being 24 mg/kg. Cardiac dimension and contractile performance were evaluated by noninvasive transthoracic echocardiography using a VisualSonics® 2100 ultrasound machine for small animal imaging and MS400 transducer (Fujifilm Visual Sonics, Inc., Toronto, Canada) before each injection and 1 week after the last injection. Functional parameters of the left ventricle were measured using standard assessment techniques as previously described [33]. All mice were sacrificed after functional measurements; hearts were removed immediately, rinsed in pre-cooled PBS, and flash frozen in liquid nitrogen for later protein analysis, or cryopreserved in OCT medium for cryosections, or fixed in 4% paraformaldehyde buffer, followed by paraffin embedding.To determine the early cytotoxic effects of doxorubicin in vivo, we have used a one-dose injury model. A single dose of doxorubicin at 10 mg/kg was administered intraperitoneally to mice 8 to 9 weeks old. Mice were sacrificed on day 2, 4, or 7 after treatment; the hearts were collected immediately as described above. To assess autophagic flux, mice received bafilomycin A1 at dose 1.5 mg/kg (Santa Cruz Biotechnology) intraperitoneally 2 hours before sample collecting. Heart homogenates were used for protein analysis by Western blot.
Histology and quantitative analysis
Total heart weight was indexed to tibial length. Cryosections or paraffin sections were stained with hematoxylin/eosin for initial evaluation, picrosirius red/Fast green to identify collagen fibers, immunostaining for laminin to measure myocyte size, and TUNEL staining to monitor cardiomyocyte apoptosis as previously described [14, 33, 34, 36]. Myocyte diameter was measured using transnuclear width at the mid-ventricular level. The quantification of collagen-stained area was performed with Image-Pro software (Media Cybernetics). Apoptosis in mouse heart sections was assayed using the fluorescent ApopTag kit according to the manufacturer’s instructions (Chemicon). These sections were also counterstained with DAPI (Molecular Probes) and mounted with Vectashield. For all quantifications, a minimum of six sections from distinct regions of each heart sample and at least four hearts per group were analyzed.
Electron microscopy analysis
Ventricular specimens (cubes less than 3 mm square) were fixed in 2.5% glutaraldehyde, underwent sectioning and heavy metal uranyl acetate staining for contrast by the Electron Microscopy Center of Indiana University School of Medicine. At least 6 separate sections from each animal strain were analyzed. Electron micrographs were acquired on a transmission electron microscope, Tecnai BioTwin (FEI) equipped with AMT CCD Camera (Advanced Microscopy Techniques).
Protein analysis
Protein samples were prepared as previously described [14, 33, 36]. Ventricular tissue fragments were disrupted with a PYREX® Potter-Elvehjem tissue grinder on ice in lysis buffer containing proteinase and phosphatase inhibitors (Roche). The homogenate was centrifuged at 15,000 x g at 4°C for 15 minutes, and the supernatant was saved for immunoblotting. The blots were probed with primary antibodies to ROCK2 (sc-5561), FAK (sc-558) from Santa Cruz Biotechnology, ROCK1 (#4035), p-FAK-Tyr397 (#3283), Bax (#2772), LC3 (#2775), Beclin 1 (#3738), p62 (#5114) and p-AMPK-Thr172 (#2535) from Cell Signaling Technology, and p-Beclin1-Thr119 (ABC118) from MilliporeSigma. All blots were normalized to GAPDH (ABS16; MilliporeSigma) or to actin (MABT523; MilliporeSigma).
Subcellular fractionation
Ventricular mitochondrial fractions were prepared as previously described [14, 36]. Samples with equal amounts of protein were analyzed by Western blot with specific antibodies. The purity of the mitochondrial fraction was assessed by Western blot analysis with GAPDH (a cytosolic protein) and cytochrome C oxidase subunit IV (COX IV) (a mitochondrial protein) (#4844; Cell Signaling Technology) as the markers, respectively.
Statistical analysis
Data are reported as mean ± SE. Comparisons between groups were analyzed by Student’s t-test or ANOVA as appropriate, with P < 0.05 considered as significant.
Authors: Ying-Min Zhang; Jacqueline Bo; George E Taffet; Jiang Chang; Jianjian Shi; Anilkumar K Reddy; Lloyd H Michael; Michael D Schneider; Mark L Entman; Robert J Schwartz; Lei Wei Journal: FASEB J Date: 2006-05 Impact factor: 5.191
Authors: Elly Barry; Jorge A Alvarez; Rebecca E Scully; Tracie L Miller; Steven E Lipshultz Journal: Expert Opin Pharmacother Date: 2007-06 Impact factor: 3.889
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Samrat Chatterjee; Shail K Chaube; Anu Chaudhary; Santosh Chauhan; Edward Chaum; Frédéric Checler; Michael E Cheetham; Chang-Shi Chen; Guang-Chao Chen; Jian-Fu Chen; Liam L Chen; Leilei Chen; Lin Chen; Mingliang Chen; Mu-Kuan Chen; Ning Chen; Quan Chen; Ruey-Hwa Chen; Shi Chen; Wei Chen; Weiqiang Chen; Xin-Ming Chen; Xiong-Wen Chen; Xu Chen; Yan Chen; Ye-Guang Chen; Yingyu Chen; Yongqiang Chen; Yu-Jen Chen; Yue-Qin Chen; Zhefan Stephen Chen; Zhi Chen; Zhi-Hua Chen; Zhijian J Chen; Zhixiang Chen; Hanhua Cheng; Jun Cheng; Shi-Yuan Cheng; Wei Cheng; Xiaodong Cheng; Xiu-Tang Cheng; Yiyun Cheng; Zhiyong Cheng; Zhong Chen; Heesun Cheong; Jit Kong Cheong; Boris V Chernyak; Sara Cherry; Chi Fai Randy Cheung; Chun Hei Antonio Cheung; King-Ho Cheung; Eric Chevet; Richard J Chi; Alan Kwok Shing Chiang; Ferdinando Chiaradonna; Roberto Chiarelli; Mario Chiariello; Nathalia Chica; Susanna Chiocca; Mario Chiong; Shih-Hwa Chiou; Abhilash I Chiramel; Valerio Chiurchiù; Dong-Hyung Cho; Seong-Kyu Choe; Augustine M K Choi; Mary E Choi; Kamalika Roy Choudhury; Norman S Chow; Charleen T Chu; Jason P Chua; John Jia En Chua; Hyewon Chung; Kin Pan Chung; Seockhoon Chung; So-Hyang Chung; Yuen-Li Chung; Valentina Cianfanelli; Iwona A Ciechomska; Mariana Cifuentes; Laura Cinque; Sebahattin Cirak; Mara Cirone; Michael J Clague; Robert Clarke; Emilio Clementi; Eliana M Coccia; Patrice Codogno; Ehud Cohen; Mickael M Cohen; Tania Colasanti; Fiorella Colasuonno; Robert A Colbert; Anna Colell; Miodrag Čolić; Nuria S Coll; Mark O Collins; María I Colombo; Daniel A Colón-Ramos; Lydie Combaret; Sergio Comincini; Márcia R Cominetti; Antonella Consiglio; Andrea Conte; Fabrizio Conti; Viorica Raluca Contu; Mark R Cookson; Kevin M Coombs; Isabelle Coppens; Maria Tiziana Corasaniti; Dale P Corkery; Nils Cordes; Katia Cortese; Maria do Carmo Costa; Sarah Costantino; Paola Costelli; Ana Coto-Montes; Peter J Crack; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Riccardo Cristofani; Tamas Csizmadia; Antonio Cuadrado; Bing Cui; Jun Cui; Yixian Cui; Yong Cui; Emmanuel Culetto; Andrea C Cumino; Andrey V Cybulsky; Mark J Czaja; Stanislaw J Czuczwar; Stefania D'Adamo; Marcello D'Amelio; Daniela D'Arcangelo; Andrew C D'Lugos; Gabriella D'Orazi; James A da Silva; Hormos Salimi Dafsari; Ruben K Dagda; Yasin Dagdas; Maria Daglia; Xiaoxia Dai; Yun Dai; Yuyuan Dai; Jessica Dal Col; Paul Dalhaimer; Luisa Dalla Valle; Tobias Dallenga; Guillaume Dalmasso; Markus Damme; Ilaria Dando; Nico P Dantuma; April L Darling; Hiranmoy Das; Srinivasan Dasarathy; Santosh K Dasari; Srikanta Dash; Oliver Daumke; Adrian N Dauphinee; Jeffrey S Davies; Valeria A Dávila; Roger J Davis; Tanja Davis; Sharadha Dayalan Naidu; Francesca De Amicis; Karolien De Bosscher; Francesca De Felice; Lucia De Franceschi; Chiara De Leonibus; Mayara G de Mattos Barbosa; Guido R Y De Meyer; Angelo De Milito; Cosimo De Nunzio; Clara De Palma; Mauro De Santi; Claudio De Virgilio; Daniela De Zio; Jayanta Debnath; Brian J DeBosch; Jean-Paul Decuypere; Mark A Deehan; Gianluca Deflorian; James DeGregori; Benjamin Dehay; Gabriel Del Rio; Joe R Delaney; Lea M D Delbridge; Elizabeth Delorme-Axford; M Victoria Delpino; Francesca Demarchi; Vilma Dembitz; Nicholas D Demers; Hongbin Deng; Zhiqiang Deng; Joern Dengjel; Paul Dent; Donna Denton; Melvin L DePamphilis; Channing J Der; Vojo Deretic; Albert Descoteaux; Laura Devis; Sushil Devkota; Olivier Devuyst; Grant Dewson; Mahendiran Dharmasivam; Rohan Dhiman; Diego di Bernardo; Manlio Di Cristina; Fabio Di Domenico; Pietro Di Fazio; Alessio Di Fonzo; Giovanni Di Guardo; Gianni M Di Guglielmo; Luca Di Leo; Chiara Di Malta; Alessia Di Nardo; Martina Di Rienzo; Federica Di Sano; George Diallinas; Jiajie Diao; Guillermo Diaz-Araya; Inés Díaz-Laviada; Jared M Dickinson; Marc Diederich; Mélanie Dieudé; Ivan Dikic; Shiping Ding; Wen-Xing Ding; Luciana Dini; Jelena Dinić; Miroslav Dinic; Albena T Dinkova-Kostova; Marc S Dionne; Jörg H W Distler; Abhinav Diwan; Ian M C Dixon; Mojgan Djavaheri-Mergny; Ina Dobrinski; Oxana Dobrovinskaya; Radek Dobrowolski; Renwick C J Dobson; Jelena Đokić; Serap Dokmeci Emre; Massimo Donadelli; Bo Dong; Xiaonan Dong; Zhiwu Dong; Gerald W Dorn Ii; Volker Dotsch; Huan Dou; Juan Dou; Moataz Dowaidar; Sami Dridi; Liat Drucker; Ailian Du; Caigan Du; Guangwei Du; Hai-Ning Du; Li-Lin Du; André du Toit; Shao-Bin Duan; Xiaoqiong Duan; Sónia P Duarte; Anna Dubrovska; Elaine A Dunlop; Nicolas Dupont; Raúl V Durán; Bilikere S Dwarakanath; Sergey A Dyshlovoy; Darius Ebrahimi-Fakhari; Leopold Eckhart; Charles L Edelstein; Thomas Efferth; Eftekhar Eftekharpour; Ludwig Eichinger; Nabil Eid; Tobias Eisenberg; N Tony Eissa; Sanaa Eissa; Miriam Ejarque; Abdeljabar El Andaloussi; Nazira El-Hage; Shahenda El-Naggar; Anna Maria Eleuteri; Eman S El-Shafey; Mohamed Elgendy; Aristides G Eliopoulos; María M Elizalde; Philip M Elks; Hans-Peter Elsasser; Eslam S Elsherbiny; Brooke M Emerling; N C Tolga Emre; Christina H Eng; Nikolai Engedal; Anna-Mart Engelbrecht; Agnete S T Engelsen; Jorrit M Enserink; Ricardo Escalante; Audrey Esclatine; Mafalda Escobar-Henriques; Eeva-Liisa Eskelinen; Lucile Espert; Makandjou-Ola Eusebio; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Francesco Facchiano; Bengt Fadeel; Claudio Fader; Alex C Faesen; W Douglas Fairlie; Alberto Falcó; Bjorn H Falkenburger; Daping Fan; Jie Fan; Yanbo Fan; Evandro F Fang; Yanshan Fang; Yognqi Fang; Manolis Fanto; Tamar Farfel-Becker; Mathias Faure; Gholamreza Fazeli; Anthony O Fedele; Arthur M Feldman; Du Feng; Jiachun Feng; Lifeng Feng; Yibin Feng; Yuchen Feng; Wei Feng; Thais Fenz Araujo; Thomas A Ferguson; Álvaro F Fernández; Jose C Fernandez-Checa; Sonia Fernández-Veledo; Alisdair R Fernie; Anthony W Ferrante; Alessandra Ferraresi; Merari F Ferrari; Julio C B Ferreira; Susan Ferro-Novick; Antonio Figueras; Riccardo Filadi; Nicoletta Filigheddu; Eduardo Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; Vittorio Fineschi; Francesca Finetti; Steven Finkbeiner; Edward A Fisher; Paul B Fisher; Flavio Flamigni; Steven J Fliesler; Trude H Flo; Ida Florance; Oliver Florey; Tullio Florio; Erika Fodor; Carlo Follo; Edward A Fon; Antonella Forlino; Francesco Fornai; Paola Fortini; Anna Fracassi; Alessandro Fraldi; Brunella Franco; Rodrigo Franco; Flavia Franconi; Lisa B Frankel; Scott L Friedman; Leopold F Fröhlich; Gema Frühbeck; Jose M Fuentes; Yukio Fujiki; Naonobu Fujita; Yuuki Fujiwara; Mitsunori Fukuda; Simone Fulda; Luc Furic; Norihiko Furuya; Carmela Fusco; Michaela U Gack; Lidia Gaffke; Sehamuddin Galadari; Alessia Galasso; Maria F Galindo; Sachith Gallolu Kankanamalage; Lorenzo Galluzzi; Vincent Galy; Noor Gammoh; Boyi Gan; Ian G Ganley; Feng Gao; Hui Gao; Minghui Gao; Ping Gao; Shou-Jiang Gao; Wentao Gao; Xiaobo Gao; Ana Garcera; Maria Noé Garcia; Verónica E Garcia; Francisco García-Del Portillo; Vega Garcia-Escudero; Aracely Garcia-Garcia; Marina Garcia-Macia; Diana García-Moreno; Carmen Garcia-Ruiz; Patricia García-Sanz; Abhishek D Garg; Ricardo Gargini; Tina Garofalo; Robert F Garry; Nils C Gassen; Damian Gatica; Liang Ge; Wanzhong Ge; Ruth Geiss-Friedlander; Cecilia Gelfi; Pascal Genschik; Ian E Gentle; Valeria Gerbino; Christoph Gerhardt; Kyla Germain; Marc Germain; David A Gewirtz; Elham Ghasemipour Afshar; Saeid Ghavami; Alessandra Ghigo; Manosij Ghosh; Georgios Giamas; Claudia Giampietri; Alexandra Giatromanolaki; Gary E Gibson; Spencer B Gibson; Vanessa Ginet; Edward Giniger; Carlotta Giorgi; Henrique Girao; Stephen E Girardin; Mridhula Giridharan; Sandy Giuliano; Cecilia Giulivi; Sylvie Giuriato; Julien Giustiniani; Alexander Gluschko; Veit Goder; Alexander Goginashvili; Jakub Golab; David C Goldstone; Anna Golebiewska; Luciana R Gomes; Rodrigo Gomez; Rubén Gómez-Sánchez; Maria Catalina Gomez-Puerto; Raquel Gomez-Sintes; Qingqiu Gong; Felix M Goni; Javier González-Gallego; Tomas Gonzalez-Hernandez; Rosa A Gonzalez-Polo; Jose A Gonzalez-Reyes; Patricia González-Rodríguez; Ing Swie Goping; Marina S Gorbatyuk; Nikolai V Gorbunov; Kıvanç Görgülü; Roxana M Gorojod; Sharon M Gorski; Sandro Goruppi; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Martin Graef; Markus H Gräler; Veronica Granatiero; Daniel Grasso; Joshua P Gray; Douglas R Green; Alexander Greenhough; Stephen L Gregory; Edward F Griffin; Mark W Grinstaff; Frederic Gros; Charles Grose; Angelina S Gross; Florian Gruber; Paolo Grumati; Tilman Grune; Xueyan Gu; Jun-Lin Guan; Carlos M Guardia; Kishore Guda; Flora Guerra; Consuelo Guerri; Prasun Guha; Carlos Guillén; Shashi Gujar; Anna Gukovskaya; Ilya Gukovsky; Jan Gunst; Andreas Günther; Anyonya R Guntur; Chuanyong Guo; Chun Guo; Hongqing Guo; Lian-Wang Guo; Ming Guo; Pawan Gupta; Shashi Kumar Gupta; Swapnil Gupta; Veer Bala Gupta; Vivek Gupta; Asa B Gustafsson; David D Gutterman; Ranjitha H B; Annakaisa Haapasalo; James E Haber; Aleksandra Hać; Shinji Hadano; Anders J Hafrén; Mansour Haidar; Belinda S Hall; Gunnel Halldén; Anne Hamacher-Brady; Andrea Hamann; Maho Hamasaki; Weidong Han; Malene Hansen; Phyllis I Hanson; Zijian Hao; Masaru Harada; Ljubica Harhaji-Trajkovic; Nirmala Hariharan; Nigil Haroon; James Harris; Takafumi Hasegawa; Noor Hasima Nagoor; Jeffrey A Haspel; Volker Haucke; Wayne D Hawkins; Bruce A Hay; Cole M Haynes; Soren B Hayrabedyan; Thomas S Hays; Congcong He; Qin He; Rong-Rong He; You-Wen He; Yu-Ying He; Yasser Heakal; Alexander M Heberle; J Fielding Hejtmancik; Gudmundur Vignir Helgason; Vanessa Henkel; Marc Herb; Alexander Hergovich; Anna Herman-Antosiewicz; Agustín Hernández; Carlos Hernandez; Sergio Hernandez-Diaz; Virginia Hernandez-Gea; Amaury Herpin; Judit Herreros; Javier H Hervás; Daniel Hesselson; Claudio Hetz; Volker T Heussler; Yujiro Higuchi; Sabine Hilfiker; Joseph A Hill; William S Hlavacek; Emmanuel A Ho; Idy H T Ho; Philip Wing-Lok Ho; Shu-Leong Ho; Wan Yun Ho; G Aaron Hobbs; Mark Hochstrasser; Peter H M Hoet; Daniel Hofius; Paul Hofman; Annika Höhn; Carina I Holmberg; Jose R Hombrebueno; Chang-Won Hong Yi-Ren Hong; Lora V Hooper; Thorsten Hoppe; Rastislav Horos; Yujin Hoshida; I-Lun Hsin; Hsin-Yun Hsu; Bing Hu; Dong Hu; Li-Fang Hu; Ming Chang Hu; Ronggui Hu; Wei Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Jinlian Hua; Yingqi Hua; Chongmin Huan; Canhua Huang; Chuanshu Huang; Chuanxin Huang; Chunling Huang; Haishan Huang; Kun Huang; Michael L H Huang; Rui Huang; Shan Huang; Tianzhi Huang; Xing Huang; Yuxiang Jack Huang; Tobias B Huber; Virginie Hubert; Christian A Hubner; Stephanie M Hughes; William E Hughes; Magali Humbert; Gerhard Hummer; James H Hurley; Sabah Hussain; Salik Hussain; Patrick J Hussey; Martina Hutabarat; Hui-Yun Hwang; Seungmin Hwang; Antonio Ieni; Fumiyo Ikeda; Yusuke Imagawa; Yuzuru Imai; Carol Imbriano; Masaya Imoto; Denise M Inman; Ken Inoki; Juan Iovanna; Renato V Iozzo; Giuseppe Ippolito; Javier E Irazoqui; Pablo Iribarren; Mohd Ishaq; Makoto Ishikawa; Nestor Ishimwe; Ciro Isidoro; Nahed Ismail; Shohreh Issazadeh-Navikas; Eisuke Itakura; Daisuke Ito; Davor Ivankovic; Saška Ivanova; Anand Krishnan V Iyer; José M Izquierdo; Masanori Izumi; Marja Jäättelä; Majid Sakhi Jabir; William T Jackson; Nadia Jacobo-Herrera; Anne-Claire Jacomin; Elise Jacquin; Pooja Jadiya; Hartmut Jaeschke; Chinnaswamy Jagannath; Arjen J Jakobi; Johan Jakobsson; Bassam Janji; Pidder Jansen-Dürr; Patric J Jansson; Jonathan Jantsch; Sławomir Januszewski; Alagie Jassey; Steve Jean; Hélène Jeltsch-David; Pavla Jendelova; Andreas Jenny; Thomas E Jensen; Niels Jessen; Jenna L Jewell; Jing Ji; Lijun Jia; Rui Jia; Liwen Jiang; Qing Jiang; Richeng Jiang; Teng Jiang; Xuejun Jiang; Yu Jiang; Maria Jimenez-Sanchez; Eun-Jung Jin; Fengyan Jin; Hongchuan Jin; Li Jin; Luqi Jin; Meiyan Jin; Si Jin; Eun-Kyeong Jo; Carine Joffre; Terje Johansen; Gail V W Johnson; Simon A Johnston; Eija Jokitalo; Mohit Kumar Jolly; Leo A B Joosten; Joaquin Jordan; Bertrand Joseph; Dianwen Ju; Jeong-Sun Ju; Jingfang Ju; Esmeralda Juárez; Delphine Judith; Gábor Juhász; Youngsoo Jun; Chang Hwa Jung; Sung-Chul Jung; Yong Keun Jung; Heinz Jungbluth; Johannes Jungverdorben; Steffen Just; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Daniel Kaganovich; Alon Kahana; Renate Kain; Shinjo Kajimura; Maria Kalamvoki; Manjula Kalia; Danuta S Kalinowski; Nina Kaludercic; Ioanna Kalvari; Joanna Kaminska; Vitaliy O Kaminskyy; Hiromitsu Kanamori; Keizo Kanasaki; Chanhee Kang; Rui Kang; Sang Sun Kang; Senthilvelrajan Kaniyappan; Tomotake Kanki; Thirumala-Devi Kanneganti; Anumantha G Kanthasamy; Arthi Kanthasamy; Marc Kantorow; Orsolya Kapuy; Michalis V Karamouzis; Md Razaul Karim; Parimal Karmakar; Rajesh G Katare; Masaru Kato; Stefan H E Kaufmann; Anu Kauppinen; Gur P Kaushal; Susmita Kaushik; Kiyoshi Kawasaki; Kemal Kazan; Po-Yuan Ke; Damien J Keating; Ursula Keber; John H Kehrl; Kate E Keller; Christian W Keller; Jongsook Kim Kemper; Candia M Kenific; Oliver Kepp; Stephanie Kermorgant; Andreas Kern; Robin Ketteler; Tom G Keulers; Boris Khalfin; Hany Khalil; Bilon Khambu; Shahid Y Khan; Vinoth Kumar Megraj Khandelwal; Rekha Khandia; Widuri Kho; Noopur V Khobrekar; Sataree Khuansuwan; Mukhran Khundadze; Samuel A Killackey; Dasol Kim; Deok Ryong Kim; Do-Hyung Kim; Dong-Eun Kim; Eun Young Kim; Eun-Kyoung Kim; Hak-Rim Kim; Hee-Sik Kim; Jeong Hun Kim; Jin Kyung Kim; Jin-Hoi Kim; Joungmok Kim; Ju Hwan Kim; Keun Il Kim; Peter K Kim; Seong-Jun Kim; Scot R Kimball; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Matthew A King; Kerri J Kinghorn; Conan G Kinsey; Vladimir Kirkin; Lorrie A Kirshenbaum; Sergey L Kiselev; Shuji Kishi; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Richard N Kitsis; Josef T Kittler; Ole Kjaerulff; Peter S Klein; Thomas Klopstock; Jochen Klucken; Helene Knævelsrud; Roland L Knorr; Ben C B Ko; Fred Ko; Jiunn-Liang Ko; Hotaka Kobayashi; Satoru Kobayashi; Ina Koch; Jan C Koch; Ulrich Koenig; Donat Kögel; Young Ho Koh; Masato Koike; Sepp D Kohlwein; Nur M Kocaturk; Masaaki Komatsu; Jeannette König; Toru Kono; Benjamin T Kopp; Tamas Korcsmaros; Gözde Korkmaz; Viktor I Korolchuk; Mónica Suárez Korsnes; Ali Koskela; Janaiah Kota; Yaichiro Kotake; Monica L Kotler; Yanjun Kou; Michael I Koukourakis; Evangelos Koustas; Attila L Kovacs; Tibor Kovács; Daisuke Koya; Tomohiro Kozako; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Anna D Krasnodembskaya; Carole Kretz-Remy; Guido Kroemer; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Sabine Kuenen; Lars Kuerschner; Thomas Kukar; Ajay Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Sharad Kumar; Shinji Kume; Caroline Kumsta; Chanakya N Kundu; Mondira Kundu; Ajaikumar B Kunnumakkara; Lukasz Kurgan; Tatiana G Kutateladze; Ozlem Kutlu; SeongAe Kwak; Ho Jeong Kwon; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert La Spada; Patrick Labonté; Sylvain Ladoire; Ilaria Laface; Frank Lafont; Diane C Lagace; Vikramjit Lahiri; Zhibing Lai; Angela S Laird; Aparna Lakkaraju; Trond Lamark; Sheng-Hui Lan; Ane Landajuela; Darius J R Lane; Jon D Lane; Charles H Lang; Carsten Lange; Ülo Langel; Rupert Langer; Pierre Lapaquette; Jocelyn Laporte; Nicholas F LaRusso; Isabel Lastres-Becker; Wilson Chun Yu Lau; Gordon W Laurie; Sergio Lavandero; Betty Yuen Kwan Law; Helen Ka-Wai Law; Rob Layfield; Weidong Le; Herve Le Stunff; Alexandre Y Leary; Jean-Jacques Lebrun; Lionel Y W Leck; Jean-Philippe Leduc-Gaudet; Changwook Lee; Chung-Pei Lee; Da-Hye Lee; Edward B Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Heung Kyu Lee; Jae Man Lee; Jason S Lee; Jin-A Lee; Joo-Yong Lee; Jun Hee Lee; Michael Lee; Min Goo Lee; Min Jae Lee; Myung-Shik Lee; Sang Yoon Lee; Seung-Jae Lee; Stella Y Lee; Sung Bae Lee; Won Hee Lee; Ying-Ray Lee; Yong-Ho Lee; Youngil Lee; Christophe Lefebvre; Renaud Legouis; Yu L Lei; Yuchen Lei; Sergey Leikin; Gerd Leitinger; Leticia Lemus; Shuilong Leng; Olivia Lenoir; Guido Lenz; Heinz Josef Lenz; Paola Lenzi; Yolanda León; Andréia M Leopoldino; Christoph Leschczyk; Stina Leskelä; Elisabeth Letellier; Chi-Ting Leung; Po Sing Leung; Jeremy S Leventhal; Beth Levine; Patrick A Lewis; Klaus Ley; Bin Li; Da-Qiang Li; Jianming Li; Jing Li; Jiong Li; Ke Li; Liwu Li; Mei Li; Min Li; Min Li; Ming Li; Mingchuan Li; Pin-Lan Li; Ming-Qing Li; Qing Li; Sheng Li; Tiangang Li; Wei Li; Wenming Li; Xue Li; Yi-Ping Li; Yuan Li; Zhiqiang Li; Zhiyong Li; Zhiyuan Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Weicheng Liang; Yongheng Liang; YongTian Liang; Guanghong Liao; Lujian Liao; Mingzhi Liao; Yung-Feng Liao; Mariangela Librizzi; Pearl P Y Lie; Mary A Lilly; Hyunjung J Lim; Thania R R Lima; Federica Limana; Chao Lin; Chih-Wen Lin; Dar-Shong Lin; Fu-Cheng Lin; Jiandie D Lin; Kurt M Lin; Kwang-Huei Lin; Liang-Tzung Lin; Pei-Hui Lin; Qiong Lin; Shaofeng Lin; Su-Ju Lin; Wenyu Lin; Xueying Lin; Yao-Xin Lin; Yee-Shin Lin; Rafael Linden; Paula Lindner; Shuo-Chien Ling; Paul Lingor; Amelia K Linnemann; Yih-Cherng Liou; Marta M Lipinski; Saška Lipovšek; Vitor A Lira; Natalia Lisiak; Paloma B Liton; Chao Liu; Ching-Hsuan Liu; Chun-Feng Liu; Cui Hua Liu; Fang Liu; Hao Liu; Hsiao-Sheng Liu; Hua-Feng Liu; Huifang Liu; Jia Liu; Jing Liu; Julia Liu; Leyuan Liu; Longhua Liu; Meilian Liu; Qin Liu; Wei Liu; Wende Liu; Xiao-Hong Liu; Xiaodong Liu; Xingguo Liu; Xu Liu; Xuedong Liu; Yanfen Liu; Yang Liu; Yang Liu; Yueyang Liu; Yule Liu; J Andrew Livingston; Gerard Lizard; Jose M Lizcano; Senka Ljubojevic-Holzer; Matilde E LLeonart; David Llobet-Navàs; Alicia Llorente; Chih Hung Lo; Damián Lobato-Márquez; Qi Long; Yun Chau Long; Ben Loos; Julia A Loos; Manuela G López; Guillermo López-Doménech; José Antonio López-Guerrero; Ana T López-Jiménez; Óscar López-Pérez; Israel López-Valero; Magdalena J Lorenowicz; Mar Lorente; Peter Lorincz; Laura Lossi; Sophie Lotersztajn; Penny E Lovat; Jonathan F Lovell; Alenka Lovy; Péter Lőw; Guang Lu; Haocheng Lu; Jia-Hong Lu; Jin-Jian Lu; Mengji Lu; Shuyan Lu; Alessandro Luciani; John M Lucocq; Paula Ludovico; Micah A Luftig; Morten Luhr; Diego Luis-Ravelo; Julian J Lum; Liany Luna-Dulcey; Anders H Lund; Viktor K Lund; Jan D Lünemann; Patrick Lüningschrör; Honglin Luo; Rongcan Luo; Shouqing Luo; Zhi Luo; Claudio Luparello; Bernhard Lüscher; Luan Luu; Alex Lyakhovich; Konstantin G Lyamzaev; Alf Håkon Lystad; Lyubomyr Lytvynchuk; Alvin C Ma; Changle Ma; Mengxiao Ma; Ning-Fang Ma; Quan-Hong Ma; Xinliang Ma; Yueyun Ma; Zhenyi Ma; Ormond A MacDougald; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; Sandra Maday; Frank Madeo; Muniswamy Madesh; Tobias Madl; Julio Madrigal-Matute; Akiko Maeda; Yasuhiro Maejima; Marta Magarinos; Poornima Mahavadi; Emiliano Maiani; Kenneth Maiese; Panchanan Maiti; Maria Chiara Maiuri; Barbara Majello; Michael B Major; Elena Makareeva; Fayaz Malik; Karthik Mallilankaraman; Walter Malorni; Alina Maloyan; Najiba Mammadova; Gene Chi Wai Man; Federico Manai; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Masoud H Manjili; Ravi Manjithaya; Patricio Manque; Bella B Manshian; Raquel Manzano; Claudia Manzoni; Kai Mao; Cinzia Marchese; Sandrine Marchetti; Anna Maria Marconi; Fabrizio Marcucci; Stefania Mardente; Olga A Mareninova; Marta Margeta; Muriel Mari; Sara Marinelli; Oliviero Marinelli; Guillermo Mariño; Sofia Mariotto; Richard S Marshall; Mark R Marten; Sascha Martens; Alexandre P J Martin; Katie R Martin; Sara Martin; Shaun Martin; Adrián Martín-Segura; Miguel A Martín-Acebes; Inmaculada Martin-Burriel; Marcos Martin-Rincon; Paloma Martin-Sanz; José A Martina; Wim Martinet; Aitor Martinez; Ana Martinez; Jennifer Martinez; Moises Martinez Velazquez; Nuria Martinez-Lopez; Marta Martinez-Vicente; Daniel O Martins; Joilson O Martins; Waleska K Martins; Tania Martins-Marques; Emanuele Marzetti; Shashank Masaldan; Celine Masclaux-Daubresse; Douglas G Mashek; Valentina Massa; Lourdes Massieu; Glenn R Masson; Laura Masuelli; Anatoliy I Masyuk; Tetyana V Masyuk; Paola Matarrese; Ander Matheu; Satoaki Matoba; Sachiko Matsuzaki; Pamela Mattar; Alessandro Matte; Domenico Mattoscio; José L Mauriz; Mario Mauthe; Caroline Mauvezin; Emanual Maverakis; Paola Maycotte; Johanna Mayer; Gianluigi Mazzoccoli; Cristina Mazzoni; Joseph R Mazzulli; Nami McCarty; Christine McDonald; Mitchell R McGill; Sharon L McKenna; BethAnn McLaughlin; Fionn McLoughlin; Mark A McNiven; Thomas G McWilliams; Fatima Mechta-Grigoriou; Tania Catarina Medeiros; Diego L Medina; Lynn A Megeney; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Alfred J Meijer; Annemarie H Meijer; Jakob Mejlvang; Alicia Meléndez; Annette Melk; Gonen Memisoglu; Alexandrina F Mendes; Delong Meng; Fei Meng; Tian Meng; Rubem Menna-Barreto; Manoj B Menon; Carol Mercer; Anne E Mercier; Jean-Louis Mergny; Adalberto Merighi; Seth D Merkley; Giuseppe Merla; Volker Meske; Ana Cecilia Mestre; Shree Padma Metur; Christian Meyer; Hemmo Meyer; Wenyi Mi; Jeanne Mialet-Perez; Junying Miao; Lucia Micale; Yasuo Miki; Enrico Milan; Małgorzata Milczarek; Dana L Miller; Samuel I Miller; Silke Miller; Steven W Millward; Ira Milosevic; Elena A Minina; Hamed Mirzaei; Hamid Reza Mirzaei; Mehdi Mirzaei; Amit Mishra; Nandita Mishra; Paras Kumar Mishra; Maja Misirkic Marjanovic; Roberta Misasi; Amit Misra; Gabriella Misso; Claire Mitchell; Geraldine Mitou; Tetsuji Miura; Shigeki Miyamoto; Makoto Miyazaki; Mitsunori Miyazaki; Taiga Miyazaki; Keisuke Miyazawa; Noboru Mizushima; Trine H Mogensen; Baharia Mograbi; Reza Mohammadinejad; Yasir Mohamud; Abhishek Mohanty; Sipra Mohapatra; Torsten Möhlmann; Asif Mohmmed; Anna Moles; Kelle H Moley; Maurizio Molinari; Vincenzo Mollace; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Costanza Montagna; Mervyn J Monteiro; Andrea Montella; L Ruth Montes; Barbara Montico; Vinod K Mony; Giacomo Monzio Compagnoni; Michael N Moore; Mohammad A Moosavi; Ana L Mora; Marina Mora; David Morales-Alamo; Rosario Moratalla; Paula I Moreira; Elena Morelli; Sandra Moreno; Daniel Moreno-Blas; Viviana Moresi; Benjamin Morga; Alwena H Morgan; Fabrice Morin; Hideaki Morishita; Orson L Moritz; Mariko Moriyama; Yuji Moriyasu; Manuela Morleo; Eugenia Morselli; Jose F Moruno-Manchon; Jorge Moscat; Serge Mostowy; Elisa Motori; Andrea Felinto Moura; Naima Moustaid-Moussa; Maria Mrakovcic; Gabriel Muciño-Hernández; Anupam Mukherjee; Subhadip Mukhopadhyay; Jean M Mulcahy Levy; Victoriano Mulero; 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Per Nilsson; Shunbin Ning; Rituraj Niranjan; Hiroshi Nishimune; Mireia Niso-Santano; Ralph A Nixon; Annalisa Nobili; Clevio Nobrega; Takeshi Noda; Uxía Nogueira-Recalde; Trevor M Nolan; Ivan Nombela; Ivana Novak; Beatriz Novoa; Takashi Nozawa; Nobuyuki Nukina; Carmen Nussbaum-Krammer; Jesper Nylandsted; Tracey R O'Donovan; Seónadh M O'Leary; Eyleen J O'Rourke; Mary P O'Sullivan; Timothy E O'Sullivan; Salvatore Oddo; Ina Oehme; Michinaga Ogawa; Eric Ogier-Denis; Margret H Ogmundsdottir; Besim Ogretmen; Goo Taeg Oh; Seon-Hee Oh; Young J Oh; Takashi Ohama; Yohei Ohashi; Masaki Ohmuraya; Vasileios Oikonomou; Rani Ojha; Koji Okamoto; Hitoshi Okazawa; Masahide Oku; Sara Oliván; Jorge M A Oliveira; Michael Ollmann; James A Olzmann; Shakib Omari; M Bishr Omary; Gizem Önal; Martin Ondrej; Sang-Bing Ong; Sang-Ging Ong; Anna Onnis; Juan A Orellana; Sara Orellana-Muñoz; Maria Del Mar Ortega-Villaizan; Xilma R Ortiz-Gonzalez; Elena Ortona; Heinz D Osiewacz; Abdel-Hamid K Osman; Rosario Osta; Marisa S Otegui; 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Francesca Pentimalli; Cláudia Mf Pereira; Gustavo J S Pereira; Lilian C Pereira; Luis Pereira de Almeida; Nirma D Perera; Ángel Pérez-Lara; Ana B Perez-Oliva; María Esther Pérez-Pérez; Palsamy Periyasamy; Andras Perl; Cristiana Perrotta; Ida Perrotta; Richard G Pestell; Morten Petersen; Irina Petrache; Goran Petrovski; Thorsten Pfirrmann; Astrid S Pfister; Jennifer A Philips; Huifeng Pi; Anna Picca; Alicia M Pickrell; Sandy Picot; Giovanna M Pierantoni; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Karolina Pierzynowska; Federico Pietrocola; Miroslawa Pietruczuk; Claudio Pignata; Felipe X Pimentel-Muiños; Mario Pinar; Roberta O Pinheiro; Ronit Pinkas-Kramarski; Paolo Pinton; Karolina Pircs; Sujan Piya; Paola Pizzo; Theo S Plantinga; Harald W Platta; Ainhoa Plaza-Zabala; Markus Plomann; Egor Y Plotnikov; Helene Plun-Favreau; Ryszard Pluta; Roger Pocock; Stefanie Pöggeler; Christian Pohl; Marc Poirot; Angelo Poletti; Marisa Ponpuak; Hana Popelka; Blagovesta Popova; Helena Porta; Soledad Porte Alcon; Eliana Portilla-Fernandez; Martin Post; Malia B Potts; Joanna Poulton; Ted Powers; Veena Prahlad; Tomasz K Prajsnar; Domenico Praticò; Rosaria Prencipe; Muriel Priault; Tassula Proikas-Cezanne; Vasilis J Promponas; Christopher G Proud; Rosa Puertollano; Luigi Puglielli; Thomas Pulinilkunnil; Deepika Puri; Rajat Puri; Julien Puyal; Xiaopeng Qi; Yongmei Qi; Wenbin Qian; Lei Qiang; Yu Qiu; Joe Quadrilatero; Jorge Quarleri; Nina Raben; Hannah Rabinowich; Debora Ragona; Michael J Ragusa; Nader Rahimi; Marveh Rahmati; Valeria Raia; Nuno Raimundo; Namakkal-Soorappan Rajasekaran; Sriganesh Ramachandra Rao; Abdelhaq Rami; Ignacio Ramírez-Pardo; David B Ramsden; Felix Randow; Pundi N Rangarajan; Danilo Ranieri; Hai Rao; Lang Rao; Rekha Rao; Sumit Rathore; J Arjuna Ratnayaka; Edward A Ratovitski; Palaniyandi Ravanan; Gloria Ravegnini; Swapan K Ray; Babak Razani; Vito Rebecca; Fulvio Reggiori; Anne Régnier-Vigouroux; Andreas S Reichert; David Reigada; Jan H Reiling; Theo Rein; Siegfried Reipert; Rokeya Sultana Rekha; Hongmei Ren; Jun Ren; Weichao Ren; Tristan Renault; Giorgia Renga; Karen Reue; Kim Rewitz; Bruna Ribeiro de Andrade Ramos; S Amer Riazuddin; Teresa M Ribeiro-Rodrigues; Jean-Ehrland Ricci; Romeo Ricci; Victoria Riccio; Des R Richardson; Yasuko Rikihisa; Makarand V Risbud; Ruth M Risueño; Konstantinos Ritis; Salvatore Rizza; Rosario Rizzuto; Helen C Roberts; Luke D Roberts; Katherine J Robinson; Maria Carmela Roccheri; Stephane Rocchi; George G Rodney; Tiago Rodrigues; Vagner Ramon Rodrigues Silva; Amaia Rodriguez; Ruth Rodriguez-Barrueco; Nieves Rodriguez-Henche; Humberto Rodriguez-Rocha; Jeroen Roelofs; Robert S Rogers; Vladimir V Rogov; Ana I Rojo; Krzysztof Rolka; Vanina Romanello; Luigina Romani; Alessandra Romano; Patricia S Romano; David Romeo-Guitart; Luis C Romero; Montserrat Romero; Joseph C Roney; Christopher Rongo; Sante Roperto; Mathias T Rosenfeldt; Philip Rosenstiel; Anne G Rosenwald; Kevin A Roth; Lynn Roth; Steven Roth; Kasper M A Rouschop; 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Alberto Sanz; Pascual Sanz; Shweta Saran; Marco Sardiello; Timothy J Sargeant; Apurva Sarin; Chinmoy Sarkar; Sovan Sarkar; Maria-Rosa Sarrias; Surajit Sarkar; Dipanka Tanu Sarmah; Jaakko Sarparanta; Aishwarya Sathyanarayan; Ranganayaki Sathyanarayanan; K Matthew Scaglione; Francesca Scatozza; Liliana Schaefer; Zachary T Schafer; Ulrich E Schaible; Anthony H V Schapira; Michael Scharl; Hermann M Schatzl; Catherine H Schein; Wiep Scheper; David Scheuring; Maria Vittoria Schiaffino; Monica Schiappacassi; Rainer Schindl; Uwe Schlattner; Oliver Schmidt; Roland Schmitt; Stephen D Schmidt; Ingo Schmitz; Eran Schmukler; Anja Schneider; Bianca E Schneider; Romana Schober; Alejandra C Schoijet; Micah B Schott; Michael Schramm; Bernd Schröder; Kai Schuh; Christoph Schüller; Ryan J Schulze; Lea Schürmanns; Jens C Schwamborn; Melanie Schwarten; Filippo Scialo; Sebastiano Sciarretta; Melanie J Scott; Kathleen W Scotto; A Ivana Scovassi; Andrea Scrima; Aurora Scrivo; David Sebastian; Salwa Sebti; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Iban Seiliez; Ekihiro Seki; Scott B Selleck; Frank W Sellke; Joshua T Selsby; Michael Sendtner; Serif Senturk; Elena Seranova; Consolato Sergi; Ruth Serra-Moreno; Hiromi Sesaki; Carmine Settembre; Subba Rao Gangi Setty; Gianluca Sgarbi; Ou Sha; John J Shacka; Javeed A Shah; Dantong Shang; Changshun Shao; Feng Shao; Soroush Sharbati; Lisa M Sharkey; Dipali Sharma; Gaurav Sharma; Kulbhushan Sharma; Pawan Sharma; Surendra Sharma; Han-Ming Shen; Hongtao Shen; Jiangang Shen; Ming Shen; Weili Shen; Zheni Shen; Rui Sheng; Zhi Sheng; Zu-Hang Sheng; Jianjian Shi; Xiaobing Shi; Ying-Hong Shi; Kahori Shiba-Fukushima; Jeng-Jer Shieh; Yohta Shimada; Shigeomi Shimizu; Makoto Shimozawa; Takahiro Shintani; Christopher J Shoemaker; Shahla Shojaei; Ikuo Shoji; Bhupendra V Shravage; Viji Shridhar; Chih-Wen Shu; Hong-Bing Shu; Ke Shui; Arvind K Shukla; Timothy E Shutt; Valentina Sica; Aleem Siddiqui; Amanda Sierra; Virginia Sierra-Torre; Santiago Signorelli; Payel Sil; Bruno J de Andrade Silva; Johnatas D Silva; Eduardo Silva-Pavez; Sandrine Silvente-Poirot; Rachel E Simmonds; Anna Katharina Simon; Hans-Uwe Simon; Matias Simons; Anurag Singh; Lalit P Singh; Rajat Singh; Shivendra V Singh; Shrawan K Singh; Sudha B Singh; Sunaina Singh; Surinder Pal Singh; Debasish Sinha; Rohit Anthony Sinha; Sangita Sinha; Agnieszka Sirko; Kapil Sirohi; Efthimios L Sivridis; Panagiotis Skendros; Aleksandra Skirycz; Iva Slaninová; Soraya S Smaili; Andrei Smertenko; Matthew D Smith; Stefaan J Soenen; Eun Jung Sohn; Sophia P M Sok; Giancarlo Solaini; Thierry Soldati; Scott A Soleimanpour; Rosa M Soler; Alexei Solovchenko; Jason A Somarelli; Avinash Sonawane; Fuyong Song; Hyun Kyu Song; Ju-Xian Song; Kunhua Song; Zhiyin Song; Leandro R Soria; Maurizio Sorice; Alexander A Soukas; Sandra-Fausia Soukup; Diana Sousa; Nadia Sousa; Paul A Spagnuolo; Stephen A Spector; M M Srinivas Bharath; Daret St Clair; Venturina Stagni; Leopoldo Staiano; Clint A Stalnecker; Metodi V Stankov; Peter B Stathopulos; Katja Stefan; Sven Marcel Stefan; Leonidas Stefanis; Joan S Steffan; Alexander Steinkasserer; Harald Stenmark; Jared Sterneckert; Craig Stevens; Veronika Stoka; Stephan Storch; Björn Stork; Flavie Strappazzon; Anne Marie Strohecker; Dwayne G Stupack; Huanxing Su; Ling-Yan Su; Longxiang Su; Ana M Suarez-Fontes; Carlos S Subauste; Selvakumar Subbian; Paula V Subirada; Ganapasam Sudhandiran; Carolyn M Sue; Xinbing Sui; Corey Summers; Guangchao Sun; Jun Sun; Kang Sun; Meng-Xiang Sun; Qiming Sun; Yi Sun; Zhongjie Sun; Karen K S Sunahara; Eva Sundberg; Katalin Susztak; Peter Sutovsky; Hidekazu Suzuki; Gary Sweeney; J David Symons; Stephen Cho Wing Sze; Nathaniel J Szewczyk; Anna Tabęcka-Łonczynska; Claudio Tabolacci; Frank Tacke; Heinrich Taegtmeyer; Marco Tafani; Mitsuo Tagaya; Haoran Tai; Stephen W G Tait; Yoshinori Takahashi; Szabolcs Takats; Priti Talwar; Chit Tam; Shing Yau Tam; Davide Tampellini; Atsushi Tamura; Chong Teik Tan; Eng-King Tan; Ya-Qin Tan; Masaki Tanaka; Motomasa Tanaka; Daolin Tang; Jingfeng Tang; Tie-Shan Tang; Isei Tanida; Zhipeng Tao; Mohammed Taouis; Lars Tatenhorst; Nektarios Tavernarakis; Allen Taylor; Gregory A Taylor; Joan M Taylor; Elena Tchetina; Andrew R Tee; Irmgard Tegeder; David Teis; Natercia Teixeira; Fatima Teixeira-Clerc; Kumsal A Tekirdag; Tewin Tencomnao; Sandra Tenreiro; Alexei V Tepikin; Pilar S Testillano; Gianluca Tettamanti; Pierre-Louis Tharaux; Kathrin Thedieck; Arvind A Thekkinghat; Stefano Thellung; Josephine W Thinwa; V P Thirumalaikumar; Sufi Mary Thomas; Paul G Thomes; Andrew Thorburn; Lipi Thukral; Thomas Thum; Michael Thumm; Ling Tian; Ales Tichy; Andreas Till; Vincent Timmerman; Vladimir I Titorenko; Sokol V Todi; Krassimira Todorova; Janne M Toivonen; Luana Tomaipitinca; Dhanendra Tomar; Cristina Tomas-Zapico; Sergej Tomić; Benjamin Chun-Kit Tong; Chao Tong; Xin Tong; Sharon A Tooze; Maria L Torgersen; Satoru Torii; Liliana Torres-López; Alicia Torriglia; Christina G Towers; Roberto Towns; Shinya Toyokuni; Vladimir Trajkovic; Donatella Tramontano; Quynh-Giao Tran; Leonardo H Travassos; Charles B Trelford; Shirley Tremel; Ioannis P Trougakos; Betty P Tsao; Mario P Tschan; Hung-Fat Tse; Tak Fu Tse; Hitoshi Tsugawa; Andrey S Tsvetkov; David A Tumbarello; Yasin Tumtas; María J Tuñón; Sandra Turcotte; Boris Turk; Vito Turk; Bradley J Turner; Richard I Tuxworth; Jessica K Tyler; Elena V Tyutereva; Yasuo Uchiyama; Aslihan Ugun-Klusek; Holm H Uhlig; Marzena Ułamek-Kozioł; Ilya V Ulasov; Midori Umekawa; Christian Ungermann; Rei Unno; Sylvie Urbe; Elisabet Uribe-Carretero; Suayib Üstün; Vladimir N Uversky; Thomas Vaccari; Maria I Vaccaro; Björn F Vahsen; Helin Vakifahmetoglu-Norberg; Rut Valdor; Maria J Valente; Ayelén Valko; Richard B Vallee; Angela M Valverde; Greet Van den Berghe; Stijn van der Veen; Luc Van Kaer; Jorg van Loosdregt; Sjoerd J L van Wijk; Wim Vandenberghe; Ilse Vanhorebeek; Marcos A Vannier-Santos; Nicola Vannini; M Cristina Vanrell; Chiara Vantaggiato; Gabriele Varano; Isabel Varela-Nieto; Máté Varga; M Helena Vasconcelos; Somya Vats; Demetrios G Vavvas; Ignacio Vega-Naredo; Silvia Vega-Rubin-de-Celis; Guillermo Velasco; Ariadna P Velázquez; Tibor Vellai; Edo Vellenga; Francesca Velotti; Mireille Verdier; Panayotis Verginis; Isabelle Vergne; Paul Verkade; Manish Verma; Patrik Verstreken; Tim Vervliet; Jörg Vervoorts; Alexandre T Vessoni; Victor M Victor; Michel Vidal; Chiara Vidoni; Otilia V Vieira; Richard D Vierstra; Sonia Viganó; Helena Vihinen; Vinoy Vijayan; Miquel Vila; Marçal Vilar; José M Villalba; Antonio Villalobo; Beatriz Villarejo-Zori; Francesc Villarroya; Joan Villarroya; Olivier Vincent; Cecile Vindis; Christophe Viret; Maria Teresa Viscomi; Dora Visnjic; Ilio Vitale; David J Vocadlo; Olga V Voitsekhovskaja; Cinzia Volonté; Mattia Volta; Marta Vomero; Clarissa Von Haefen; Marc A Vooijs; Wolfgang Voos; Ljubica Vucicevic; Richard Wade-Martins; Satoshi Waguri; Kenrick A Waite; Shuji Wakatsuki; David W Walker; Mark J Walker; Simon A Walker; Jochen Walter; Francisco G Wandosell; Bo Wang; Chao-Yung Wang; Chen Wang; Chenran Wang; Chenwei Wang; Cun-Yu Wang; Dong Wang; Fangyang Wang; Feng Wang; Fengming Wang; Guansong Wang; Han Wang; Hao Wang; Hexiang Wang; Hong-Gang Wang; Jianrong Wang; Jigang Wang; Jiou Wang; Jundong Wang; Kui Wang; Lianrong Wang; Liming Wang; Maggie Haitian Wang; Meiqing Wang; Nanbu Wang; Pengwei Wang; Peipei Wang; Ping Wang; Ping Wang; Qing Jun Wang; Qing Wang; Qing Kenneth Wang; Qiong A Wang; Wen-Tao Wang; Wuyang Wang; Xinnan Wang; Xuejun Wang; Yan Wang; Yanchang Wang; Yanzhuang Wang; Yen-Yun Wang; Yihua Wang; Yipeng Wang; Yu Wang; Yuqi Wang; Zhe Wang; Zhenyu Wang; Zhouguang Wang; Gary Warnes; Verena Warnsmann; Hirotaka Watada; Eizo Watanabe; Maxinne Watchon; Anna Wawrzyńska; Timothy E Weaver; Grzegorz Wegrzyn; Ann M Wehman; Huafeng Wei; Lei Wei; Taotao Wei; Yongjie Wei; Oliver H Weiergräber; Conrad C Weihl; Günther Weindl; Ralf Weiskirchen; Alan Wells; Runxia H Wen; Xin Wen; Antonia Werner; Beatrice Weykopf; Sally P Wheatley; J Lindsay Whitton; Alexander J Whitworth; Katarzyna Wiktorska; Manon E Wildenberg; Tom Wileman; Simon Wilkinson; Dieter Willbold; Brett Williams; Robin S B Williams; Roger L Williams; Peter R Williamson; Richard A Wilson; Beate Winner; Nathaniel J Winsor; Steven S Witkin; Harald Wodrich; Ute Woehlbier; Thomas Wollert; Esther Wong; Jack Ho Wong; Richard W Wong; Vincent Kam Wai Wong; W Wei-Lynn Wong; An-Guo Wu; Chengbiao Wu; Jian Wu; Junfang Wu; Kenneth K Wu; Min Wu; Shan-Ying Wu; Shengzhou Wu; Shu-Yan Wu; Shufang Wu; William K K Wu; Xiaohong Wu; Xiaoqing Wu; Yao-Wen Wu; Yihua Wu; Ramnik J Xavier; Hongguang Xia; Lixin Xia; Zhengyuan Xia; Ge Xiang; Jin Xiang; Mingliang Xiang; Wei Xiang; Bin Xiao; Guozhi Xiao; Hengyi Xiao; Hong-Tao Xiao; Jian Xiao; Lan Xiao; Shi Xiao; Yin Xiao; Baoming Xie; Chuan-Ming Xie; Min Xie; Yuxiang Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Congfeng Xu; En Xu; Haoxing Xu; Jing Xu; JinRong Xu; Liang Xu; Wen Wen Xu; Xiulong Xu; Yu Xue; Sokhna M S Yakhine-Diop; Masamitsu Yamaguchi; Osamu Yamaguchi; Ai Yamamoto; Shunhei Yamashina; Shengmin Yan; Shian-Jang Yan; Zhen Yan; Yasuo Yanagi; Chuanbin Yang; Dun-Sheng Yang; Huan Yang; Huang-Tian Yang; Hui Yang; Jin-Ming Yang; Jing Yang; Jingyu Yang; Ling Yang; Liu Yang; Ming Yang; Pei-Ming Yang; Qian Yang; Seungwon Yang; Shu Yang; Shun-Fa Yang; Wannian Yang; Wei Yuan Yang; Xiaoyong Yang; Xuesong Yang; Yi Yang; Ying Yang; Honghong Yao; Shenggen Yao; Xiaoqiang Yao; Yong-Gang Yao; Yong-Ming Yao; Takahiro Yasui; Meysam Yazdankhah; Paul M Yen; Cong Yi; Xiao-Ming Yin; Yanhai Yin; Zhangyuan Yin; Ziyi Yin; Meidan Ying; Zheng Ying; Calvin K Yip; Stephanie Pei Tung Yiu; Young H Yoo; Kiyotsugu Yoshida; Saori R Yoshii; Tamotsu Yoshimori; Bahman Yousefi; Boxuan Yu; Haiyang Yu; Jun Yu; Jun Yu; Li Yu; Ming-Lung Yu; Seong-Woon Yu; Victor C Yu; W Haung Yu; Zhengping Yu; Zhou Yu; Junying Yuan; Ling-Qing Yuan; Shilin Yuan; Shyng-Shiou F Yuan; Yanggang Yuan; Zengqiang Yuan; Jianbo Yue; Zhenyu Yue; Jeanho Yun; Raymond L Yung; David N Zacks; Gabriele Zaffagnini; Vanessa O Zambelli; Isabella Zanella; Qun S Zang; Sara Zanivan; Silvia Zappavigna; Pilar Zaragoza; Konstantinos S Zarbalis; Amir Zarebkohan; Amira Zarrouk; Scott O Zeitlin; Jialiu Zeng; Ju-Deng Zeng; Eva Žerovnik; Lixuan Zhan; Bin Zhang; Donna D Zhang; Hanlin Zhang; Hong Zhang; Hong Zhang; Honghe Zhang; Huafeng Zhang; Huaye Zhang; Hui Zhang; Hui-Ling Zhang; Jianbin Zhang; Jianhua Zhang; Jing-Pu Zhang; Kalin Y B Zhang; Leshuai W Zhang; Lin Zhang; Lisheng Zhang; Lu Zhang; Luoying Zhang; Menghuan Zhang; Peng Zhang; Sheng Zhang; Wei Zhang; Xiangnan Zhang; Xiao-Wei Zhang; Xiaolei Zhang; Xiaoyan Zhang; Xin Zhang; Xinxin Zhang; Xu Dong Zhang; Yang Zhang; Yanjin Zhang; Yi Zhang; Ying-Dong Zhang; Yingmei Zhang; Yuan-Yuan Zhang; Yuchen Zhang; Zhe Zhang; Zhengguang Zhang; Zhibing Zhang; Zhihai Zhang; Zhiyong Zhang; Zili Zhang; Haobin Zhao; Lei Zhao; Shuang Zhao; Tongbiao Zhao; Xiao-Fan Zhao; Ying Zhao; Yongchao Zhao; Yongliang Zhao; Yuting Zhao; Guoping Zheng; Kai Zheng; Ling Zheng; Shizhong Zheng; Xi-Long Zheng; Yi Zheng; Zu-Guo Zheng; Boris Zhivotovsky; Qing Zhong; Ao Zhou; Ben Zhou; Cefan Zhou; Gang Zhou; Hao Zhou; Hong Zhou; Hongbo Zhou; Jie Zhou; Jing Zhou; Jing Zhou; Jiyong Zhou; Kailiang Zhou; Rongjia Zhou; Xu-Jie Zhou; Yanshuang Zhou; Yinghong Zhou; Yubin Zhou; Zheng-Yu Zhou; Zhou Zhou; Binglin Zhu; Changlian Zhu; Guo-Qing Zhu; Haining Zhu; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Yanping Zhu; Yushan Zhu; Haixia Zhuang; Xiaohong Zhuang; Katarzyna Zientara-Rytter; Christine M Zimmermann; Elena Ziviani; Teresa Zoladek; Wei-Xing Zong; Dmitry B Zorov; Antonio Zorzano; Weiping Zou; Zhen Zou; Zhengzhi Zou; Steven Zuryn; Werner Zwerschke; Beate Brand-Saberi; X Charlie Dong; Chandra Shekar Kenchappa; Zuguo Li; Yong Lin; Shigeru Oshima; Yueguang Rong; Judith C Sluimer; Christina L Stallings; Chun-Kit Tong Journal: Autophagy Date: 2021-02-08 Impact factor: 13.391
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