c-cbl inhibition improves cardiac function and survival in...

2031

Myocardial infarction (MI) due to coronary artery occlu-sion is a leading cause of death worldwide.1 The loss

of blood flow after MI results in loss of cardiac myocytes in the ischemic zone, which diminishes cardiac contractility and impedes angiogenesis and repair. Although much is known about the pathways that promote the pathological remodel-ing responses, mechanisms that promote myocyte loss and impair cardiac function have not been as clearly defined. The ubiquitin proteasome system is largely responsible for the degradation of misfolded and damaged proteins as well as proteins involved in the control of components of the con-tractile apparatus and hypertrophic gene expression, thereby regulating cardiac hypertrophy and remodeling.2 Activation of the ubiquitin proteasome system in heart reduces muscle mass as occurs during muscle atrophy,3,4 promotes myocyte hyper-trophy, and impairs cardiac remodeling.5

Clinical Perspective on p 2043

The formation of ubiquitin-protein conjugates involves 3 components that participate in a cascade of ubiquitin transfer reactions: a ubiquitin-activation enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) that acts at the last step of the cascade and presents the greatest tissue and substrate specificity.6,7 Once a protein has been marked with polyubiquitin conjugates, it is destined for degradation by the multicatalytic protease, the 26S protea-some complex.8 The proto-oncogene Casitas b-lineage lym-phoma (c-Cbl) contains a highly conserved helical region and an adjacent RING finger domain that together bind an E2; as such, c-Cbl acts as a RING type ubiquitin ligase (E3) and negatively regulates receptor and nonreceptor tyrosine kinases by promoting their ubiquitination and lysosomal/pro-teasomal degradation.9 In addition to a tyrosine kinase–bind-ing domain for binding to activated tyrosine kinases, c-Cbl also contains multiple protein interaction motifs; a tyrosine kinase binding domain that encompasses a 4-helical bundle,

Background—The proto-oncogene Casitas b-lineage lymphoma (c-Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor and nonreceptor tyrosine kinases, resulting in their ubiquitination and downregulation. However, the function of c-Cbl in the control of cardiac function is currently unknown. In this study, we examined the role of c-Cbl in myocyte death and cardiac function after myocardial ischemia.

Methods and Results—We show increased c-Cbl expression in human ischemic and dilated cardiomyopathy hearts and in response to pathological stress stimuli in mice. c-Cbl–deficient mice demonstrated a more robust functional recovery after myocardial ischemia/reperfusion injury and significantly reduced myocyte apoptosis and improved cardiac function. Ubiquitination and downregulation of key survival c-Cbl targets, epidermal growth factor receptors and focal adhesion kinase, were significantly reduced in c-Cbl knockout mice. Inhibition of c-Cbl expression or its ubiquitin ligase activity in cardiac myocytes offered protection against H

2O

2 stress. Interestingly, c-Cbl deletion reduced the risk of death and

increased cardiac functional recovery after chronic myocardial ischemia. This beneficial effect of c-Cbl deletion was associated with enhanced neoangiogenesis and increased expression of vascular endothelial growth factor-a and vascular endothelial growth factor receptor type 2 in the infarcted region.

Conclusions—c-Cbl activation promotes myocyte apoptosis, inhibits angiogenesis, and causes adverse cardiac remodeling after myocardial infarction. These findings point to c-Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling after myocardial ischemia. (Circulation. 2014;129:2031-2043.)

Key Words: angiogenesis ◼ apoptosis ◼ myocardial ischemia ◼ ubiquitin

© 2014 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.113.007004

Received October 19, 2013; accepted February 21, 2014.From the Cardiovascular Research Center and Department of Physiology, Temple University School of Medicine, Philadelphia, PA (K.R., M.A.K., R.S.,

J.G., X.G., Z.Q., D.Y., S.R.H., A. Sabri); Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha (B.M., N.Z., W.A., H.B.); and Department of Surgery, University of Connecticut Health Center, Farmington (A. Sanjay).

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA. 113.007004/-/DC1.

Correspondence to Abdelkarim Sabri, PhD, Cardiovascular Research Center, Temple University, MERB 1045, 3500 N Broad St, Philadelphia, PA 19140. E-mail [email protected]

c-Cbl Inhibition Improves Cardiac Function and Survival in Response to Myocardial Ischemia

Khadija Rafiq, PhD; Mikhail A. Kolpakov, MD, PhD; Rachid Seqqat, PhD; Jianfen Guo, PhD; Xinji Guo, PhD; Zhao Qi, MD; Daohai Yu, PhD; Bhopal Mohapatra, DVM; Neha Zutshi, MS;

Wei An, BS; Hamid Band, MD, PhD; Archana Sanjay, PhD; Steven R. Houser, PhD; Abdelkarim Sabri, PhD

by guest on July 14, 2018http://circ.ahajournals.org/

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http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.113.007004/-/DC1

http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.113.007004/-/DC1

mailto:[email protected]

http://circ.ahajournals.org/


























2032 Circulation May 20, 2014

an EF hand, a Src homology domain 2, and a proline-rich motif.10 These domains/motifs allow the interaction of c-Cbl with multiple signal transducers, including the p85 subunit of phosphoinositide-3 kinase, the Crk and Grb2 adaptor proteins, and Src family kinases, suggesting that c-Cbl acts as a dock-ing protein to integrate signaling pathways.9 Although c-Cbl phosphorylation in response to a variety of cell surface recep-tors has been documented, the precise biological function of c-Cbl is unclear, and both negative and positive roles for c-Cbl have been proposed.9 In the heart, c-Cbl expression increases in response to right ventricular pressure overload stimuli,11 and 1 study linked c-Cbl to reduced insulin responsiveness in adult rat cardiomyocytes in vitro.12 However, specific func-tions of c-Cbl in myocyte growth and cardiac function are still not well understood, and no loss-of-function cardiac pheno-types of c-Cbl gene have yet been described.

We recently showed that activation of c-Cbl ubiquitin ligase by neutrophil-derived serine proteases leads to focal adhesion protein turnover and myofibril degeneration.13 In the pres-ent study, we show that c-Cbl expression increases in human cardiomyopathies and in response to myocardial stress in mice. Mice lacking c-Cbl display normal cardiac function but exhibit less myocyte loss and cardiac dysfunction in response to ischemia/reperfusion (IR) injury. c-Cbl–deficient mice are also protected against pathological remodeling of the heart after chronic MI. This protection appears to be mediated, at least in part, to neoangiogenesis in the infarcted region. We conclude that c-Cbl negatively regulates ventricular myocyte survival and angiogenesis after myocardial ischemia injury.

MethodsSubjects and Tissue Preparation Human left ventricular (LV) myocardial tissue was obtained at the time of cardiac transplantation from patients with end-stage heart failure ischemic cardiomyopathy (ICM; n=5) and dilated cardiomy-opathy (DCM; n=5). LV tissue was also obtained from nonfailing hearts (n=5) of brain-dead organ donors that could not be used for transplantation. Relevant clinical data were collected from all subjects providing heart tissue (Table I in the online-only Data Supplement). Our protocol was approved by the Temple University institutional review board, according to the guidelines noted in our instructions to authors. All hearts were arrested in situ with cold, blood-containing, cardioplegia solution and promptly transported to the laboratory in Krebs-Henseleit buffer solution, as described previously.14 LV tissue slices were immediately snap-frozen in liquid nitrogen and stored at −80°C or fixed in formalin.

Myocardial Ischemia and IR Injury Procedure All mice were maintained and studied with the use of proto-cols approved by the Animal Care and Use Committee of Temple University. c-Cbl knockout (KO) and Cblflox/flox mice (provided by Dr Hua Gu, Institut de Recheche Clinique de Montreal, Montreal, Quebec, Canada) were genotyped as described previously.15,16 Generation of α-myosin heavy chain (α-MHC)-MerCreMer/c-Cblflox/

flox mice is described in Methods in the online-only Data Supplement (Figure I in the online-only Data Supplement). For easier reading, we refer to these mice as CM-Cbl KO mice. CM-Cbl KO mice were paired with age- and sex-matched c-Cblflox/flox littermates for all experiments. Ten- to 12-week-old wild-type (WT) control, c-Cbl KO, CM-Cbl KO, and c-Cblflox/flox mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) to perform a left thoracotomy under mechanical ventilation. The body temperatures of the mice were maintained by a heated surgical platform, and body

temperature was monitored with a rectal sensor during the surgi-cal procedure. A 6-0 suture with a slipknot was tied around the left anterior descending coronary artery to produce ischemia. Consistent elevation of the ST segment was observed in lead II tracings after occlusion of the left anterior descending coronary vessel. One group of mice was revived for a 30-minute ischemia period, after which the knot was released, and reperfusion in the heart occurred. The chest wall was closed with 8-0 silk, and then the animal was removed from the ventilator and kept warm in the cage maintained at 37°C over-night. A sham procedure constituted the surgical incision without left anterior descending coronary artery ligation. Hearts were harvested after 2, 7, or 30 days (for animals subjected to myocardial ischemia) or after 24 hours of reperfusion (for animal subjected to IR injury). All mice were randomized to the aforementioned experimental protocol.

Data Analysis Summary data are presented as mean±SEM. For comparisons of >2 groups, 1-way ANOVA or, more generally, the generalized lin-ear regression approach was used for normal distributions, and the Kruskal-Wallis test was used for nonnormal or small sample situa-tions. Two-group comparisons were analyzed by the 2-sample t test or nonparametric Wilcoxon rank test, whenever appropriate (eg, when the sample size was small or the distribution was not normal). Bonferroni post hoc test adjustments were used for multiple pairwise group comparisons after the overall F or Kruskal Wallis test showed a statistical significance. The exact testing was used when the sample size was small (eg, when all group sizes were <10). The survival time was analyzed by the Kaplan-Meier product-limit approach and com-pared by the log-rank test. To make the plot and interpretation of the fold increase over WT sham easier to understand, we scaled the data value from each animal in each of the 4 groups, including individ-ual sham values, using the mean of the WT sham group. All in vitro experiments were performed at least 3 times from 3 different cultures, and the data values were scaled to controls. A value of P<0.05 was considered statistically significant.

An expanded Methods section is included in the online-only Data Supplement.

Resultsc-Cbl Expression Is Developmentally DownregulatedTo determine the temporal pattern of c-Cbl expression dur-ing normal cardiac development in the mouse, we analyzed total protein extracts from series of fetal and postnatal time points. c-Cbl protein was highly expressed in fetal hearts at 12.5 days, and this expression decreased gradually throughout fetal life (−17±3% at E19.5 compared with E12.5; Figure 1A). After birth, c-Cbl expression decreased significantly at post-natal days 3 and 7 (−30±2% at 1 day, −37±2% at 3 days, and −46±3% at 7 days compared with E12.5) and reached low but detectable levels in adult hearts (−80±5% compared with E12.5). This decrease in c-Cbl expression in adult hearts was associated with a decrease in c-Cbl expression in adult iso-lated myocytes (Figure 1B).

c-Cbl Expression Is Upregulated in Failing Human MyocardiumCardiac explants from human donors (clinical characteristics of heart donors are summarized in Table I in the online-only Data Supplement) were assessed for c-Cbl expression with the use of Western blot and immunohistochemistry. Figure 1C shows increased c-Cbl immunostaining in both ICM and DCM compared with nonfailing hearts. This c-Cbl accumu-lation was detected mainly in myocytes, and no appreciable


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Rafiq et al c-Cbl Deletion Is Cardioprotective 2033

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Figure 1. Proto-oncogene Casitas b-lineage lymphoma (c-Cbl) expression is upregulated in human cardiomyopathies. A and B, Representative immunoblots of cardiac lysates from indicated embryonic (E), postnatal day (P), or adult (Ad) Sprague-Dawley rats (A) or cardiomyocytes isolated from 1 day postnatal or adult rat hearts (B). GAPDH was shown as a loading control. Western blots are representative of 3 separate experiments. C, Representative immunostainings of paraffin-embedded nonfailing (NF), ischemic cardiomyopathy (ICM), and dilated cardiomyopathy (DCM) human heart sections stained for c-Cbl and counterstained with hematoxylin. Bar, 40 μm. *P<0.05 vs NF controls. D, Immunoblot analysis of whole lysates from NF (n=5), ICM (n=5), and DCM (n=5) human hearts. E, Lysates from neonatal rat cardiomyocytes untreated or treated with isoproterenol (Iso; 10 μmol/L), norepinephrine (NE; 10 μmol/L), thrombin (Thr; 1 U/mL), or tumor necrosis factor-α (TNFα; 100 ng/mL) for 48 hours. Ctrl indicates control. Top, c-Cbl immunoblot with GAPDH taken as a loading control. Bottom, Quantification of experiments expressed as mean±SE from 3 separate cultures. *P<0.05 vs control.


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labeling was observed at the level of fibroblasts in the fibrotic zone, smooth muscle cells of medial arteries, or endothelial cells. Some c-Cbl immunostaining was detected at the medial layer of small arterioles (data not shown). c-Cbl accumula-tion in failing human heart samples was 2.3-fold and 1.5-fold higher in the ICM and DCM human hearts, respectively, com-pared with signals in nonfailing hearts (Figure 1D).

To explore the mechanism of induction of c-Cbl, we exam-ined the effect of stress stimuli on c-Cbl expression in neonatal rat cardiomyocytes. Myocytes challenged with hypertrophic agonists norepinephrine (primarily an α-adrenergic receptor agonist), isoproterenol (a nonselective β-adrenergic receptor agonist), thrombin, or tumor necrosis factor-α for 48 hours showed an increase in c-Cbl accumulation compared with controls (Figure 1E). These data together show the effect of hypertrophic agonists and inflammatory cytokines in modulat-ing c-Cbl expression in cardiomyocytes.

c-Cbl Ablation Protects the Heart From IR InjuryTo explore the functional effects associated with c-Cbl induc-tion in failing hearts, c-Cbl KO mice and their WT controls were subjected to transient left anterior descending coro-nary artery ligation for 30 minutes followed by reperfusion for 24 hours. c-Cbl KO mice did not differ from WT mice with regard to their baseline ratio of heart weight to body weight, heart rate (WT, 457±10 bpm; c-Cbl KO, 471±24 bpm), LV end-systolic pressure (WT, 89±9 mm Hg; c-Cbl KO, 86±5 mm Hg), LV end-diastolic pressure (WT, 5.2±0.8 mm Hg; c-Cbl KO, 6.3±0.9 mm Hg), and maximal dP/dt (WT, 8517±780 mm Hg·min–1; c-Cbl KO, 7517±395 mm Hg·min–1); hemodynamic data were obtained in n=8 WT mice and n=8 c-Cbl KO mice. Interestingly, c-Cbl KO mice subjected to IR injury showed significantly less impairment of cardiac func-tion as assessed by LV ejection fraction and fractional short-ening percentage compared with WT mice (Figure 2A and 2B and Table). Ablation of c-Cbl also reduced the percentage of infarcted area normalized to area at risk (22±3%) compared with WT mice (34±5%; Figure 2C and 2D). Apoptotic cell death in the area at risk, as evaluated by terminal deoxynu-cleotidyl transferase dUTP nick end-labeling (TUNEL) stain-ing, was significantly lower in c-Cbl KO mice compared with WT mice (Figure 2E and 2F). Under baseline conditions, the proportion of TUNEL-positive cardiomyocytes was very low (<0.1%) in c-Cbl KO and WT mice. Consistent with TUNEL staining, caspase-3 activity was also lower in c-Cbl KO infarcted hearts compared with WT, thus confirming a lesser propensity for myocyte death by apoptosis in c-Cbl KO mice subjected to IR (Figure 2G). Circulating plasma levels of the cardiac-specific isoform of troponin-I were evaluated as an additional marker of myocardial injury (Figure 2H). Serum samples from c-Cbl KO mice subjected to IR injury were found to contain lower levels of cardiac-specific isoform of troponin-I compared with WT mice (43.2±2.2 in WT versus 28.1±3.5 ng/mL per milligram protein in c-Cbl KO), thus con-firming the cytoprotective effect of c-Cbl deletion.

c-Cbl Deletion Exhibits Protective SignalingActivation of c-Cbl enables it to act as a multivalent adaptor for a plethora of Src homology domain 2– or Src homology

domain 3–containing signaling proteins to positively or negatively regulate signaling pathways and cell growth.9 Therefore, we next examined the molecular mechanisms responsible for c-Cbl–mediated cardioprotection. Relative to WT samples, c-Cbl KO heart samples showed increases in the levels of phospho-Akt(S473) (3.2±0.2-fold), phos-pho-Bad(S112) (3.3±0.4-fold), Bad (2.9±0.5-fold), XIAP (1.8±0.2-fold), and phospho-Erk

1/2 (1.7±0.2-fold; Figure 2I).

However, we observed no significant increase in the expres-sion of total-Akt or c-IAP1 in c-Cbl KO compared with WT control heart samples. We also examined these signal-ing molecules 1 day after IR injury; relative to sham-oper-ated controls, WT mice after IR injury showed significant increases in phospho-Akt(S473) (2.2±0.2-fold) and phospho-Erk

1/2 (2.4±0.2-fold), along with a significant decrease in

the expression of phospho-Bad(S112) (−0.78±0.07-fold), c-IAP-1 (−0.70±0.1-fold), and XIAP (−0.80±0.14-fold). In comparison, c-Cbl KO mice subjected to IR injury showed a stronger increase in phospho-Bad(S112), c-IAP, and XIAP expression compared with WT mice subjected to IR injury. No significant effect on total Akt expression or phosphoryla-tion between WT and c-Cbl KO mice subjected to IR injury was detected. Thus, compared with WT hearts, c-Cbl KO hearts show significantly more elevation in the levels of anti-apoptotic signaling molecules.

Cardiac-Specific Deletion of c-Cbl Is CardioprotectiveIn light of the cardioprotective effect of global c-Cbl deletion, we generated mice homozygous for a loxP-flanked c-Cbl allele and positive for tamoxifen-inducible Cre recombi-nase driven by the cardiomyocyte-specific α-MHC promoter (α-MHC-MerCreMer/c-Cblflox/flox). Tamoxifen injections at the age of 8 weeks for 5 days induced Cre-mediated recom-bination after 4 weeks, and Western analysis of protein lysates confirmed that c-Cbl protein was reduced by 90% in cardiac tissue (Figure I in the online-only Data Supplement). To assess the potential consequences of cardiac c-Cbl loss of function on IR injury, we subjected CM-Cbl KO and c-Cblflox/

flox mice to IR for 24 hours. Echocardiography before sur-gery showed no difference in cardiac function and geom-etry between the groups (Table II in the online-only Data Supplement). Twenty-four hours after surgery, CM-Cbl KO mice showed significantly improved ejection fraction and fractional shortening compared with c-Cblflox/flox mice (Figure IIA and IIB in the online-only Data Supplement). This was associated with reduced LV internal diameter (Figure IIC and IID in the online-only Data Supplement). Cardiac myo-cyte–specific deletion of c-Cbl also reduced the percentage of infarcted area normalized to area at risk (25±4%; n=6) compared with c-Cblflox/flox hearts (38±3%; n=5; Figure IIE in the online-only Data Supplement), reduced occurrence of TUNEL-positive cells in the infarcted hearts (Figure IIIA and IIIB in the online-only Data Supplement), and attenuated caspase-3 activity in infarcted hearts compared with c-Cblflox/flox mice (Figure IIIC in the online-only Data Supplement). These data collectively demonstrate that dele-tion of c-Cbl in cardiomyocytes offers cardioprotection in response to IR injury.


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Figure 2. Proto-oncogene Casitas b-lineage lymphoma (c-Cbl) ablation protects against ischemia/reperfusion (IR) injury. The left anterior descending coronary artery was ligated for 30 minutes to induce ischemia, and it subsequently was reperfused for 24 hours. A and B, Echocardiography measurement of ejection fraction (A) and fractional shortening (B) in wild-type (WT) and c-Cbl knockout (KO) animals (n=8 for sham groups; n=9 for IR groups). C, Representative cross sections were stained with triphenyltetrazolium chloride and Evans blue to determine the extent of injury. D, Quantification of infarct area (IA) vs area at risk (AAR) after IR injury in the indicated groups (n=6 per group). E, Left ventricular (LV) tissue sections were assessed for apoptosis with the use of the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (green), tropomyosin (red), and DAPI (4′,6-diamidino-2-phenylindole; blue) staining. Bars, 40 μm (a through d) or 20 μm (e and f). F, The number of TUNEL-positive myocytes in the ischemic area was expressed as a percentage of total nuclei detected by DAPI staining. G, Quantification of caspase-3 activity in LV with the use of caspase-3–specific fluorogenic substrate. RFU indicates relative fluorescence units. H, Serum levels of cardiac troponin I (cTnI) after IR injury in WT and c-Cbl KO mice (n=6 in each group). I, Representative immunoblots of LV lysates from WT or c-Cbl KO animals. GAPDH was included as a loading control. Left, Representative autoradiogram (with each lane from a single gel exposed for the same duration). Right, Fold induction (n=6 in each group). *P<0.05 vs WT shams; †P<0.05 vs WT IR.


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c-Cbl Deletion Prevents Epidermal Growth Factor Receptor and Focal Adhesion Kinase Protein Ubiquitination and Degradation Induced After IR Injuryc-Cbl proteins contain a RING domain through which they interact with E2 ubiquitin-conjugating enzymes and which is required for their ubiquitin ligase activity.9 Immunoprecipitation analysis coupled with in vitro ubiquiti-nation assay revealed that c-Cbl ubiquitin ligase activity was increased in WT heart samples subjected to IR injury com-pared with WT controls (Figure 3A). c-Cbl tyrosine phos-phorylation at Y774 residue, a site involved in c-Cbl interaction with phosphoinositide-3 kinase, was also increased after IR. No change in c-Cbl expression was detectable after IR injury.

To explore the significance of c-Cbl activation in vivo, we assessed next whether c-Cbl mediates ubiquitination of epi-dermal growth factor receptor (EGFR) and focal adhesion kinase (FAK), 2 key signaling targets of c-Cbl that control myocyte hypertrophy and survival.17,18 We found that IR injury induced an increase in c-Cbl interaction with EGFR and FAK (Figure 3B), along with an increase in their ubiquitination at lysine 48 residue, a ubiquitin chain structure that is usually required for substrate targeting to the 26S proteasome for degradation (Figure 3C and 3D).19 Notably, EGFR and FAK tyrosine phosphorylation and expression levels decreased sig-nificantly in WT mice subjected to IR injury compared with shams, suggesting a link between EGFR/FAK ubiquitination and degradation. c-Cbl deletion attenuated EGFR and FAK ubiquitination and degradation induced by IR injury com-pared with WT. No significant difference in EGFR or FAK protein accumulation was detected between c-Cbl KO and WT shams, although a slight increase in basal ubiquitination of EGFR was detected in c-Cbl KO compared with WT sham hearts. This effect of c-Cbl deletion on basal EGFR ubiq-uitination could result from decreased EGFR degradation, increased EGFR association with c-Cbl homolog Cbl-b,20 or both. Collectively, these data show that c-Cbl activation after

IR mediates ubiquitination and degradation of signaling mol-ecules involved in myocyte survival.

c-Cbl Knockdown Induces Protection Against Oxidative Stress–Induced Myocyte DeathTo directly demonstrate that the cardioprotective effect of c-Cbl deletion after IR results from endogenous expres-sion of c-Cbl in cardiomyocytes, we investigated the role of c-Cbl in oxidative stress–induced cardiac myocyte death. Treatment of neonatal rat cardiomyocytes with H

2O

2 induced

an increase in c-Cbl ubiquitin ligase activity as assessed by in vitro auto-ubiquitination assay (Figure 4A). This increase was rapid, was sustained for >60 minutes after H

2O

2 treatment,

and was potent compared with cells treated with EGF for 5 minutes. H

2O

2 also caused a transient increase in c-Cbl Y774

phosphorylation, with maximum phosphorylation observed at 30 minutes after H

2O

2 treatment. Concomitant with increased

c-Cbl auto-ubiquitination, H2O

2 treatment increased c-Cbl

interaction with EGFR along with increased EGFR ubiqui-tination (Figure 4B), suggesting that c-Cbl mediates EGFR ubiquitination. Transduction of adenovirus carrying shRNA c-Cbl (Ad-shCbl), which reduced c-Cbl expression by ≈75% to 85% compared with cells infected with shRNA control (Ad-shCtrl), significantly decreased EGFR ubiquitination and p38 mitogen-activated protein kinase and AKT phos-phorylation induced by H

2O

2 or EGF treatment for 10 minutes

(Figure 4C and Figure IV in the online-only Data Supplement). Expression of Ad-shCbl enhanced the basal phosphorylation of prosurvival signaling molecules ERK

1/2 and AKT compared

with Ad-shCtrl–infected cells. Furthermore, H2O

2 treatment

did not further increase or induce ERK1/2

or JNK phosphory-lation, respectively. Ad-shCbl–infected cells also showed a significantly attenuated phosphorylation of these 2 kinases in response to EGF treatment compared with Ad-shCtrl–infected cells. These data show that inhibition of c-Cbl expression enhances prosurvival signaling and suggest that c-Cbl–depen-dent and –independent pathways are involved in H

2O

2-induced

downstream signaling.To further demonstrate the role of c-Cbl ligase activity

on EGFR ubiquitination and EGFR downstream signaling activation, we infected myocytes with recombinant adenovi-ruses expressing Lac.Z or 70Z-Cbl, a c-Cbl mutant in which a 17–amino acid deletion in the helix linker and RING fin-ger domain abolishes the ubiquitin ligase activity, resulting in a dominant-negative mutant.21 Overexpression of 70Z-Cbl significantly attenuated EGFR ubiquitination and p38 mitogen-activated protein kinase phosphorylation induced by H

2O

2 or EGF treatment compared with Lac.Z-infected cells

(Figure V in the online-only Data Supplement).To assess whether c-Cbl activation is involved in

H2O

2-induced myocyte apoptosis, myocytes infected with

recombinant adenovirus expressing shRNA c-Cbl, shRNA control, Lac.Z, or 70Z-Cbl were evaluated for apoptotic signaling. Overexpression of c-Cbl shRNA or 70Z-Cbl sig-nificantly attenuated myocyte apoptosis induced by H

2O

2

treatment for 16 hours, as demonstrated by a decrease in the percentage of TUNEL-positive myocytes and DNA fragmen-tation (Figure 4D and 4E and Figure V in the online-only Data Supplement). No effect of Ad-shCbl or 70Z-Cbl expression

Table. Echocardiographic Measurements in WT and c-Cbl Knockout Mice Subjected or Not to Ischemia/Reperfusion Injury

Sham Ischemia/Reperfusion

WT(n=8)

c-Cbl Knockout(n=9)

WT(n=8)

c-Cbl Knockout(n=9)

HR, bpm 480±10 471±24 457±12 497±37

HW, mg 126±1 117±10 135±4 129±10

BW, g 24.5±0.9 21.6±0.9 24.2±0.6 22.5±0.9

HW/BW, mg/g 5.3±0.1 5.5±0.3 5.6±0.2 5.8±0.3

LVEDD, mm 3.34±0.11 3.15±0.15 3.66±0.09 3.29±0.09

LVESD, mm 2.19±0.1 2.00±0.12 2.85±0.04* 2.32±0.07*†

LVPWTd, mm 0.98±0.01 0.94±0.01 0.96±0.02 0.92±0.03

LVPWTs, mm 1.45±0.04 1.33±0.03 1.42±0.08 1.30±0.11

BW indicates body weight; c-Cbl, proto-oncogene Casitas b-lineage lymphoma; HR, heart rate; HW, heart weight; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; LVPWTd, left ventricular posterior wall thickness diastole; LVPWTs, left ventricular posterior wall thickness systole; and WT, wild-type.

*P<0.05 vs WT sham group. †P<0.05 vs WT ischemia/reperfusion group.


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on apoptosis was observed in control myocytes. These results suggest that inhibition of endogenous c-Cbl during oxidative stress is protective and that the protective effect of c-Cbl is cardiomyocyte cell autonomous.

Mice Lacking c-Cbl Are Protected Against Pathological Remodeling Induced After Myocardial IschemiaTo explore the potential influence of c-Cbl on cardiac remod-eling responses after myocardial ischemia, we examined c-Cbl localization and expression 2, 7, and 30 days after MI. Figure 5A shows increased c-Cbl immunostaining in border-zone cardiomyocytes and infiltrating inflammatory cells at 7 and 30 days after MI. c-Cbl was not immunode-tected in fibroblasts or myofibroblasts of the infarcted area. Quantification of c-Cbl accumulation in shams and the

border zone of the infarct showed a 2.7±0.6-, 3.5±0.2-, and 3.7±0.3-fold increase over sham control at 2, 7, and 30 days after MI, respectively (Figure 5B).

Survival up to 4 weeks after MI was comparable in WT and c-Cbl KO shams (97% versus 98%, respectively). However, c-Cbl–deficient mice displayed remarkably improved postinfarction survival compared with WT (85% in c-Cbl KO mice compared with 55% in WT mice; P<0.05), especially in the first week after the insult (Figure 5C). Echocardiography 1 and 4 weeks after MI surgery indicated that LV end-diastolic dimension, LV end-systolic dimen-sion, and LV ejection fraction improved significantly in Cbl KO compared with WT mice, whereas there were no dif-ferences in the sham-operated animals (Figure 5D and 5E and Tables III and IV in the online-only Data Supplement). Morphometric analysis at 4 weeks after MI indicated the

Sham IR

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Figure 3. Proto-oncogene Casitas b-lineage lymphoma (c-Cbl) ablation protects against epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) ubiquitination and downregulation induced after ischemia/reperfusion (IR) injury. The left anterior descending coronary artery was ligated for 30 minutes to induce ischemia, and it subsequently was reperfused for 24 hours. A and B, c-Cbl immunoprecipitates (IP) were assayed for auto-ubiquitination assay and immunoblot analysis. C and D, EGFR or FAK immunoprecipitates from left ventricular lysates of shams and mice subjected to IR for 24 hours were immunoblotted with ubiquitin-Lys48, phosphotyrosine (P-Tyr), EGFR, or FAK antibodies. Top, Representative autoradiogram (with each lane from a single gel exposed for the same duration). Bottom, Fold induction (n=6 in each group). *P<0.05 compared with wild-type (WT) shams; †P<0.05 compared with WT IR. KO indicates knockout.


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presence of cardiac hypertrophy and concomitant signs of cardiac failure, such as an increase in lung weight, in WT mice, whereas this pathological remodeling response was lacking in the c-Cbl KO mice (Figure 5F and 5G). Myocyte cross-sectional area, an index of myocyte size, mirrored the changes in cardiac hypertrophy and was significantly attenuated in c-Cbl KO compared with WT infarcted hearts (Figure VI in the online-only Data Supplement). Infarct size measurements as percentage of LV circumference indicated that 45±5% of the LV was affected by the infarct in WT, whereas the infarct accounted for 29±4% of the LV in c-Cbl KO mice (Figure 5H). Examination of cell death in the bor-der zone indicated fewer TUNEL-positive cells in the c-Cbl KO mice compared with WT animals subjected to MI (data not shown). Thus, the integrity of the infarcted area was bet-ter maintained after MI in c-Cbl KO mice and likely pre-vented the heart from dilating to the same extent as in the

WT mice, which results in a better maintenance of cardiac function after MI.

Upregulation of Vascular Endothelial Growth Factor a/b and Vascular Endothelial Growth Factor Receptor 2 in the Infarcted Region of c-Cbl KO MiceOne of the beneficial effects of c-Cbl deficiency on the cardio-vascular system may be through an improvement in myocar-dial perfusion by the formation of new capillaries and by the enlargement of preexisting collateral vessels. One key regu-lator of this proangiogenic response is vascular endothelial growth factor (VEGF)a, which encodes an extracellular ligand for its corresponding endothelial receptors VEGF receptor (VEGFr)1 and VEGFr2.22,23 VEGFa, VEGFb, and VEGFr2 levels were significantly upregulated in c-Cbl KO compared with WT hearts in both sham and MI groups (Figure 6A and

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Figure 4. Proto-oncogene Casitas b-lineage lymphoma (c-Cbl) deletion protects myocytes from H2O2-induced apoptosis. A, Neonatal rat cardiac myocytes treated with H2O2 (100 μmol/L) for the indicated time were evaluated for auto-ubiquitination assay and immunoblot analysis. The result of densitometric analyses is shown. Ctrl indicates control. B and C, Cardiac myocytes were transduced with adenoviruses expressing shRNA Cbl (Ad-shCbl; 10 pfu per cell) or shRNA control (Ad-shCtrl; 10 pfu per cell) for 48 hours and then untreated or treated with 100 ng/mL epidermal growth factor (EGF) or 100 μmol/L H2O2 for 10 minutes. Cell lysates were assayed for EGF receptor (EGFR) immunoprecipitation (IP) assays (B) or immunoblot analysis (C). D and E, Myocyte apoptosis induced by H2O2 as assessed by the percentage of terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)–positive myocytes in culture (D) or DNA fragmentation enzyme-linked immunosorbent assay (E). Results are expressed as (OD410-OD500)/mg DNA (D) for triplicate determinations from a single experiment (mean±SE). All experiments were performed at least 3 times from 3 different cultures, and the data values were scaled to untreated controls. *P<0.05 vs control; †P<0.05 vs treated myocytes.


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6B). This observation is consistent with the fact that VEGFa induces expression of its receptors to create a positive feed-forward loop, allowing endothelial cells to become responsive to and activated by VEGF.24 Immunohistochemistry 7 days after MI indicated that VEGFr2 expression was significantly induced in the infarct area and the border zone, which was even more pronounced in the c-Cbl KO mice (Figure 6C). Erk

1/2

and AKT phosphorylation was also upregulated in c-Cbl KO compared with WT hearts in shams (Figure VIIA and VIIB in the online-only Data Supplement). However, no change in Erk

1/2 and AKT phosphorylation levels was observed between

c-Cbl KO and WT mice after MI. Collectively, these data indi-cate that removal of c-Cbl increases the expression of VEGFa and its downstream receptor, which likely promotes cardio-protection after ischemic damage.

We next visualized vasculature using anti-CD31 as an endo-thelial surface marker and smooth muscle α-actin as a marker for smooth muscle cells. There was a regular distribution of capillaries around cardiomyocytes in both sham-operated groups, with no detectable differences in vessel density (Figure 6D and 6E). Four weeks after MI, the border zone of the infarcted area contained regions of low vascularity in the

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Figure 5. Proto-oncogene Casitas b-lineage lymphoma (c-Cbl) knockout (KO) mice are protected against post–myocardial infarction (MI) cardiac remodeling. A, Representative micrographs of paraffin-embedded sections from mouse hearts subjected to permanent left coronary artery ligation for 2, 7, and 30 days. Bar, 40 μm. B, top, Representative immunoblot of left ventricular heart lysates for c-Cbl. Sh indicates sham. B, bottom: Fold induction of c-Cbl accumulation (n=5 for sham groups; n=6 for MI groups). C, Comparison of post-MI mortality between wild-type (WT; n=18) and c-Cbl knockout (KO; n=16) mice. c-Cbl KO mice showed an increase in survival after MI compared with WT mice (P<0.05). D and E, Cardiac function was measured by echocardiography 4 weeks after MI. The data demonstrate cardiac dilation and loss of contractile function in WT mice, as indicated by left ventricular end-systolic dimension (LVESD; D) and ejection fraction (E), whereas the loss of cardiac function was substantially attenuated in c-Cbl KO mice (n=6 for sham groups; n=8 for MI groups). F and G, c-Cbl deletion attenuated MI-induced increase in the ratio of heart weight (HW) to tibia length (TL; F) and the ratio of lung weight (LW) to TL (G). H, Infarct size 4 weeks after MI expressed as a fraction of the total cross-sectional circumference of the left ventricle indicates that the infarct size in c-Cbl KO mice is significantly smaller than the infarct size in WT. *P<0.05 compared with WT sham group; †P<0.05 compared with WT MI group. by guest on July 14, 2018

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WT animals, with a more pronounced reduction in the infarct. However, both the border zone and the infarcted region of the c-Cbl KO appeared to be highly vascularized, with enlarged thin-walled vessels (Figure 6C and 6D). The number of capil-laries counted in the remote region showed no significant dif-ference between WT and c-Cbl KO mice after MI, whereas this number was significantly increased in the border zone of the c-Cbl KO mice (Figure 6E). These findings suggest that

the cardioprotection observed in Cbl KO mice is attributable, at least in part, to an increase in vessel formation after MI, which might involve VEGFa and its receptor VEGFr2.23

DiscussionIn this study, we identified a novel role for the E3 ubiquitin ligase and adaptor molecule c-Cbl in mediating early stress responses in the heart. c-Cbl expression was strongly induced

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Figure 6. Proto-oncogene Casitas b-lineage lymphoma (c-Cbl) deletion enhances angiogenic response in infarcted region after myocardial infarction (MI). A, Immunoblot analysis for vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFr)2 indicates an increased angiogenic response in the infarcted region of the c-Cbl knockout (KO) mice vs the wild-type (WT) mice 7 days after MI. Sh indicates sham. B, Data are represented as fold change compared with WT animals (n=5 for sham groups; n=6 for MI groups). C, Immunohistochemistry analysis reveals an increase in VEGFr2 expression and smooth muscle (SM) α-actin–positive blood vessels at the border zone of the infarcted region 7 days after MI, which is more pronounced in the Cbl KO mice. Bar, 40 μm. D, Representative sections stained for CD31 labeling show an equal distribution of capillaries surrounding cardiomyocytes in the noninfarcted, remote areas, whereas the ischemic region shows irregular patterning of vasculature with additional and enlarged vessels in the border zone and the infarcted region of the Cbl KO mice compared with the WT 30 days after MI. Bars, 40 μm. E, Semiquantitative analysis of capillary density in the myocardium indicates an increase in vessel density in the border zone of c-Cbl KO mice compared with WT. The capillary count of these sections is expressed as number of vessels per microscopic field (n=5 for sham groups; n=6 for MI groups). *P<0.05 compared with WT sham group; †P<0.05 compared with WT MI group.


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in human failing hearts, and its inhibition provided protection against IR injury, evidenced by decreased cardiac apoptosis and improved LV function. c-Cbl directly ubiquitinates EGFR and FAK such that decreased EGFR and FAK ubiquitination in c-Cbl KO mice reduced their degradation and attenuated cardiomyocyte death induced after IR. More interestingly, c-Cbl deletion attenuated LV hypertrophy, enhanced angio-genesis, and improved LV contractility after chronic ischemic insult. This seems to be related, in part, to increased VEGFa and VEGFr2 expression. These data highlight c-Cbl as one of the important signaling molecules that is specifically involved in pathological cardiac remodeling.

Using c-Cbl KO mice, we provide the first evidence, to our knowledge, that this multifaceted protein is involved in the early responses induced by IR injury. Adult c-Cbl–deficient mice have virtually normal hearts under nonstressed, base-line conditions. Moreover, baseline heart rate, blood pressure, and maximal dP/dt were comparable in c-Cbl–deficient and WT mice. However, c-Cbl–deficient mice had smaller infarct sizes, reduced cardiomyocyte apoptosis in the infarct border zone after IR injury, and preserved cardiac contractile function compared with WT controls, indicating that endogenous c-Cbl mediates myocardial tissue damage in vivo. The cardiopro-tective effects of c-Cbl deletion correlate with the induction of XIAP expression as well as the phosphorylation of Bad, 2 molecules known to prevent myocyte death.25 We also found high basal levels of Akt and Erk

1/2 phosphorylation in c-Cbl–

deficient mice compared with WT controls, suggesting cardio-protective effects of increased Akt and Erk

1/2 phosphorylation

in these mice. Interestingly, these beneficial effects of c-Cbl deletion on myocyte apoptosis and cardiac function induced after IR injury occurred independently of c-Cbl effects on the immune system, as shown by the lack of significant changes in leukocyte infiltration and activation between c-Cbl KO and WT mice (Figure VIII in the online-only Data Supplement). Rather, deletion of c-Cbl in cardiac myocytes conferred this protection against IR injury. Using CM-Cbl KO mice, we dem-onstrated that endogenous deletion of c-Cbl in cardiac myo-cytes offered cardioprotection against IR injury that mirrored the effect of global c-Cbl deletion. CM-Cbl KO mice showed less myocyte apoptosis, reduced infarct size, and improved cardiac function in response to IR injury. Furthermore, inhibi-tion of c-Cbl expression or of its E3 ubiquitin ligase activity markedly attenuated myocyte apoptosis induced in response to oxidative stress, suggesting that endogenous expression of c-Cbl in myocytes plays an important role in mediating the deleterious effect of IR injury.

c-Cbl activation occurs in response to a variety of stimuli, including activated EGFR, cytokines, and hormones.9 We describe in the present report that c-Cbl ubiquitin ligase activ-ity and its tyrosine phosphorylation were increased in hearts of mice subjected to IR injury and in cultured myocytes after H

2O

2 stimulation. Furthermore, inhibition of c-Cbl expression

by shRNA or inhibition of its ubiquitin ligase activity signifi-cantly reduced myocyte apoptosis in response to H

2O

2. Given

that H2O

2 is a potent inducer of myocyte death that is released

as a result of IR injury, it may account for the c-Cbl activation and myocyte death in the early IR injury. However, stimula-tion from other factors released after IR injury could not be

excluded. In this regard, we showed recently that c-Cbl ubiq-uitin ligase activity is involved in mediating focal adhesion, myofibril protein degradation, and myocyte death induced by neutrophil-derived serine proteases.13 Together, these findings show that activation of c-Cbl after IR increases myocyte apop-tosis and that a similar mechanism may be critical in other settings of cardiac stress.

An important finding of the present study is the identifica-tion of the molecular mechanisms by which the c-Cbl exerts its cardioprotective effect. c-Cbl RING finger has intrin-sic E3 ligase activity and can recruit E2s for the transfer of ubiquitin to substrates.9 Activation of c-Cbl ligase activity has been shown to target several activated receptor and non-receptor tyrosine kinases and mediates their downregulation, thus providing a means by which signaling processes can be negatively regulated.9,26 In this study, we found that c-Cbl acti-vation after IR injury correlated with c-Cbl interaction with EGFR and FAK, 2 known targets of c-Cbl.9 Interestingly, EGFR and FAK ubiquitination and degradation were sig-nificantly decreased in c-Cbl KO mice, suggesting that c-Cbl activation presumably mediates EGFR and FAK ubiquitina-tion and degradation induced by IR injury. To our knowledge, this is the first demonstration that EGFR and FAK are being ubiquitinated and downregulated in vivo. A similar decrease in EGFR and FAK expression was observed in human fail-ing hearts, but the mechanisms of such downregulation have never been elucidated.27,28 Because selective downregulation of EGFR and focal adhesion proteins appears to be critical for reduction in myocyte survival,17,18 they might be directly linked to reduced myocyte loss and enhanced cardiac contrac-tility observed after IR injury in c-Cbl KO mice. These data extend our previous findings showing the role of c-Cbl ubiq-uitin ligase activity in FAK and myofibril protein degradation and myocyte death in response to neutrophil-derived serine proteases.13 Although our data that deletion of c-Cbl or inhibi-tion of its ubiquitin ligase activity results in reduced EGFR ubiquitination and attenuated myocyte apoptosis in response to H

2O

2 clearly support the established model that c-Cbl is a

negative regulator of tyrosine kinase signaling, the implica-tion of the phosphoinositide-3 kinase interacting domain of c-Cbl (located at Y731,Y770,Y774)9 remains to be elucidated. In this regard, c-Cbl has been suggested to function as a positive regulator of signaling pathways through its adaptor function to recruit phosphoinositide-3 kinase and other Src homology domain 2–containing molecules in some systems.9,26 It is note-worthy that hearts from c-Cbl KO mice showed high basal levels of Akt and Erk

1/2 phosphorylation compared with WT

control mice. However, this basal increase in Akt and Erk1/2

phosphorylation did not affect heart weight in c-Cbl KO mice. The explanation for this lack of hypertrophy in c-Cbl KO mice needs further investigation. However, it has been reported that c-Cbl KO mice exhibit elevated energy expenditure and improved insulin action,29 which could, in an organ with high energy demands like the heart, play a role in maintaining car-diac structure and function and prevent cardiac hypertrophy.

In addition to its role in protecting myocyte death, our results show that c-Cbl deficiency also affected the levels of VEGFa and its receptor VEGFr2 in response to chronic myo-cardial ischemia. Because the angiogenic activity of VEGF


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is dependent on its expression levels,30 this increase in total VEGF levels would be expected to result in vascular devel-opment/angiogenesis. However, c-Cbl KO mice showed no obvious defects in vascular development in shams despite increased levels of VEGFa and VEGFr2. Early studies have demonstrated that loss of c-Cbl alone was dispensable for normal embryonic development,15 whereas loss of both c-Cbl and Cbl-b was embryonically lethal.16 Therefore, it is tempting to speculate that c-Cbl activity is not stringently required during development, and the function of Cbl fam-ily proteins may be compensatory in their ability to regulate the activity of target proteins. These data are consistent with previous studies showing normal embryonic and postnatal angiogenesis but impaired ischemia-induced angiogen-esis in several gene KO mouse models,31,32 suggesting that c-Cbl selectively plays a role in pathological angiogenesis. Moreover, c-Cbl inactivation has been shown to enhance endothelial cell proliferation and tube formation in response to VEGF in vitro and results in increased tumor angiogen-esis and retinal neovascularization in vivo.33,34 This latter effect of c-Cbl is mediated by a direct regulation of VEGFr2 expression through its phosphorylation on Y1052 and Y1057 res-idues, as well as indirectly through PLCγ1 activation.33,34 In addition, it is notable that the enhancement of overall vascu-larization may not come from neoangiogenesis exclusively but also from protecting preexisting vascular cells. In fact, c-Cbl deletion protects cells from undergoing apoptosis after MI. Irrespective of whether c-Cbl removal enhances cardiac protection through upregulation of angiogenesis, through an increase in protection of preexisting vascular cells, or a combination of both, our findings show that c-Cbl is a nega-tive regulator for angiogenesis by functioning as a molecular switch to fine-tune angiogenic events during pathological conditions such as MI.

The role of muscle-specific protein ubiquitin ligases as molecules that affect the severity of cardiac diseases is com-plex and often ambiguous. Activation of E3 ligases CHIP and MDM2 protected myocardium from IR injury,35,36 whereas activation of Atrogin1 and MuRF1 attenuated cardiac hyper-trophy.37,38 The present study adds c-Cbl to the list of E3 ubiquitin ligases that play a role in cardiac protection after myocardial ischemia. Expression of c-Cbl was increased in human hearts with ICM and DCM, and its activation provided a means by which signaling emanating from EGFR and FAK can be negatively regulated. Inactivation of ubiquitin ligase activity of c-Cbl or inhibition of its expression may lead to enhanced and prolonged signaling, a function that can explain the increased survival signaling in myocytes expressing a ligase-deficient mutant of c-Cbl or in c-Cbl KO hearts, respec-tively. These data, along with the recent findings linking muta-tion in c-Cbl RING finger domain to Noonan syndrome, one of the most common genetic syndromes associated with con-genital heart disease in humans,39 suggest that c-Cbl inhibition may constitute a novel therapy offering cardioprotection.

In conclusion, the results of this study suggest that c-Cbl deletion protects the heart against IR injury by decreasing/pre-venting ubiquitination and subsequent degradation of EGFR and FAK and myocyte death in response to injury. These find-ings provide evidence for an important role of c-Cbl in direct

modulation of EGFR and focal adhesion protein turnover in the heart. Given that EGFR and FAK signaling downregula-tion also underlie cardiac hypertrophy and other form of heart disease, it is likely that c-Cbl plays a role in a variety of stress settings. Thus, modulating c-Cbl activation to attenuate myo-cyte death and to enhance angiogenesis may have potential implications in the treatment of cardiac disease.

Sources of FundingThis work was supported by the National Institutes of Health (HL076799 and HL088626 to Dr Sabri and CA105489, CA99163, CA87986, and CA116552 to Dr Band). Dr Rafiq is a recipient of an American Heart Association award (240052).

DisclosuresNone.

References 1. Members WG, Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry

JD, Borden WB, Bravata DM, Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Soliman EZ, Sorlie PD, Sotoodehnia N, Turan TN, Virani SS, Wong ND, Woo D, Turner MB. Heart disease and stroke statistics “2012 update.” Circulation. 2012; 125:e2–e220.

2. Wang X, Robbins J. Heart failure and protein quality control. Circ Res. 2006;99:1315–1328.

3. Razeghi P, Sharma S, Ying J, Li YP, Stepkowski S, Reid MB, Taegtmeyer H. Atrophic remodeling of the heart in vivo simultaneously acti-vates pathways of protein synthesis and degradation. Circulation. 2003;108:2536–2541.

4. Glass DJ. Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol. 2003;5:87–90.

5. Depre C, Wang Q, Yan L, Hedhli N, Peter P, Chen L, Hong C, Hittinger L, Ghaleh B, Sadoshima J, Vatner DE, Vatner SF, Madura K. Activation of the cardiac proteasome during pressure overload promotes ventricular hypertrophy. Circulation. 2006;114:1821–1828.

6. Ciechanover A, Orian A, Schwartz AL. The ubiquitin-mediated proteo-lytic pathway: mode of action and clinical implications. J Cell Biochem Suppl. 2000;34:40–51.

7. Friedman J, Xue D. To live or die by the sword: the regulation of apoptosis by the proteasome. Dev Cell. 2004;6:460–461.

8. Coux O, Tanaka K, Goldberg AL. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem. 1996;65:801–847.

9. Schmidt MH, Dikic I. The Cbl interactome and its functions. Nat Rev Mol Cell Biol. 2005;6:907–918.

10. Meng W, Sawasdikosol S, Burakoff SJ, Eck MJ. Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase. Nature. 1999;398:84–90.

11. Balasubramanian S, Mani S, Shiraishi H, Johnston RK, Yamane K, Willey CD, Cooper G 4th, Tuxworth WJ, Kuppuswamy D. Enhanced ubiqui-tination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases. J Mol Cell Cardiol. 2006;41:669–679.

12. Rosenblatt-Velin N, Lerch R, Papageorgiou I, Montessuit C. Insulin resistance in adult cardiomyocytes undergoing dedifferentiation: role of GLUT4 expression and translocation. FASEB J. 2004;18:872–874.

13. Rafiq K, Guo J, Vlasenko L, Guo X, Kolpakov MA, Sanjay A, Houser SR, Sabri A. c-Cbl ubiquitin ligase regulates focal adhesion protein turnover and myofibril degeneration induced by neutrophil protease cathepsin G. J Biol Chem. 2012;287:5327–5339.

14. Chaudhary KW, Rossman EI, Piacentino V III, Kenessey A, Weber C, Gaughan JP, Ojamaa K, Klein I, Bers DM, Houser SR, Margulies KB. Altered myocardial Ca2+ cycling after left ventricular assist device sup-port in the failing human heart. J Am Coll Cardiol. 2004;44:837–845.

15. Murphy MA, Schnall RG, Venter DJ, Barnett L, Bertoncello I, Thien CB, Langdon WY, Bowtell DD. Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice. Mol Cell Biol. 1998;18:4872–4882.


Dow

nloaded from



16. Naramura M, Jang IK, Kole H, Huang F, Haines D, Gu H. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat Immunol. 2002;3:1192–1199.

17. Pentassuglia L, Sawyer DB. The role of neuregulin-1beta/ErbB signaling in the heart. Exp Cell Res. 2009;315:627–637.

18. Franchini KG. Focal adhesion kinase: the basis of local hypertrophic sig-naling domains. J Mol Cell Cardiol. 2012;52:485–492.

19. Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, Gonda DK, Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989;243:1576–1583.

20. Pennock S, Wang Z. A tale of two Cbls: interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation. Mol Cell Biol. 2008;28:3020–3037.

21. Joazeiro CAnP, Wing SS, Huang H-k, Leverson JD, Hunter T, Liu Y-C. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science. 1999; 286:309–312.

22. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676.

23. Dor Y, Djonov V, Abramovitch R, Itin A, Fishman GI, Carmeliet P, Goelman G, Keshet E. Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy. EMBO J. 2002;21:1939–1947.

24. Hervé MA, Meduri G, Petit FG, Domet TS, Lazennec G, Mourah S, Perrot-Applanat M. Regulation of the vascular endothelial growth fac-tor (VEGF) receptor Flk-1/KDR by estradiol through VEGF in uterus. J Endocrinol. 2006;188:91–99.

25. Dorn GW II. Apoptotic and non-apoptotic programmed cardiomyocyte death in ventricular remodeling. Cardiovasc Res. 2009; 81:465–473.

26. Thien CB, Langdon WY. Cbl: many adaptations to regulate protein tyro-sine kinases. Nat Rev Mol Cell Biol. 2001;2:294–307.

27. Pfister R, Acksteiner C, Baumgarth J, Burst V, Geissler HJ, Margulies KB, Houser S, Bloch W, Flesch M. Loss of beta1D-integrin function in human ischemic cardiomyopathy. Basic Res Cardiol. 2007;102:257–264.

28. Rohrbach S, Niemann B, Silber R-E, Holtz J. Neuregulin recep-tors erbB2 and erbB4 in failing human myocardium. Bas Res Cardiol. 2005;100:240–249.

29. Molero JC, Jensen TE, Withers PC, Couzens M, Herzog H, Thien CB, Langdon WY, Walder K, Murphy MA, Bowtell DD, James DE, Cooney GJ. c-Cbl-deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action. J Clin Invest. 2004;114:1326–1333.

30. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling

J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel devel-opment and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435–439.

31. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T, DiPalma T, Dewerchin M, Noel A, Stalmans I, Barra A, Blacher S, VandenDriessche T, Ponten A, Eriksson U, Plate KH, Foidart JM, Schaper W, Charnock-Jones DS, Hicklin DJ, Herbert JM, Collen D, Persico MG. Synergism between vas-cular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med. 2001;7:575–583.

32. Chen J, Somanath PR, Razorenova O, Chen WS, Hay N, Bornstein P, Byzova TV. Akt1 regulates pathological angiogenesis, vascular matura-tion and permeability in vivo. Nat Med. 2005;11:1188–1196.

33. Meyer RD, Husain D, Rahimi N. c-Cbl inhibits angiogenesis and tumor growth by suppressing activation of PLC[gamma]1. Oncogene. 2011; 30:2198–2206.

34. Singh AJ, Meyer RD, Navruzbekov G, Shelke R, Duan L, Band H, Leeman SE, Rahimi N. A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCgamma1 activation and angiogenesis. Proc Natl Acad Sci U S A. 2007;104:5413–5418.

35. Zhang C, Xu Z, He XR, Michael LH, Patterson C. CHIP, a cochaperone/ubiquitin ligase that regulates protein quality control, is required for maxi-mal cardioprotection after myocardial infarction in mice. Am J Physiol. 2005;288:H2836–H2842.

36. Toth A, Nickson P, Qin LL, Erhardt P. Differential regulation of cardio-myocyte survival and hypertrophy by MDM2, an E3 ubiquitin ligase. J Biol Chem. 2006;281:3679–3689.

37. Arya R, Kedar V, Hwang JR, McDonough H, Li HH, Taylor J, Patterson C. Muscle RING finger protein-1 inhibits PKC{epsilon} activation and prevents cardiomyocyte hypertrophy. J Cell Biol. 2004;167:1147–1159.

38. Li HH, Kedar V, Zhang C, McDonough H, Arya R, Wang DZ, Patterson C. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex. J Clin Invest. 2004;114:1058–1071.

39. Niemeyer CM, Kang MW, Shin DH, Furlan I, Erlacher M, Bunin NJ, Bunda S, Finklestein JZ, Sakamoto KM, Gorr TA, Mehta P, Schmid I, Kropshofer G, Corbacioglu S, Lang PJ, Klein C, Schlegel PG, Heinzmann A, Schneider M, Starý J, van den Heuvel-Eibrink MM, Hasle H, Locatelli F, Sakai D, Archambeault S, Chen L, Russell RC, Sybingco SS, Ohh M, Braun BS, Flotho C, Loh ML. Germline CBL mutations cause develop-mental abnormalities and predispose to juvenile myelomonocytic leuke-mia. Nat Genet. 2010;42:794–800.

CLINICAL PERSPECTIVEMyocardial infarction due to coronary artery occlusion is one of the leading causes of death worldwide. The loss of blood flow after myocardial infarction results in loss of cardiac myocytes in the ischemic zone, which diminishes cardiac con-tractility and impedes angiogenesis and repair. Although much is known about the pathways that promote the pathologi-cal remodeling responses, mechanisms that promote myocyte loss and impair cardiac function have not been as clearly defined. The ubiquitin proteasome system is largely responsible for the degradation of misfolded and damaged proteins as well as proteins involved in the control of components of the contractile apparatus and hypertrophic gene expression, thereby regulating cardiac hypertrophy and remodeling. In the present study, we show that expression of proto-oncogene Casitas b-lineage lymphoma (c-Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor and nonreceptor tyrosine kinases, is increased in human failing hearts. Inhibition of c-Cbl in mice reduces myocyte death, enhances angiogenesis, and improves cardiac function and survival in response to myocardial ischemia. Thus, modulating c-Cbl activation to attenuate myocyte death and to enhance angiogenesis may have potential implications in the treatment of cardiac disease.


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and Abdelkarim SabriYu, Bhopal Mohapatra, Neha Zutshi, Wei An, Hamid Band, Archana Sanjay, Steven R. Houser Khadija Rafiq, Mikhail A. Kolpakov, Rachid Seqqat, Jianfen Guo, Xinji Guo, Zhao Qi, Daohai

Ischemiac-Cbl Inhibition Improves Cardiac Function and Survival in Response to Myocardial

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2014 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.113.007004

2014;129:2031-2043; originally published online February 28, 2014;Circulation.

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CIRCULATIONAHA/2012/145946/R2

1

SUPPLEMENTAL MATERIAL

Supplemental MATERIALS & METHODS

Materials: Monoclonal antibodies to c-Cbl and ubiquitin were obtained from BD Biosciences,

and polyclonal antibody for FAK was from Santa Cruz Biotechnology. Phospho-c-Cbl Y774,

EGFR receptor (EGFR), Bcl2, phospho-Bad-S112, Bad, c-IAP1, and XIAP were from Cell

Signaling. Monoclonal antibodies for phosphotyrosine and ubiquitin Lys48 were from Upstate

Biotechnology and anti-troponin I was from Sigma. All other chemicals were from standard

suppliers.

Generation of cardiac myocyte-specific c-Cbl knockout mice: Conditional mice bearing floxed c-

Cbl alleles (c-Cblflox/flox) have previously been described.1 Transgenic mice overexpressing Cre-

recombinase protein fused to 2 mutant estrogen receptor ligand-binding domains under the

control of the α-myosin heavy chain (α-MHC) promoter (α-MHC-MerCreMer)2 were received

from The Jackson Laboratory (JAX Mice and Services, Bar Harbor, Me). Homozygous mice

with the floxed c-Cbl alleles were crossed with αMHC-MerCreMer mice and bred back to c-

Cblflox/flox to produce αMHC-MerCreMer×c-Cblflox/flox mice. c-Cblflox/flox littermate were taken as

controls. All animals were bred and maintained on a C57Bl/6 background.

To induce Cre-dependent recombination, tamoxifen (20 mg/kg body weight, Sigma-Aldrich) was

injected into 8-weeks-old animals intraperitoneally (i.p.) for 5 days. Tissue-specific c-Cbl

deficiency was achieved 4 weeks after injection as verified by western blot analysis.2

Genotyping of mice: The c-Cblflox/flox and c-Cblwt/wt alleles and the Cre transgene were detected

in mouse tail DNA by PCR. Primers for c-Cblwt/wt and c-Cblflox/flox result in a 600bp and a 700bp

product, respectively. The primer sets used are: Cre (Forward 5'-


2

TCCAATTTACTGACCGTACACCAA -3'; Reverse, 5'- CCTGATCCTGGCAATTTCGGCTA

-3'); c-Cblflox/flox (Forward 5’-GTGGTGGCTTGCAATTATAATCCTACCACTTAGG-3’;

Reverse 5’-GTTTGAGATGTCTGGCTGTGTGTACACGCG-3’), c-Cbl-/- (Forward 5'-

TCCCCTCCCCTTCCCATGTTTTTAATAGACTC-3’; Reverse 5’-

TGGCTGGACGTAAACTCCTCTTCAGACCTAATAAC-3’), and c-Cblwt/wt (Forward 5'-

GACGATAGTCCCGTGGAAGAGCTTTCGACA-3’, Reverse 5’-

CCTAAGTGGTAGGATTATAATTGCAAGCCACCAC-3’).

Assessment of area at risk and infarct size: After 24 hours of reperfusion, the slipknot was retied

and the right carotid artery was cannulated to allow injection of KCL (40 mEq/L) followed by

1% Evan's blue dye for identification of the area at risk (AR). The hearts were excised, rinsed

briefly in PBS, and sliced transversely. Slices were then incubated with 1% triphenyltretrazolium

chloride (TTC, pH 7.4 in phosphate buffer) at 37°C for 30 minutes, fixed in 10% formalin,

photographed, and the images were used to quantify IR-induced myocardial infarction using

Bioquant software. The Evan's blue stained area defined the perfused area, whereas the Evan's

blue unstained area defined the area at risk (AAR). The area lacking the red TTC staining within

the AAR was considered as the infarct area (IA). Both the surgeon and the evaluator of infarct

size were blinded to mouse genotypes.

Heart function: Echocardiographic measurements were taken before surgery and at 24 hours

after IR injury or 4 weeks after MI to determine the baseline heart function and ventricular

dimensions in the experimental groups. Briefly, following light sedation with 1% Isoflurane, the

mice were placed on a heated platform in the left lateral decubitus position for imaging. A

Visualsonic Ultrasound System (Vevo770) containing a 40 Mhz variable frequency probe was

used to capture the echocardiogram. Standard long and short axis M-Mode views were recorded


3

when the mouse possessed a target heart rate between 450 and 550 beats per minute. End-

diastolic and end-systolic interventricular septum (IVSd, IVDs), posterior wall thickness (PWTd,

PWTs) and left ventricular internal diameters (LVEDD, LVESD) were calculated and averaged

from 4 consecutive contractions using manufacturer’s software. Percent fractional shortening

was calculated using: % FS= [(LVEDD-LVESD)/LVEDD] x 100. LVEF was calculated by the

cubed method as follows: LVEF = [(LVEDD)3 – (LVESD)3]/(LVEDD)3.

Hemodynamic measurements were performed using a 1.0 F catheter tip micromanometer (Millar

PVR-1035). Following calibration, the catheter (connected to a Powerlab 40/3 data acquisition

box; ADInstruments) was inserted through the right carotid artery of anesthetized mice. The first

derivative of left ventricular pressure (dP/dT) was assessed using PVAN 3.6 software (Millar).

Neonatal rat cardiomyocyte isolation: Myocytes were isolated from the ventricles of neonatal

Sprague-Dawley rats by collagenase digestion as previously described.3 After 30 minutes of

preplating (to eliminate non-myocyte cell contamination), myocytes were plated in collagen

precoated dishes or in fibronectin (BD bioscience) precoated glass coverslips at a density of

160,000/cm2 in 10% fetal bovine serum DMEM supplemented with 1 mmol/liter L-glutamine,

antibiotic/antimycotic solution, and 100 µmol/L 5-bromo-2-deoxyuridine (BrdU). Under these

high density conditions, the myocytes form cell-cell contacts and display spontaneous contractile

activity within 24 hours of plating.

Cardiac troponin-I ELISA assay: Mice were exsanguinated after IR injury and the serum levels

of the cardiac-specific isoform of troponin-I were assessed using ELISA assay (Life Diagnostics,

PA).

Immunoprecipitation and immunoblot analysis: Extraction of proteins from heart tissue samples

was performed as described previously.3 Briefly, lysates were cleared by centrifugation at


4

12,000 rpm and the supernatants (800 µg of protein/ml) were subjected to immunoprecipitation

with corresponding antibodies. After overnight incubation at 4 °C, protein A- or G-agarose

beads were added and left for an additional 3 hours. Immunocomplexes were then subjected to

SDS-PAGE followed by Western blot analysis according to methods published previously or to

the manufacturer's instructions.3 Each panel in each figure represents results from a single gel

exposed for a uniform duration, with bands detected by enhanced chemiluminescence and

quantified by laser scanning densitometry.

c-Cbl auto-ubiquitination assay: c-Cbl ubiquitin ligase activity was performed using auto-

ubiquitination assay kit from Enzo Life Sciences with minor modification. Briefly, c-Cbl

immunoprecipitates were washed twice with lysis buffer, once with ubiquitination buffer and

then were incubated with 30 µL of ligase reaction buffer mixture containing ubiquitination

buffer, 1 mmol/L DTT, 20 U/ml inorganic pyrophosphatase, 5 mmol/L Mg-ATP, 2.5 µM

ubiquitin, 100 nmol/L E1, and 2.5 µM at 37°C for 1 h. The reaction was quenched by adding 30

µl of 2x non-reducing gel loading buffer and heating at 95°C for 5 minutes. Finally, the samples

were assessed for immunoblotting with anti-ubiquitin for detecting ubiquitinated c-Cbl and for c-

Cbl as a loading control.

Expression of adenoviral vectors: Production of recombinant adenovirus expressing Lac-Z,

ligase deficient c-Cbl (70Z-Cbl) , a mutant that lacks 17 amino acid in the RING finger domain

necessary for ubiquitin ligase activity, was described elsewhere.3,4 The 70Z Cbl plasmids were a

gift from Tsyganov A (Temple University, USA). Adenoviral vectors were purified using a kit

from Virapur and titrated using BD Adeno-X rapid titer kit (BD Bioscience). After 24 hours of

plating, NRCMs were infected with adenoviruses expressing Lac-Z (10 pfu/cell), or 70Z-Cbl-Cbl

(5 pfu/cell) in DMEM for 2 h, then 5% fetal bovine serum DMEM was added, and cells were


5

incubated for an additional 48 h. Serum-free DMEM/F-12 medium was changed 1 hour before

the start of the experiments.

Preparation of adenoviral shRNA: The generation of adenovirus harboring shRNA constructs of

c-Cbl and control has been described previously.5 Briefly, DNA sequences that encoded short

hairpin RNA to c-Cbl (Ad-shCbl) 5′-

CACCGCAGATGGCTCAAGAGACACGAATGTCTCTTGAGCCATCTGC-3′ or an inactive

randomized control RNA (Ad-shCtrol) 5′-

CACCGCCTCCAGCACTGTCGTTAACGAATTAACGACAGTGCTGGAGG-3′ were cloned

into pENTRU6 vector, recombined into pAd/BLOCKit vector (Gateway system; Invitrogen), and

packaged into recombinant adenoviruses expressing Ad-shCbl or Ad-Ctrl by using U293 cells

(Invitrogen) following the manufacturer's instructions. Adenovirus was added to neonatal rat

cardiomyocytes after 1 day of culture on collagen at 10 plaque forming unit (PFU)/cell, which

resulted in a 75-85% down-regulation of c-Cbl relative to uninfected or control-shRNA-infected

myocytes.

Immunostaining. Paraffin heart sections were deparaffinized in xylene and re-hydrated. Antigen

retrieval was achieved by boiling the slides in citrate solution for 12-15 minutes and slides were

then washed with phosphate-buffered Saline (PBS). After quenching endogenous tissue

peroxidase activity with 3% H2O2 for 20 minutes, slides were then washed in PBS and samples

blocked in PBS containing 5% bovine serum albumin (BSA) at room temperature for 30

minutes. Primary antibodies to detect c-Cbl (BD Biosciences), CD31 (Santa-Cruz

Biotechnology), SM α-actin (Sigma), and VEGFr2 (Cell Signaling) were applied overnight at

4°C in PBS containing 2% BSA. The next day, samples were washed in PBS and then

sequentially incubated with Vectastain Elite ABC Kit (Avidin/Biotin/Horseradish Peroxidase-


6

System (Vector Laboratories) or with fluorophore-secondary antibody conjugates (Molecular

Probes). The peroxidase reaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride

(DAB) and slides were counterstained with Hematoxylin.

Cathepsin G activity in LV tissue lysates. Snap frozen LV tissues were homogenized in ice-cold

buffer containing 100 mmol/L Hepes, pH 7.5, 1 mol/L NaOH, 50 mmol/L CaCl2, and 0.01%

Igepal CA-630 in presence or absence of Cat.G inhibitor. After centrifugation, supernatants

containing 100 µg proteins were used for Cat.G activity assay by measurement of the rate of

cleavage of fluorogenic conjugated substrate Suc-Ala2-Pro-Phe-Amc (R&D Systems).

Measurement of myeloperoxidase activity. MPO activity in LV homogenates was determined

using MPO peroxidation assay kit from Cayman Chemicals. Briefly, LV samples were

suspended in lysis buffer provided in the kit and homogenates were cleared by centrifuging at

10,000 rpm at 4°C. The samples were incubated with hydrogen peroxide and the substrate

ADHP (10-acetyl-3,7-dihydroxyphenoxazine). Fluorescence was then analyzed with an

excitation wavelength of 540 nm and emission wavelength of 595 nm. The specificity of the

assay was confirmed by addition of a MPO inhibitor (4-aminobenzhydrazide) in the reaction

mixture prior to the assay.

Terminal deoxynucleotidyl transferase (TdT) and tropomyosin immunolabeling. Three sections

from each LV cut perpendicularly to the major axis of the heart were sampled. TdT assay was

performed using kit from Promega. Positive cells were counted throughout the LV and were

expressed as percentage of the total number of nuclei as determined by DAPI (Molecular probes)

staining.


7

Apoptotic Cell Death ELISA: A cell death detection ELISA kit (Roche Applied Science) was

used to quantitatively determine the apoptotic DNA fragmentation by measuring the cytosolic

histone-associated mono- and oligo-nucleosomes fragments associated with apoptotic cell death.


8

Supplemental Table 1. Demographic and clinical data.

Non-failing

(n=5)

DCM

(n=5)

ICM

(n=5)

Age (years) 62 ± 9 49 ± 11 52 ± 9

Gender (male/female) 1/4 2/3 5/0

Heart Weight (g) 418 ± 68 561 ± 218 514 ± 165

Ejection Fraction (%) 60 ± 5 14 ± 6 * 24 ± 22 *

Inotropes (yes/no) 1/4 2/3 2/3

ACE inhibitors (yes/no) 0/5 5/0 3/2

β-blockers (yes/no) 1/4 2/3 3/2

DCM: dilated cardiomyopathy; ICM: ischemic cardiomyopathy; ACE: angiotensin II converting

enzyme. *p<0.001 compared to non-failing group.


9

Supplemental Table 2. Echocardiographic measurements in CM-Cbl KO and Cblflox/flox

littermates subjected or not to IR injury.

Sham IR

Cblflox/flox

(n=5)

CM-Cbl KO

(n=6)

Cblflox/flox

(n=5)

CM-Cbl KO

(n=6)

HR (bpm) 465 ±18 453 ±14 473 ±24 485 ±32

HW (mg) 111 ±10 108 ±10 110 ±8 104 ±7

BW (g) 21 ±2 21 ±3 21 ±2 19 ±2

HW/BW (mg/g) 5.3 ±0.2 5.1 ±0.1 5.4 ±0.2 5.5 ±0.3

LVEDD (mm) 3.66 ±0.13 3.43 ±0.12 3.97 ±0.11* 3.63 ±0.12†

LVESD (mm) 2.34 ±0.12 2.18 ±0.14 3.12 ±0.13* 2.60 ±0.15*†

LVPWTd (mm) 0.78 ±0.01 0.83 ±0.02 0.76 ±0.04 0.86 ±0.06

LVPWTs (mm) 1.12 ±0.05 1.17 ±0.08 0.94 ±0.06 1.07 ±0.10

HR indicates heart rate; HW, heart weight; BW, body weight; LVEDD, LV end diastolic

dimension; LVESD, LV end systolic dimension; LVPWTd, LV posterior wall thickness diastole;

and LVPWTs, LV posterior wall thickness systole. *P<0.05 vs. WT shams, †P<0.05 vs. WT IR


10

Supplemental Table 3. Echocardiographic measurements in WT and c-Cbl KO mice subjected

or not to 7d MI injury.

Sham 7d MI

WT

(n=5)

c-Cbl KO

(n=5)

WT

(n=6)

c-Cbl KO

(n=6)

HR (bpm) 428 ±13 471 ±24 482 ±26 484 ±32

HW (mg) 137 ±5 117 ±10 199 ±10* 173 ±11*†

BW (g) 24 ±2 25 ±2 21 ±1 22 ±2

HW/BW (mg/g) 5.7 ±0.3 5.5 ±0.3 9.5 ±0.3* 7.9 ±0.4*†

LVEF (%) 65 ±2 64 ±2 20 ±3* 44 ±3*†

LVFS (FS) 36 ±2 34 ±3 10 ±2* 21 ±2*†

LVEDD (mm) 3.39 ±0.10 3.15 ±0.15 4.93 ±0.27* 3.81 ±0.2*†

LVESD (mm) 2.19 ±0.1 2.00 ±0.12 4.48 ±0.26* 2.98 ±0.17*†

LVPWTd (mm) 0.98 ±0.01 0.94 ±0.01 0.59 ±0.04* 0.85 ±0.04*†

LVPWTs (mm) 1.45 ±0.04 1.49 ±0.03 0.70 ±0.03* 1.25 ±0.09*†

HR indicates heart rate; HW, heart weight; BW, body weight; LVEF, LV ejection fraction;

LVFS, LV fractional shortening; LVEDD, LV end diastolic dimension; LVESD, LV end

systolic dimension; LVPWTd, LV posterior wall thickness diastole; and LVPWTs, LV

posterior wall thickness systole. *P<0.05 vs. WT shams, †P<0.05 vs. WT MI


11

Supplemental Table 4. Echocardiographic measurements in WT and c-Cbl KO mice subjected

or not to 30d MI injury.

Sham 30d MI

WT

(n=6)

c-Cbl KO

(n=6)

WT

(n=8)

c-Cbl KO

(n=8)

HR (bpm) 439 ±13 471 ±24 477 ±16 484 ±32

HW (mg) 135 ±5 117 ±10 209 ±10* 193 ±21*†

BW (g) 25 ±2 25 ±2 23 ±1 25 ±2

HW/BW (mg/g) 5.2 ±0.2 5.6 ±0.4 9.17 ±0.3* 8.14 ±0.3*†

LVEF (%) 63 ±2 64 ±2 30 ±2* 45 ±3*†

LVFS (FS) 34 ±2 35 ±2 15 ±1* 23 ±2*†

LVEDD (mm) 3.41 ±0.10 3.23 ±0.16 4.83 ±0.27* 4.09 ±0.2*†

LVESD (mm) 2.10 ±0.1 2.04 ±0.20 4.19 ±0.26* 3.30 ±0.05*†

LVPWTd (mm) 0.99 ±0.01 0.93 ±0.02 0.74 ±0.04* 0.98 ±0.04†

LVPWTs (mm) 1.58 ±0.04 1.38 ±0.04 0.92 ±0.03* 1.26 ±0.09*†

HR indicates heart rate; HW, heart weight; BW, body weight; LVEF, LV ejection fraction;

LVFS, LV fractional shortening; LVEDD, LV end diastolic dimension; LVESD, LV end systolic

dimension; LVPWTd, LV posterior wall thickness diastole; and LVPWTs, LV posterior wall

thickness systole. *P<0.05 vs. WT shams, †P<0.05 vs. WT MI


12

SUPLLENTARY FIGURE LEGEND

Supplemental Figure S1. Targeting strategy for specific disruption of c-Cbl in cardiac

myocytes. (A) Generation of c-Cbl floxed mutant mice has been previously described.1 In brief,

the floxed Cbl allele was generated by introducing two loxP sequences (triangles) into the introns

flanking one of the exons of c-Cbl (corresponding to nucleotides 681−837 of the mouse c-Cbl

cDNA) by gene targeting. Removal of the floxed exon by Cre-loxP-mediated DNA

recombination generated transcripts out-of-frame for translation. PTB, phosphotyrosine binding

domain; RF, RING-finger domain; LZ, leucine-zipper domain; S, SphI site. (B) Cardiac

myocyte-specific deletion of c-Cbl mediated by αMHC-MerCreMer transgene expression.

Genomic DNA samples from heart tissues obtained from Cblflox/flox and CM-Cbl KO mice (α-

MHC-MerCreMer×c-Cblflox/flox) were digested with SphI and hybridized with the probe in (A).

(C) Immunoblot analysis of c-Cbl deletion in the heart. Heart lysates were immunoblotted with

c-Cbl antibodies. The same membrane was reprobed for GAPDH as a loading control.

Supplemental Figure S2: Cardiac myocyte-specific deletion of c-Cbl improves cardiac

function. The left anterior descending artery was ligated for 30 minutes to induce ischemia and it

subsequently was reperfused for 24 hours. Cardiac function was measured by echocardiography

after IR injury. The data demonstrate improved left ventricular ejection fraction (A) and

fractional shortening (B) in CM-Cbl KO compared to c-Cblflox/flox mice (n=7 in sham groups; n=8

in MI groups). (C and D) Cardiac-myocyte deletion of c-Cbl attenuated IR-induced increase in

LV end-systolic (C) and end-diastolic dimension (D). (E) Quantification of infarct area (IA) vs.

area at risk (AAR) after IR injury shows significantly reduced infarct in CM-Cbl KO compared

to c-Cblflox/flox mice (n=6 per group). *P<0.05 vs. c-Cblflox/flox shams. †P<0.05 vs. c-Cblflox/flox IR.


13

Supplemental Figure S3: Cardiac myocyte-specific deletion of c-Cbl reduces apoptotic cell

death. The left anterior descending artery was ligated for 30 minutes to induce ischemia and it

subsequently was reperfused for 24 hours. (A) LV tissue sections from Cblflox/flox (a, c) or CM-

Cbl KO (b, d) were assessed for apoptosis using TUNEL assay (green), tropomyosin (red), and

DAPI (4',6-diamidino-2-phenylindole) (blue) staining. Arrows indicate apoptotic myocytes.

Bar=40 µm (a, b) or 20 µm (c, d). (B) The number of TUNEL-positive myocytes in the ischemic

area was expressed as a percentage of total nuclei detected by DAPI staining. (C) Quantification

of caspase-3 activity in the LV using caspase-3 specific fluorogenic substrate. *P<0.05 vs.

Cblflox/flox shams. †P<0.05 vs. Cblflox/flox IR.

Supplemental Figure S4. c-Cbl deletion attenuates H2O2-induced AKT , but not JNK,

phosphorylation. Neonatal rat cardiac myocytes were transduced with adenoviruses expressing

shRNA c-Cbl or shRNA control for 48 hours and then untreated or treated with 100 ng/ml EGF

or 100 µM H2O2 for 10 minutes. Cell lysates were assayed for immunoblot analysis.

Experiments were performed at least three times from three different cultures and the data values

were scaled to untreated Ad-shCtrl. *P<0.05 vs. control; †P<0.05 vs. treated myocytes.

Supplemental Figure S5. c-Cbl ubiquitin ligase activity is involved in H2O2-induced

myocyte apoptosis. (A) Neonatal rat cardiac myocytes were transduced with adenoviruses

expressing Lac.Z or ligase deficient c-Cbl mutant 70Z-Cbl for 48 hours and then untreated or

treated with 100 ng/ml EGF or 100 µM H2O2 for 10 minutes. Cell lysates were assayed for

EGFR immunoprecipitation assays (A) or immunoblot analysis (B). Experiments were


14

performed at least three times from three different cultures and the data values were scaled to

untreated Ad-shCtrl. (C-D) Myocyte apoptosis induced by H2O2 as assessed by percentage of

TUNEL-positive myocytes in culture (C) or DNA fragmentation assay (D). Results are

expressed as (OD410-OD500)/mg DNA (D) for triplicate determinations from a single

experiment (mean ± SE). *P<0.05 vs. control; †P<0.05 vs. treated myocytes.

Supplemental Figure S6. Deletion of c-Cbl reduces myocyte hypertrophy 4 weeks after MI.

c-Cbl or WT mice were subjected to MI for 4 weeks. (A) Histological assessment of cellular

pathology of c-Cbl KO or WT mice by Masson’s trichrome staining. Scale Bar: 40 µm. (B)

Histological analysis of myocyte cross-sectional area from sham and infarcted LV. Values are

mean ±SE. *p<0.05 vs. WT sham; †P<0.05 vs. WT MI.

Supplemental Figure S7. Effect of c-Cbl deletion on Erk1/2 and AKT phosphorylation in

infarcted LV. Top: Immunoblot analysis indicates a similar increase in Erk1/2 and AKT

phosphorylation in the infarcted region of the c-Cbl KO mice and the WT 7 days post-MI.

Bottom: Data are represented as fold change compared to WT animals (n=6 per group;

means±SEM). *P<0.05 compared with the WT sham group, †P<0.05 compared with the WT MI

group.

Supplemental Figure S8. Leukocyte infiltration and activation induced after IR injury is

minimally affected by c-Cbl deletion. The left anterior descending artery was ligated for 30

minutes to induce ischemia and it subsequently was reperfused for 24 hours. (A) Representative

immunolabeling of paraffin-embedded heart sections stained for MPO and counterstained with


15

hematoxylin. Scale Bar: 40 µm. (B) Quantification of MPO positive cells in the ischemia

reperfused area. (C-D) LV homogenates from shams or mice subjected to IR for 24 hours were

assessed for myeloperoxidase (MPO) (C) or cathepsin G (Cat.G)(D) activity assay using specific

fluorogenic substrates. Results are expressed as relative fluorescence unit (RFU)/min/g protein.

Values are mean ±SEM. *p<0.05; **p<0.01 vs. WT sham.

Cbl

GAPDH

Cblflox/f

lox

CM-Cbl-K

O

LZRFPTB Pro-rich

SS S S 1 kb

loxPSS

S 1 kbneo

S S 1 kb

S S 1 kb

c-Cbl

Wild type

Targeted

Floxed

Deleted

A

B

C

Cblflo

x/flox

Hea

rt

Mus

cle

Live

r

Lung

Cbl+/+

CM-Cbl-K

O

Hea

rt

Hea

rt

Supplemental Figure S1

A BLV

ES

D (m

m)

0

1

2

3

4 * ShamIR

* †

Cblflox/flox CM-Cbl KO

LVE

DD

(mm

)

1

2

3

4

5*

ShamIR

* †


C D


Are

a (%

)

AAR/LV

†

0

10

20

30

40

50

IA/AAR

IA/LV

†

Cblflox/flox

CM-Cbl KO

E

Frac

tiona

l Sho

rteni

ng (%

)

ShamIR

Cblflox/flox CM-Cbl KO0

10

20

30

40

** †

50E

ject

ion

Frac

tion

(%)


ShamIR

0

20

40

60*

* †

80

(5) (5) (5) (6) (5) (5) (5) (6)

(5) (5) (5) (6) (5) (5) (5) (6)

A

B


Sham

IR

10

20

30

40 *

* †

Cas

pase

-3 a

ctiv

ity x

103

(RFU

/min

/mg

prot

ein)


ShamIR

C

Cblflox/flox

CM-Cbl KO

a

b

c

dTU

NE

L po

sitiv

e m

yocy

tes

(%)

0

1

2

3

4

5

* †

*


(4) (6) (4) (6)

(5) (6) (5) (6)

p-AKT

AKT

c-Cbl

p-JNK

JNK

Ctrl H 2O 2

EGFCtrl H 2

O 2EGF

Ad.shCtrl Ad.shCbl

CtrlH2O2EGF

Ad-shCtrl

AK

T ph

osph

oryl

atio

n(F

old

activ

atio

n ov

er b

asal

)*

*

Ad-shCbl

*

* †

0

1

23

45

* †JN

K p

hosp

hory

latio

n(F

old

activ

atio

n ov

er b

asal

)

**

01234

Ad-shCtrl Ad-shCbl

56

* †

*


TUN

EL

posi

tive

moc

ytes

(%)

Lac.Z 70Z-Cbl0

5

10

15

20

25

* †

Control100 µM H2O2

30 *

Lac.Z 70Z-Cbl

IP: EGFR

Ctrl H 2O 2

EGFCtrl H 2

O 2EGF

Ub

P-Tyr

EGFR

p-ERK1/2

p-p38

p38

Lac.Z 70Z-Cbl

Ctrl H 2O 2

EGFCtrl H 2

O 2EGF

c-Cbl

ERK1/2

DN

A F

ragm

enta

tion

(OD

410

-OD

500/

mg

DN

A)

0

0.1

0.2

0.3

0.4

0.5

* †

Control100 µM H2O2

*

Lac.Z 70Z-Cbl

ER

K1/

2ph

osph

oryl

atio

n(F

old

activ

atio

n ov

er b

asal

)

** *

Lac.Z01234

†*

70Z-Cbl

5

p38

phos

phor

ylat

ion

(Fol

d ac

tivat

ion

over

bas

al)

01234

*

††

*

Lac.Z 70Z-Cbl

56

C D

A BCtrlH2O2EGF


100

150

200

250

Myo

cyte

cro

ss-s

ectio

nal a

rea

(µm

2 )

Sham MI infarct area

MI remote area

*

* †*

†

A

B


WT

c-Cbl KO

sham MI

WTc-Cbl KO

(5) (5) (5) (6) (5) (6)

p-Erk1/2

Erk1/2

Sham MI Sham MI

WT c-Cbl KO

*

AK

T ph

osph

oryl

atio

n(fo

ld in

crea

se o

ver W

T sh

am)

0

1

2

3

WT c-Cbl KO

*

*

*

Erk

1/2

phos

phor

ylat

ion

(fold

incr

ease

ove

r WT

sham

)

0

1

2

3

WT c-Cbl KO

*

*


p-AKT

AKT

Sham MI Sham MI

WT c-Cbl KOA B

ShamMI

ShamMI

(6) (6) (6) (6) (6) (6) (6) (6)

A B

C D

WT c-Cbl KOa b

c d

Sham

IR

Sham

200

400

600

800

1000

1200

WT c-Cbl KO

MP

O a

ctiv

ity x

103

(RFU

/mg/

g pr

otei

n)

(5) (6) (6) (6)

** **

*

IR 24hr

ShamIR 24hr

MP

O p

ositi

ve c

ells

(num

ber/m

m2 )

WT c-Cbl KO0

2

4

6

8

10 **

(5) (5)(5) (5)

ShamIR 24hr

Cat

.Gac

tivity

x10

3

(RFU

/mg/

g pr

otei

n)

** *

*

50

100

150

200

250

WT c-Cbl KO

(5) (6) (6) (6)


SUPPLEMENTAL REFERENCES

1. Naramura M, Jang I-K, Kole H, Huang F, Haines D, Gu H. c-Cbl and Cbl-b regulate T

cell responsiveness by promoting ligand-induced TCR down-modulation. Nature

Immunol. 2002; 3:1192-1199.

2. Sohal DS, Nghiem M, Crackower MA, Witt SA, Kimball TR, Tymitz KM, Penninger

JM, Molkentin JD. Temporally regulated and tissue-specific gene manipulations in the

adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ Res. 2001;

89:20-25.

3. Rafiq K, Kolpakov MA, Abdelfettah M, Streblow DN, Hassid A, Dell'Italia LJ, Sabri A.

Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation

during neutrophil cathepsin G-induced cardiomyocytes anoikis. J Biol Chem. 2006;

281:19781-19792.

4. Rafiq K, Hanscom M, Valerie K, Steinberg S, Sabri A. Novel mode for neutrophil

protease cathepsin G-mediated signaling: Membrane shedding of epidermal grwoth factor

is required for cardiomyocyte anoikis. Circ Res. 2008; 102:32-41.

5. Purev E, Neff L, Horne WC, Baron R. c-Cbl and Cbl-b act redundantly to protect

osteoclasts from apoptosis and to displace HDAC6 from β-tubulin, stabilizing

microtubules and podosomes. Mol Biol Cell. 2009; 20:4021-4030.

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