c-Cbl inhibition improves cardiac function and survival in response to myocardial ischemia

Abstract

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 H2O2 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.

Keywords: angiogenesis; apoptosis; myocardial ischemia; ubiquitin.

Figure 1

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 post-natal 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 non failing (NF), ischemic (ICM) and dilated cardiomyopathy (DCM) human heart sections stained for c-Cbl and counterstained with hematoxylin. Scale 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 NRCMs untreated or treated with isoproterenol (Iso, 10 µM), norepinephrine (NE, 10 µM), thrombin (Thr, 1 U/ml), or TNFα (100 ng/ml) for 48 hours. Top, c-Cbl immunoblot with GAPDH taken as a loading control. Bottom, Quantification of experiments expressed as mean ± S.E from three separate cultures. *P<0.05 vs. control.

Figure 2

c-Cbl ablation protects against IR injury. The left anterior descending artery was ligated for 30 minutes to induce ischemia and it subsequently was reperfused for 24 hours. (A-B) Echocardiography measurement of ejection fraction (A) and fractional shortening (B) in WT and c-Cbl KO animals (n=8 for sham groups, n=9 for IR groups). (C) Representative cross-sections were stained with triphenyl tetrazolium 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) LV tissue sections were assessed for apoptosis using TUNEL assay (green), tropomyosin (red), and DAPI (4′,6-diamidino-2-phenylindole) (blue) staining. Scale bars: 40 µm (a-d) or 20 µm (e, 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 using caspase-3 specific fluorogenic substrate. (H) Serum levels of cardiac troponin I after IR injury in WT and c-Cbl KO mice (n=6 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 each group). *P<0.05 vs. WT shams, †P<0.05 vs. WT IR.

Figure 2

c-Cbl ablation protects against IR injury. The left anterior descending artery was ligated for 30 minutes to induce ischemia and it subsequently was reperfused for 24 hours. (A-B) Echocardiography measurement of ejection fraction (A) and fractional shortening (B) in WT and c-Cbl KO animals (n=8 for sham groups, n=9 for IR groups). (C) Representative cross-sections were stained with triphenyl tetrazolium 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) LV tissue sections were assessed for apoptosis using TUNEL assay (green), tropomyosin (red), and DAPI (4′,6-diamidino-2-phenylindole) (blue) staining. Scale bars: 40 µm (a-d) or 20 µm (e, 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 using caspase-3 specific fluorogenic substrate. (H) Serum levels of cardiac troponin I after IR injury in WT and c-Cbl KO mice (n=6 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 each group). *P<0.05 vs. WT shams, †P<0.05 vs. WT IR.

Figure 3

c-Cbl ablation protects against EGFR and FAK ubiquitination and downregulation induced after IR injury. The left anterior descending artery was ligated for 30 minutes to induce ischemia and it subsequently was reperfused for 24 hours. (A-B) c-Cbl immunoprecipitates were assayed for auto-ubiquitination assay and immunoblot analysis. (C-D) EGFR or FAK immunoprecipitates from LV 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 each group). *P<0.05 compared to WT shams, †P<0.05 compared to WT IR.

Figure 4

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. (B-C) Cardiac myocytes were transduced with adenoviruses expressing shRNA Cbl (Ad-shCbl, 10 pfu/cell) or shRNA control (Ad-shCtrl, 10 pfu/cell) 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 (B) or immunoblot analysis (C). (D-E) Myocyte apoptosis induced by H₂O₂ as assessed by the percentage of TUNEL-positive myocytes in culture (D) or DNA fragmentation ELISA assay (E). Results are expressed as (OD410-OD500)/mg DNA (D) for triplicate determinations from a single experiment (mean ± S.E). All experiments were performed at least three times from three different cultures and the data values were scaled to untreated controls. *P<0.05 vs. control; †P<0.05 vs. treated myocytes.

Figure 5

c-Cbl KO mice are protected against post-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. Scale bar: 40 µm. (B) Top: Representative immunoblot of LV heart lysates for c-Cbl. Bottom: Fold induction of c-Cbl accumulation (n=5 for sham groups; n=6 for MI groups). (C) Comparison of post-MI mortality between WT (n=18) and c-Cbl KO (n=16) mice. c-Cbl KO mice showed an increase in survival after MI compared to WT (p<0.05). (D-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 the heart weight (HW) to tibia length (TL) (F) and the ratio of lung weight (LW) to TL (G). (H) The infarct size 4 weeks post-MI expressed as a fraction of the total cross-sectional circumference of the LV 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 the WT MI group.

Figure 5

c-Cbl KO mice are protected against post-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. Scale bar: 40 µm. (B) Top: Representative immunoblot of LV heart lysates for c-Cbl. Bottom: Fold induction of c-Cbl accumulation (n=5 for sham groups; n=6 for MI groups). (C) Comparison of post-MI mortality between WT (n=18) and c-Cbl KO (n=16) mice. c-Cbl KO mice showed an increase in survival after MI compared to WT (p<0.05). (D-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 the heart weight (HW) to tibia length (TL) (F) and the ratio of lung weight (LW) to TL (G). (H) The infarct size 4 weeks post-MI expressed as a fraction of the total cross-sectional circumference of the LV 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 the WT MI group.

Figure 6

c-Cbl deletion enhances angiogenic response in infarcted region after MI. (A) Immunoblot analysis for VEGF and VEGFr2 indicates an increased angiogenic response in the infarcted region of the c-Cbl KO mice vs. the WT 7 days post-MI. (B) Data are represented as fold change compared to WT animals (n=5 for sham groups, n=6 for MI groups). (C) Immunohistochemistry analysis reveals an increase in VEGFr2 expression and smooth muscle α-actin (SM α-actin) positive blood vessels at the border zone of the infarcted region 7 days post-MI, which is more pronounced in the Cbl KO mice. Scale bar: 40 μm. (D) Representative sections stained for CD31 labeling show an equal distribution of capillaries surrounding cardiomyocytes in the non-infarcted, 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 to the WT 30 days post-MI. Scale bars: 40 μm. (E) Semi-quantitative analysis of capillary density in the myocardium indicates an increase in vessel density in the border zone of c-Cbl KO mice compared to 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 the WT sham group, †P<0.05 compared with the WT MI group.