Cardiomyocyte Specific Ablation of p53 Is Not Sufficient to Block Doxorubicin Induced Cardiac Fibrosis and Associated Cytoskeletal Changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity

Signaling Mechanisms Regulating Fibroblast Activation, Phenoconversion and Fibrosis in the Heart

476-482Cardiac fibroblasts (CFs) maintain the cardiac extracellular matrix (ECM) through myocardial remodelling. The remodelling process can become dysregulated during various forms of heart disease which leads to an overall accumulation of ECM. This results in cardiac fibrosis which increases the risk of heart failure in many patients. During heart disease, quiescent CFs undergo phenoconversion to an activated cell type called cardiac myofibroblasts (CMFs). Factors influencing phenoconversion include transforming growth factor β (TGF-β) which via SMADs (small mothers against decapentaplegic) activates the myofibroblast marker gene αSMA (α smooth muscle actin). Signaling molecules as diverse as NAD(P)H oxidase 4 (Nox4) and Wnt have been found to interact with TGF-β signalling via SMADs. Pathways, including FAK/TAK/JNK and PI3K/Akt/rac have also been implicated in activating phenoconversion of fibroblasts. Another major contributor is mechanical stress exerted on CFs by ECM changes, which involves activation of ERK and subsequent αSMA expression. Other factors, such as the mast cell protease tryptase and the seeding density also affect the phenoconversion of fibroblast cultures in vitro. Further, reversal of myofibroblast phenotype has been reported by a negative regulator of TGF-β, Ski, as well as the hormone relaxin and the second messenger cAMP. Targeting the signaling molecules involved in promoting phenoconversion of CFs to CMFs presents a possible method of controlling cardiac fibrosis. Here, we provide a brief review of signaling mechanisms responsible for phenoconversion and identify critical targets for the treatment of cardiac fibrosis

Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells

Characterization of growth suppressive functions of a splice variant of cyclin D2.

We have recently cloned a novel splice variant of cyclin D2 termed as cycD2SV. CycD2SV overexpression in several immortalized cell lines led to formation of ubiquitinated protein aggregates accompanied by a significant decrease in cell proliferation. Based on immuno co-localization and ultrastructural analysis experiments, cycD2SV protein aggregates were frequently found in various subcellular compartments such as endosomes, autophagosomes, lysosomes and the microtubule organizing centre. Secondary structure analysis revealed that the amino terminal α-helix in cycD2SV is not tightly packed with the cyclin box suggesting a misfolded conformation compared to other cyclins. Deletion analysis suggests that 1-53 amino acid region of cycD2SV may be required for protein aggregation and 54-136 amino acid region may mediate cell cycle inhibition. Based on co-immunoprecipitation experiments, we have shown that cycD2SV binds to cycD2 as well as CDK4. In addition, gene expression analysis demonstrated an upregulation in GADD45α and dynamin 2 mRNA levels in cycD2SV overexpressing cells. These two proteins are known to play critical roles in the DNA damage response and apoptosis pathways. TUNEL experiments were negative for apoptosis, however, cycD2SV expressing cells were more sensitive to cell death induced by external stressors such as trypsinization. Collectively our results suggest that cycD2SV mediates cell cycle inhibition by sequestering endogenous cell cycle proteins, such as cycD2 and CDK4, and possibly targeting them for ubiquitin mediated protein degradation

Three-dimensional (3D) protein structure predictions for cycD2 and cycD2SV.

<p>Protein structures were determined by the iterative threading assembly refinement (I-TASSER) server, an internet based 3D protein structure prediction engine. The N-terminus of the presented protein structures is denoted by blue and the C-terminus is denoted by red.</p

Interaction of cycD2SV with CDK4.

<p>HEK293 cells transfected with cycD2SVmyc (A–C) processed for myc (A), CDK4 (B) immunostaining and nuclear stain (C). Interaction of transfected cycD2SV with endogenous CDK4 was determined by CDK4 immunoprecipitation (D). Endogenous CDK4 was immunoprecipitated from pcDNA (negative control), cycD2myc (positive control) and D2SVmyc transfected cells using CDK4 antibodies. An additional immunoprecipitation negative control was completed using rabbit non-immune serum (RNIS, control) on cycD2SVmyc transfected HEK293 cells. Immunoprecipitated samples were resolved by western blot and the nitrocellulose blot was probed with myc and CDK4 antibodies. Western blot analysis of HEK293 cells transfected with pcDNA (control), empty vector, and D2SVmyc using cycD2SV antibodies (E). IP, immunoprecipitation; WB, western blot; End., endogenous.</p

Interaction of cycD2SV with cyclin D2.

<p>Overexpressed cycD2SV co-localizes with endogenous and co-transfected cycD2. HEK293 cells transfected with cycD2SVmyc alone (A–C) or co-transfected with cycD2myc (D–F) were processed for myc (A, E), D2SV (D), cycD2 (B) immunostaining and nuclear stain (C, F). Scale bar is 20 µm. Immunoprecipitation analysis of interactions between cycD2SV and cycD2 (G). Western blot (WB) analysis performed on HEK293 cells transfected with pcDNA, cycD2myc, cycD2SVmyc and cycD2SVΔCTmyc using myc and D2SV antibodies (H) as well as cycD2 and α-tubulin antibodies (I). Results in panels H and I indicate the specificity of cycD2SV and cycD2 antibodies and rule out any cross-reactivity.</p

Effects of cycD2SV 54–136 and cycD2SVΔCT overexpression on cell cycle regulation.

<p>A schematic representation of D2SVΔCT, D2SV 1–53 and D2SV 54–136 deletions in comparison to full length D2SV (A). Shaded box (136–156 amino acids) in cycD2SV represents the unique CT sequence. HEK293 cells transfected with D2SVΔCTmyc (B, C), D2SV 1–53 (D, E) and D2SV 54–136 myc (F, G) were processed for myc (B, D, F) immunostaining and nuclear stain (C, E, G). Scale bar is 20 µm. HEK293 cells transfected with D2SV, D2SVΔCT and D2SV 54–136 were labeled with [³H]-thymidine and processed for immunostaining and [³H]-thymidine autoradiography. The percentage of cells positive for protein aggregation (H) and [³H]-thymidine (I) were quantified and expressed as a percent of total transfected cells. Cells transfected with pcDNA 3.1 vector were used as a control (cont). Values are expressed as mean ± SEM. One way ANOVA, *p<0.05 compared to control, approximately 1000 cells were counted for each group from three independent experiments (N = 3).</p