Volume Load-Induced Right Ventricular Failure in Rats Is Not Associated With Myocardial Fibrosis

Background Right ventricular (RV) function and failure are key determinants of morbidity and mortality in various cardiovascular diseases. Myocardial fibrosis is regarded as a contributing factor to heart failure, but its importance in RV failure has been challenged. This study aims to assess whether myocardial fibrosis drives the transition from compensated to decompensated volume load-induced RV dysfunction. Methods Wistar rats were subjected to aorto-caval shunt (ACS, n = 23) or sham (control, n = 15) surgery, and sacrificed after 1 month, 3 months, or 6 months. Echocardiography, RV pressure-volume analysis, assessment of gene expression and cardiac histology were performed. Results At 6 months, 6/8 ACS-rats (75%) showed clinical signs of RV failure (pleural effusion, ascites and/or liver edema), whereas at 1 month and 3 months, no signs of RV failure had developed yet. Cardiac output has increased two- to threefold and biventricular dilatation occurred, while LV ejection fraction gradually decreased. At 1 month and 3 months, RV end-systolic elastance (Ees) remained unaltered, but at 6 months, RV Ees had decreased substantially. In the RV, no oxidative stress, inflammation, pro-fibrotic signaling (TGF beta 1 and pSMAD2/3), or fibrosis were present at any time point. Conclusions In the ACS rat model, long-term volume load was initially well tolerated at 1 month and 3 months, but induced overt clinical signs of end-stage RV failure at 6 months. However, no myocardial fibrosis or increased pro-fibrotic signaling had developed. These findings indicate that myocardial fibrosis is not involved in the transition from compensated to decompensated RV dysfunction in this model

BMP-2 Up-Regulates PTEN Expression and Induces Apoptosis of Pulmonary Artery Smooth Muscle Cells under Hypoxia

To investigate the role of bone morphogenetic protein 2 (BMP-2) in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and apoptosis of pulmonary artery smooth muscle cells (PASMCs) under hypoxia.Normal human PASMCs were cultured in growth medium (GM) and treated with BMP-2 from 5-80 ng/ml under hypoxia (5% CO(2)+94% N(2)+1% O(2)) for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of PTEN, AKT and phosph-AKT (pAKT) were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic) (PTEN inhibitor) and GW9662 (PPARγ antagonist) were used to determine the signalling pathways.Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%). BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic) and GW9662 (PPARγ inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate.BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway

PlGF Repairs Myocardial Ischemia through Mechanisms of Angiogenesis, Cardioprotection and Recruitment of Myo-Angiogenic Competent Marrow Progenitors

Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1(+)/Lin(-) (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease

Deficiency of Thioredoxin Binding Protein-2 (TBP-2) Enhances TGF-β Signaling and Promotes Epithelial to Mesenchymal Transition

Transforming growth factor beta (TGF-β) has critical roles in regulating cell growth, differentiation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) of various cancer cells. TGF-β-induced EMT is an important step during carcinoma progression to invasion state. Thioredoxin binding protein-2 (TBP-2, also called Txnip or VDUP1) is downregulated in various types of human cancer, and its deficiency results in the earlier onset of cancer. However, it remains unclear how TBP-2 suppresses the invasion and metastasis of cancer.In this study, we demonstrated that TBP-2 deficiency increases the transcriptional activity in response to TGF-β and also enhances TGF-β-induced Smad2 phosphorylation levels. Knockdown of TBP-2 augmented the TGF-β-responsive expression of Snail and Slug, transcriptional factors related to TGF-β-mediated induction of EMT, and promoted TGF-β-induced spindle-like morphology consistent with the depletion of E-Cadherin in A549 cells.Our results indicate that TBP-2 deficiency enhances TGF-β signaling and promotes TGF-β-induced EMT. The control of TGF-β-induced EMT is critical for the inhibition of the invasion and metastasis. Thus TBP-2, as a novel regulatory molecule of TGF-β signaling, is likely to be a prognostic indicator or a potential therapeutic target for preventing tumor progression

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction

<div><p>Background</p><p>Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium.</p><p>Results</p><p>Wildtype and Wt1^CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21^lacZ/+ reporter mice and PDGFRα^-/-;Tcf21^LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-^LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα^-/-;Tcf21^LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-^LacZ + cells and reduced ventricular compaction.</p><p>Conclusions</p><p>During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-^LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.</p></div

Pim1 maintains telomere length in mouse cardiomyocytes by inhibiting TGFβ signalling.

AimsTelomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFβ) signalling pathway where inhibiting TGFβ signalling maintains telomere length. The relationship between Pim1 and TGFβ has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFβ signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes.Methods and resultsTelomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFβ signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFβ signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFβ pathway genes.ConclusionPim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFβ pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function

WT1-derived cells and Tcf21^lacZ/+ show regional differences in intramyocardial distribution of at E14.5.

<p>A-E: WT1^CreERT2/+; Rosa26^fsLz/+ mouse section (A,B,D) and control WT-1 staining (C,E) showing differences between RV and LV in WT-1+ cells. Red tissue in A,B,D indicates β-gal staining. A is an overview of RV and LV, B and D are enlargements of RV (B) and LV (D). C and E show WT-1+ cells comparable to B and D. F-I: H: Tcf21^lacZ/+ mouse section stained for LacZ in blue. Anterior (Ant) and posterior (Pos) are indicated. The upper box at the RV (H) corresponds to the enlargement in F of the anterior RV, the lower box at the RV (H) corresponds to the enlargement (G) of the posterior RV. The box of the LV in H corresponds to enlargement (I). Tcf21^lacZ/+ demonstrates the same distribution pattern, i.e. more lacZ+ cells in LV than RV (compare G with I) and more lacZ+ cells in posterior part of RV than anterior (compare G with F). Bars: I,N: 200 μm, other bars: 50 μm.</p

Ventricular compaction and wall thickness in sequential stages E11.5–15.5.

<p>A-E. Myocardial staining for myosin light chain 2a (MLC-2a) shows myocardial compaction and thickening of the anterior part of right ventricle (RV). F-J: Myocardial compaction and thickening of posterior part of RV. K-O: Myocardial compaction and thickening of the left ventricle (LV). The LV wall is thicker than RV, and the RV posterior wall appears thicker than anterior (compare sections E, J and O). P-R depict the myocardial wall thickness measurements of the RV and LV. Two distinction can be made in the measurements. One is the <i>anatomical region</i> of each ventricle: the anterior portion of the RV, the posterior portion of the RV or the posterior portion of the LV. The second distinction is the <i>location in the myocardial wall</i>: the lateral wall or the apical wall. P. The RV shows a significantly thicker posterior than anterior wall at E15.5. Q,R: RV and LV wall thickness. From E13.5, LV lateral wall (Q) is significantly thicker than RV, except for the latest stage (E15.5). For the apical wall (R) this difference is significant from E12.5. Grey line with circle (°) represents significant difference in LV thickness between E11.5-E13.5 and E14.5-E15.5, showing a gradual growth during development. Grey line with square (□) represents significant difference in RV thickness between 11.5–14.5 and E15.5, indicating a gradual ventricular growth during development. Grey line with alpha (α) represents significant difference in LV thickness between E11.5-E12.5 and E15.5. *p<0.05; **p<0.01; ***p<0.001. Bars: 50 μm.</p

Regional differences in intramyocardial WT-1 expression at E13.5.

<p>A-D: Control, transverse sections. A. Anterior section, stained for WT-1. B: Enlargement of the box in A with few WT-1+ cells in the right ventricle (RV) and more WT-1+ cells in the left ventricle (LV). C. Posterior section, stained for WT-1. D: Enlargement of RV in D with more WT-1+ cells than anterior and enlargement of the LV with overall more WT-1+ cells than in the RV (compare with panel B). E-J: fluorescence sequential sagittal sections from RV (E-F, H-I) towards LV (G,J) double stained for WT-1 (green) and cTnI (red). Arrows indicate areas with abundant WT-1+ cells. Anterior (A) and posterior (P) are indicated. K-N: Tcf21^lacZ/+ mouse sections stained for LacZ in blue. K. Anterior section of the heart. L: Enlargement of RV with no LacZ+ cells and of LV with sporadic WT-1+ cells (arrowheads). M. Posterior section of the heart. N: Enlargement RV with sporadic LacZ+ cells (arrowheads) and of LV with overall more LacZ+ cells than in the RV (arrowheads). Bars: A,C,E,G: 200 μm, B,D,F,H: 50 μm, I-N: 100 μm.</p

E12.5. Early intramyocardial WT-1 positive cells.

<p>A-C. Control, transverse sections. B. E12.5 MLC-2a staining of right ventricle (RV) and left ventricle (LV) with complete epicardial covering and a visible subepicardial space in the LV and in the atrioventricular and interventricular sulcus (arrows). A, C: Enlargements of adjacent WT-1 stained sections of boxes in B, showing RV (A) and LV (C). The subepicardial layer is slightly broader on the LV side. WT-1+ cells are found in RV lateral wall (B, arrowheads) but not in the LV lateral wall (C). D-F: Fluorescent double stainings of WT-1 (green) and cTnI (red) at E12.5 confirm the first WT-1+ cells in the RV lateral wall, in the apical LV wall and the interventricular septum. G-L: WT1^CreERT2/+, E12.5. There is a subepicardial layer in both ventricles with WT-1+ cells in the RV lateral wall (arrows in G and J), but not at the LV lateral wall (I and L). Some WT-1+ cells present at the base of both ventricles, and in the interventricular septum (IVS) (arrowheads in H). M-O: Tcf21^lacZ/+ mouse sections stained for LacZ in blue. N. LacZ staining of RV and LV comparable to B, showing epicardial covering and subepicardial space in the LV and in the atrioventricular and interventricular sulcus (arrows). M,O: Enlargements of boxed areas in E, showing RV (M) and LV (O). As in C, the subepicardial layer is slightly broader compared to the RV. No LacZ + cells where found in the RV lateral wall. Bars: B,E: 200μm, H, K: 500 μm, other bars: 50 μm.</p