Dioxygen and Metabolism; Dangerous Liaisons in Cardiac Function and Disease

The heart must consume a significant amount of energy to sustain its contractile activity. Although the fuel demands are huge, the stock remains very low. Thus, in order to supply its daily needs, the heart must have amazing adaptive abilities, which are dependent on dioxygen availability. However, in myriad cardiovascular diseases, “fuel” depletion and hypoxia are common features, leading cardiomyocytes to favor low-dioxygen-consuming glycolysis rather than oxidation of fatty acids. This metabolic switch makes it challenging to distinguish causes from consequences in cardiac pathologies. Finally, despite the progress achieved in the past few decades, medical treatments have not improved substantially, either. In such a situation, it seems clear that much remains to be learned about cardiac diseases. Therefore, in this review, we will discuss how reconciling dioxygen availability and cardiac metabolic adaptations may contribute to develop full and innovative strategies from bench to bedside

Regulation of Connective Tissue Growth Factor and Cardiac Fibrosis by an SRF/MicroRNA-133a Axis

International audienceMyocardial fibrosis contributes to the remodeling of heart and the loss of cardiac function leading to heart failure. SRF is a transcription factor implicated in the regulation of a large variety of genes involved in cardiac structure and function. To investigate the impact of an SRF overexpression in heart, we developed a new cardiac-specific and tamoxifen-inducible SRF overexpression mouse model by the Cre/loxP strategy. Here, we report that a high level over-expression of SRF leads to severe modifications of cardiac cytoarchitecture affecting the balance between cardiomyocytes and cardiac fibroblasts and also a profound alteration of cardiac gene expression program. The drastic development of fibrosis was characterized by intense sirius red staining and associated with an increased expression of genes encoding extracellular matrix proteins such as fibronectin, procollagen type 1α1 and type 3α1 and especially connective tissue growth factor (CTGF). Furthermore miR-133a, one of the most predominant cardiac miRNAs, is strongly downregulated when SRF is overexpressed. By comparison a low level overexpression of SRF has minor impact on these different processes. Investigation with miR-133a, antimiR-133a and AdSRF-VP16 experiments in H9c2 cardiac cells demonstrated that: 1)–miR-133a acts as a repressor of SRF and CTGF expression ; 2)–a simultaneous overexpression of SRF by AdSRF-VP16 and inhibition of miR-133a by a specific antimiR increase CTGF expression; 3)–miR-133a overexpression can block the upregulation of CTGF induced by AdSRF-VP16. Taken together, these findings reveal a key role of the SRF/CTGF/miR-133a axis in the regulation of cardiac fibrosis

A defective mechanosensing pathway affects fibroblast-to-myofibroblast transition in the old male mouse heart

Summary: The cardiac fibroblast interacts with an extracellular matrix (ECM), enabling myofibroblast maturation via a process called mechanosensing. Although in the aging male heart, ECM is stiffer than in the young mouse, myofibroblast development is impaired, as demonstrated in 2-D and 3-D experiments. In old male cardiac fibroblasts, we found a decrease in actin polymerization, α-smooth muscle actin (α-SMA), and Kindlin-2 expressions, the latter an effector of the mechanosensing. When Kindlin-2 levels were manipulated via siRNA interference, young fibroblasts developed an old-like fibroblast phenotype, whereas Kindlin-2 overexpression in old fibroblasts reversed the defective phenotype. Finally, inhibition of overactivated extracellular regulated kinases 1 and 2 (ERK1/2) in the old male fibroblasts rescued actin polymerization and α-SMA expression. Pathological ERK1/2 overactivation was also attenuated by Kindlin-2 overexpression. In contrast, old female cardiac fibroblasts retained an operant mechanosensing pathway. In conclusion, we identified defective components of the Kindlin/ERK/actin/α-SMA mechanosensing axis in aged male fibroblasts

Effects of cardiac-specific overexpression of SRF on heart dilation and cardiomyocyte architecture.

<p>(A) Western blot analysis of SRF protein (10 μg of total protein per lane). The blot was also probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. (B) Quantification of SRF protein level (n = 4). Data are presented as means ± s.e.m. *** indicates significant difference at P < 0.001, respectively versus the control group. (C) Hematoxylin-eosin staining of paraffin-embedded heart sections. The dilation of the ventricles is less marked in SRF-LL than in SRF-HL samples. LV: left ventricle; RV: right ventricle. These data are representative of three independent experiments. Scale bar: 1 mm. (D) Sections of mouse hearts were stained with Sirius red. The presence of endomyocardial fibrotic regions was observed in SRF-HL mice (white arrows). These data are representative of three independent experiments. Scale bar: 100 μm. (E) Identical fields than in D visualized with polarized light. Collagen fibers are highly birefrigent with fine fibers appearing green, and thicker fibers appearing yellow or orange (white arrows). (F) Confocal microscopy of cardiac sections labeled with anti-SRF antibody (red), anti-vinculin FITC (green) for cardiomyocyte membranes and DAPI (blue) for nuclei. SRF staining (white arrows) showing higher labeled cardiomyocyte nuclei in the SRF-HL than in the SRF-LL and in the control groups corroborating overexpression of the SRF gene. In the same way, intercalated discs (orange arrows) are substantially enlarged and irregularly shaped in the SRF-HL group compared with the SRF-LL and the control groups. These results are representative of three independent experiments. Scale bar: 80 μm. (G) Distribution of cardiomyocyte lengths and widths in the three groups of mice; n = 180 for each group (three different mice per group). SRF-LL: low level of SRF; SRF-HL: high level of SRF.</p

Involvement of SRF and of miR-133a in CTGF regulation.

<p>(A) ChIP was performed using H9c2 cells and antibodies specific to SRF, RNApol2 and IgG. Bound SRF or CTGF promoters was amplified by qRT-PCR and normalized to input and to an additional negative control (primers spanning the first intron of Il4). Data are mean± s.e.m. ** and *** indicate significant difference at P < 0.01 and P < 0.001, respectively versus IgG. Data are representative of 4 independent experiments. (B) qRT-PCR assays of miR-133a in cardiac tissue from control (n = 7), SRF-LL (n = 6) and SRF-HL (n = 6) mouse miRNA, using miR–16 as internal control. Data are presented as means ± s.e.m. * indicates significant difference at P < 0.05, respectively versus the control group. (C) H9c2 cells were not treated (n = 7) or treated by miR-133a (50 nM) (n = 7) or antimiR-133a (50 nM) (n = 7) for 48 hours then scratched and total RNAs were extracted. Data are presented as means ± s.e.m. *, ** and *** indicate significant difference at P < 0.05, P < 0.01 and P < 0.001, respectively versus the control group. (D) Western blot analysis of SRF, CTGF and GAPDH proteins (20 μg of total protein per lane). This blot is representative of three independent experiments. (E) H9c2 cells were transduced with AdGFP (n = 5) or AdSRF-VP16 (n = 5) adenoviruses for 8 hours, the medium was changed and treated or not by miR-133a (50 nM) or antimiR-133a (50 nM). 48 hours later, cells were scratched and total RNAs extraction was done. Data are presented as means ± s.e.m. ** and *** indicate significant difference at P < 0.01 and P < 0.001, respectively versus AdGFP; §§§ indicates significant difference at P < 0.001, respectively versus AdSRF-VP16. (F) H9c2 cells were transduced with AdGFP (n = 5) or AdSRF-VP16 (n = 5) adenoviruses for 8 hours then fresh medium was added. 48 hours later, cells were scratched and total RNAs extraction was done. Data are presented as means ± s.e.m. *** indicates significant difference at P < 0.001, respectively versus AdGFP.</p

Impact of cardiac-specific overexpression of SRF on gene expression.

<p>qRT-PCR analysis of control (n = 7), SRF-LL (n = 6) and SRF-HL (n = 6) mouse mRNA, using cyclophilin as internal control. Data are presented as means ± s.e.m. *, ** and *** indicate significant difference at P < 0.05, P < 0.01 and P < 0.001, respectively versus the control group. (A) SRF target genes. (B) Genes involved in cardiac fibrosis. SRF-LL: low level of SRF; SRF-HL: high level of SRF.</p

Impact of the level of SRF overexpression on cardiac fibrosis development.

<p>(A) Western blot analysis of vimentin protein. (B) Quantification of vimentin protein level (n = 4). Data are presented as means ± s.e.m. ** indicates significant difference at P < 0.01, respectively versus the control group. (C) Vizualisation of cardiac fibroblasts by vimentin staining of heart sections from control, SRF-LL and SRF-HL mice. Longitudinal frozen section showing the localization of cardiac fibroblasts stained with vimentin (red) and polymerized actin in myocytes labeled with Alexa-Fluor-488-phalloidin (green). These data are representative of three independent experiments. Scale bar: 80 μm. (D) Confocal microscopic view of Ki67 labeling (pink), nuclei (blue) and vinculin (green). Ki67 labeling is stronger in the SRF-HL than the SRF-LL and control mice and is exclusively located in the interstitial cells. The enlargement is of twice. These data are representative of three independent experiments. Scale bar: 80 μm. (E) Confocal microscopic view of Ki67 labeling (pink), vimentin (green) and nuclei (blue) of heart sections from SRF-HL and control mice. Top panel, scale bar: 100 μm; bottom panel corresponding to the square indicated in the top panel, scale bar: 10 μm. These data are representative of two independent experiments.</p

Inducible cardiac-specific SRF overexpression in adult mice.

<p>Schematic representation of the genetic strategy used to obtain a cardiac-specific SRF overexpression in adult mice. Tamoxifen injections to the double transgenic mice (α-MHC-MerCreMer/CAG-flCAT-SRF) induce excision of the floxed CAT gene that allows the expression of SRF gene.</p

Cardiac CTGF expression induced by a high SRF overexpression.

<p>(A) Western blot analysis of CTGF protein. The GAPDH blot represented here is the same used for vimentin in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139858#pone.0139858.g004" target="_blank">Fig 4</a> since CTGF and vimentin were blot on the same membrane. (B) Quantification of CTGF protein level (n = 4). Data are presented as means ± s.e.m. * and *** indicate significant difference at P < 0.05 and P < 0.001, respectively versus the control group. (C) Immunoflurescence labeling of SRF (red), vinculin (green) and nuclei (blue) (upper line), and CTGF (orange), vinculin (green) and nuclei (blue) (lower line) on serial heart sections of control, SRF-LL and SRF-HL mice. The white arrows indicate SRF-positive/CTGF-null cardiomyocytes while the orange arrows indicate SRF-positive/CTGF-positive cardiomyocytes. All these data presented in this figure are representative of three independent experiments. Scale bar: 80 μm.</p

A scheme illustrating the loop of regulation between SRF, CTGF and miR-133a in low and high SRF expression level in cardiomyocytes.

<p>A scheme illustrating the loop of regulation between SRF, CTGF and miR-133a in low and high SRF expression level in cardiomyocytes.</p