Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes.

Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF-MRTF-Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF-MRTFA-actin axis in cardiac remodeling.

Nitrate consumption preserves HFD-induced skeletal muscle mitochondrial ADP sensitivity and lysine acetylation: A potential role for SIRT1.

Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent Km), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH2O2) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH2O2, protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated.

CD38-NADase is a new major contributor to Duchenne muscular dystrophic phenotype.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration. Two important deleterious features are a Ca2+ dysregulation linked to Ca2+ influxes associated with ryanodine receptor hyperactivation, and a muscular nicotinamide adenine dinucleotide (NAD+ ) deficit. Here, we identified that deletion in mdx mice of CD38, a NAD+ glycohydrolase-producing modulators of Ca2+ signaling, led to a fully restored heart function and structure, with skeletal muscle performance improvements, associated with a reduction in inflammation and senescence markers. Muscle NAD+ levels were also fully restored, while the levels of the two main products of CD38, nicotinamide and ADP-ribose, were reduced, in heart, diaphragm, and limb. In cardiomyocytes from mdx/CD38-/- mice, the pathological spontaneous Ca2+ activity was reduced, as well as in myotubes from DMD patients treated with isatuximab (SARCLISA® ) a monoclonal anti-CD38 antibody. Finally, treatment of mdx and utrophin-dystrophin-deficient (mdx/utr-/- ) mice with CD38 inhibitors resulted in improved skeletal muscle performances. Thus, we demonstrate that CD38 actively contributes to DMD physiopathology. We propose that a selective anti-CD38 therapeutic intervention could be highly relevant to develop for DMD patients.

Impacts of a high-fat diet on the metabolic profile and the phenotype of atrial myocardium in mice.

AIMS: Obesity, diabetes, and metabolic syndromes are risk factors of atrial fibrillation (AF). We tested the hypothesis that metabolic disorders have a direct impact on the atria favouring the formation of the substrate of AF. METHODS AND RESULTS: Untargeted metabolomic and lipidomic analysis was used to investigate the consequences of a prolonged high-fat diet (HFD) on mouse atria. Atrial properties were characterized by measuring mitochondria respiration in saponin-permeabilized trabeculae, by recording action potential (AP) with glass microelectrodes in trabeculae and ionic currents in myocytes using the perforated configuration of patch clamp technique and by several immuno-histological and biochemical approaches. After 16 weeks of HFD, obesogenic mice showed a vulnerability to AF. The atrial myocardium acquired an adipogenic and inflammatory phenotypes. Metabolomic and lipidomic analysis revealed a profound transformation of atrial energy metabolism with a predominance of long-chain lipid accumulation and beta-oxidation activation in the obese mice. Mitochondria respiration showed an increased use of palmitoyl-CoA as energy substrate. APs were short duration and sensitive to the K-ATP-dependent channel inhibitor, whereas K-ATP current was enhanced in isolated atrial myocytes of obese mouse. CONCLUSION: HFD transforms energy metabolism, causes fat accumulation, and induces electrical remodelling of the atrial myocardium of mice that become vulnerable to AF.

Cardioprotective effects of α-cardiac actin on oxidative stress in a dilated cardiomyopathy mouse model.

The expression of α-cardiac actin, a major constituent of the cytoskeleton of cardiomyocytes, is dramatically decreased in a mouse model of dilated cardiomyopathy triggered by inducible cardiac-specific serum response factor (Srf) gene disruption that could mimic some forms of human dilated cardiomyopathy. To investigate the consequences of the maintenance of α-cardiac actin expression in this model, we developed a new transgenic mouse based on Cre/LoxP strategy, allowing together the induction of SRF loss and a compensatory expression of α-cardiac actin. Here, we report that maintenance of α-cardiac actin within cardiomyocytes temporally preserved cytoarchitecture from adverse cardiac remodeling through a positive impact on both structural and transcriptional levels. These protective effects were accompanied in vivo by the decrease of ROS generation and protein carbonylation and the downregulation of NADPH oxidases NOX2 and NOX4. We also show that ectopic expression of α-cardiac actin protects HEK293 cells against oxidative stress induced by H2 O2 . Oxidative stress plays an important role in the development of cardiac remodeling and contributes also to the pathogenesis of heart failure. Taken together, these findings indicate that α-cardiac actin could be involved in the regulation of oxidative stress that is a leading cause of adverse remodeling during dilated cardiomyopathy development.

Mitochondria: a central target for sex differences in pathologies.

It is increasingly acknowledged that a sex and gender specificity affects the occurrence, development, and consequence of a plethora of pathologies. Mitochondria are considered as the powerhouse of the cell because they produce the majority of energy-rich phosphate bonds in the form of adenosine tri-phosphate (ATP) but they also participate in many other functions like steroid hormone synthesis, reactive oxygen species (ROS) production, ionic regulation, and cell death. Adequate cellular energy supply and survival depend on mitochondrial life cycle, a process involving mitochondrial biogenesis, dynamics, and quality control via mitophagy. It appears that mitochondria are the place of marked sexual dimorphism involving mainly oxidative capacities, calcium handling, and resistance to oxidative stress. In turn, sex hormones regulate mitochondrial function and biogenesis. Mutations in genes encoding mitochondrial proteins are the origin of serious mitochondrial genetic diseases. Mitochondrial dysfunction is also an important parameter for a large panel of pathologies including neuromuscular disorders, encephalopathies, cardiovascular diseases (CVDs), metabolic disorders, neuropathies, renal dysfunction etc. Many of these pathologies present sex/gender specificity. Here we review the sexual dimorphism of mitochondria from different tissues and how this dimorphism takes part in the sex specificity of important pathologies mainly CVDs and neurological disorders.

Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer.

Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In order to identify the pathways involved in the physical activity-mediated rescue of skeletal muscle mass and function, we investigated the effects of voluntary exercise on cachexia in colon carcinoma (C26)-bearing mice. Voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the autophagic flux is overloaded in skeletal muscle of both colon carcinoma murine models and patients, but not in running C26-bearing mice, thus suggesting that exercise may release the autophagic flux and ultimately rescue muscle homeostasis. Treatment of C26-bearing mice with either AICAR or rapamycin, two drugs that trigger the autophagic flux, also rescued muscle mass and prevented atrogene induction. Similar effects were reproduced on myotubes in vitro, which displayed atrophy following exposure to C26-conditioned medium, a phenomenon that was rescued by AICAR or rapamycin treatment and relies on autophagosome-lysosome fusion (inhibited by chloroquine). Since AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia, we believe autophagy-triggering drugs may be exploited to treat cachexia in conditions in which exercise cannot be prescribed.

Nicotinamide adenine dinucleotide homeostasis and signalling in heart disease: Pathophysiological implications and therapeutic potential.

Heart failure is a highly morbid syndrome generating enormous socio-economic costs. The failing heart is characterized by a state of deficient bioenergetics that is not currently addressed by classical clinical approaches. Nicotinamide adenine dinucleotide (NAD(+)/NADH) is a major coenzyme for oxidoreduction reactions in energy metabolism; it has recently emerged as a signalling molecule with a broad range of activities, ranging from calcium (Ca(2+)) signalling (CD38 ectoenzyme) to the epigenetic regulation of gene expression involved in the oxidative stress response, catabolic metabolism and mitochondrial biogenesis (sirtuins, poly[adenosine diphosphate-ribose] polymerases [PARPs]). Here, we review current knowledge regarding alterations to myocardial NAD homeostasis that have been observed in various models of heart failure, and their effect on mitochondrial functions, Ca(2+), sirtuin and PARP signalling. We highlight the therapeutic approaches that are currently in use or in development, which inhibit or stimulate NAD(+)-consuming enzymes, and emerging approaches aimed at stimulating NAD biosynthesis in the failing heart.

CTIP2 is a negative regulator of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the ß-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.

SRF selectively controls tip cell invasive behavior in angiogenesis.

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Locally expressed IGF1 propeptide improves mouse heart function in induced dilated cardiomyopathy by blocking myocardial fibrosis and SRF-dependent CTGF induction.

Cardiac fibrosis is critically involved in the adverse remodeling accompanying dilated cardiomyopathies (DCMs), which leads to cardiac dysfunction and heart failure (HF). Connective tissue growth factor (CTGF), a profibrotic cytokine, plays a key role in this deleterious process. Some beneficial effects of IGF1 on cardiomyopathy have been described, but its potential role in improving DCM is less well characterized. We investigated the consequences of expressing a cardiac-specific transgene encoding locally acting IGF1 propeptide (muscle-produced IGF1; mIGF1) on disease progression in a mouse model of DCM [cardiac-specific and inducible serum response factor (SRF) gene disruption] that mimics some forms of human DCM. Cardiac-specific mIGF1 expression substantially extended the lifespan of SRF mutant mice, markedly improved cardiac functions, and delayed both DCM and HF. These protective effects were accompanied by an overall improvement in cardiomyocyte architecture and a massive reduction of myocardial fibrosis with a concomitant amelioration of inflammation. At least some of the beneficial effects of mIGF1 transgene expression were due to mIGF1 counteracting the strong increase in CTGF expression within cardiomyocytes caused by SRF deficiency, resulting in the blockade of fibroblast proliferation and related myocardial fibrosis. These findings demonstrate that SRF plays a key role in the modulation of cardiac fibrosis through repression of cardiomyocyte CTGF expression in a paracrine fashion. They also explain how impaired SRF function observed in human HF promotes fibrosis and adverse cardiac remodeling. Locally acting mIGF1 efficiently protects the myocardium from these adverse processes, and might thus represent a therapeutic avenue to counter DCM.

Serum response factor is required for sprouting angiogenesis and vascular integrity.

Serum response factor (SRF) is a transcription factor that controls the expression of cytoskeletal proteins and immediate early genes in different cell types. Here, we found that SRF expression is restricted to endothelial cells (ECs) of small vessels such as capillaries in the mouse embryo. EC-specific Srf deletion led to aneurysms and hemorrhages from 11.5 days of mouse development (E11.5) and lethality at E14.5. Mutant embryos presented a reduced capillary density and defects in EC migration, with fewer numbers of filopodia in tip cells and ECs showing defects in actin polymerization and intercellular junctions. We show that SRF is essential for the expression of VE-cadherin and beta-actin in ECs both in vivo and in vitro. Moreover, knockdown of SRF in ECs impaired VEGF- and FGF-induced in vitro angiogenesis. Taken together, our results demonstrate that SRF plays an important role in sprouting angiogenesis and small vessel integrity in the mouse embryo.

Inducible mouse model of chronic intestinal pseudo-obstruction by smooth muscle-specific inactivation of the SRF gene.

BACKGROUND & AIMS: Serum response factor (SRF) regulates the expression of muscle genes and immediate early genes. We investigated the consequences of inactivating this transcription factor SRF in adult gastrointestinal smooth muscle cells. METHODS: SRF-floxed mice were crossed with SM-CreER(T2)(ki) mice expressing a tamoxifen-inducible recombinase in smooth muscle cells. Tamoxifen was injected into 12-week-old animals to activate the CreER(T2) and excise the SRF gene. RESULTS: SRF was down-regulated in the smooth muscle cells of the gastrointestinal tract, urinary bladder, and aorta. The mutant mice developed severe dilation of the intestinal tract associated with food stasis and air-fluid levels in the lumen 13 days after tamoxifen treatment. Mutant mice displayed cachexia and died between days 13 and 22. The dilation was associated with a thinning of the muscularis propria and was also observed in the urinary bladder. Ex vivo colonic contraction induced by electric field stimulation and carbachol was impaired in the mutant mice before the occurrence of the dilation phenotype. The expression of several genes, including those encoding smooth muscle actin, the heavy chain of smooth muscle myosin, and smoothelin, was 60% to 70% lower in mutants than in controls, and mutants also had a lower F/G actin ratio. CONCLUSIONS: SRF plays a central role in maintaining visceral smooth muscle contractile function in adults. Mice with a smooth muscle cell-specific SRF mutation develop a severe motility disorder resembling chronic intestinal pseudo-obstruction in humans and may be used as an inducible model of this disorder.

Diethylstilbestrol exposure in utero: a paradigm for mechanisms leading to adult disease.

The synthetic estrogen diethylstilbestrol (DES) was administered to pregnant women between the 1940s and the mid-1970s and is believed to be responsible for numerous uterine/cervical/vaginal malformations and cancers that appeared after birth and in young adult life. This medical tragedy has served as one of the prototypical examples of a phenomenon known as "endocrine disruption," in which either environmental agents or other compounds disrupt normal hormonal signaling in the body. Whereas DES signals through estrogen receptors, the subsequent molecular targets were largely unknown. We had identified Wnt7a as a target in this pathway and have used genetic analyses of mutant mice to demonstrate that disruption of Wnt7a is the key event leading to the DES phenotypes and cancers. We find that Wnt7a expression is only transiently deregulated in response to DES exposure, leading to the conclusion that critical events during early reproductive tract development results in a permanent change or "reprogramming" in subsequent development.

SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells.

Recent evidence infers a contribution of smooth muscle cell (SMC) progenitors and stromal cell-derived factor (SDF)-1alpha to neointima formation after arterial injury. Inhibition of plaque area and SMC content in apolipoprotein E-deficient mice repopulated with LacZ+ or CXCR4-/- BM or lentiviral transfer of an antagonist reveals a crucial involvement of local SDF-1alpha and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1alpha expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1alpha binds to platelets at the site of injury, triggers CXCR4- and P-selectin-dependent arrest of progenitor cells on injured arteries or matrix-adherent platelets, preferentially mobilizes and recruits c-kit-/platelet-derived growth factor receptor (PDGFR)-beta+/lineage-/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1alpha/CXCR4 axis is pivotal for vascular remodeling by recruiting a subset of SMC progenitors in response to apoptosis and in concert with platelets, epitomizing its importance for tissue repair and identifying a prime target to limit lesion development.

Wnt5a is required for proper epithelial-mesenchymal interactions in the uterus.

Epithelial-mesenchymal interactions play a crucial role in the correct patterning of the mammalian female reproductive tract (FRT). Three members of the Wnt family of growth factors are expressed at high levels in the developing FRT in the mouse embryo. The expression of Wnt genes is maintained in the adult FRT, although levels fluctuate during estrous. Wnt4 is required for Müllerian duct initiation, whereas Wnt7a is required for subsequent differentiation. In this study, we show that Wnt5a is required for posterior growth of the FRT. We further demonstrate that the mutant FRT has the potential to form the posterior compartments of the FRT using grafting techniques. Postnatally, Wnt5a plays a crucial role in the generation of uterine glands and is required for cellular and molecular responses to exogenous estrogens. Finally, we show that Wnt5a participates in a regulatory loop with other FRT patterning genes including Wnt7a, Hoxa10 and Hoxa11. Data presented provide a mechanistic basis for how uterine stroma mediates both developmental and estrogen-mediated changes in the epithelium and demonstrates that Wnt5a is a key component in this process. The similarities of the Wnt5a and Wnt7a mutant FRT phenotypes to those described for the Hoxa11 and Hoxa13 mutant FRT phenotypes reveal a mechanism whereby Wnt and Hox genes cooperate to pattern the FRT along the anteroposterior axis.

The receptor tyrosine kinase regulator Sprouty1 is a target of the tumor suppressor WT1 and important for kidney development.

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.