Mathias Mericskay

SDF-1α/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)-1&agr; 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-1&agr; and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1&; expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1&agr; 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)-&bgr;+/lineage−/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1&agr;/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.

Temporally Controlled Onset of Dilated Cardiomyopathy Through Disruption of the SRF Gene in Adult Heart

Background— Serum response factor (SRF) is a cardiac transcription factor involved in cell growth and differentiation. We have shown, using the Cre/loxP system, that cardiac-specific disruption of SRF gene in the embryonic heart results in lethal cardiac defects. The role of SRF in adult heart is unknown. Methods and Results— We disrupted SRF in the adult heart using a heart-specific tamoxifen-inducible Cre recombinase. This disruption led to impaired left ventricular function with reduced contractility, subsequently progressing to dilated cardiomyopathy, as demonstrated by serial echocardiography, including tissue Doppler imaging. The cytoarchitecture of cardiomyocytes was altered in the intercalated disks. All mutant mice died from heart failure 10 weeks after treatment. These functional and structural defects were preceded by early alterations in the cardiac gene expression program: major decreases in mRNA levels for cardiac &agr;-actin, muscle creatine kinase, and calcium-handling genes. Conclusions— SRF is crucial for adult cardiac function and integrity. We suggest that the rapid progression to heart failure in SRF mutant mice results primarily from decreased expression of proteins involved in force generation and transmission, low levels of polymerized actin, and changes in cytoarchitecture, without hypertrophic compensation. These cardiac-specific SRF-deficient mice have the morphological and clinical features of acquired dilated cardiomyopathy in humans and may therefore be used as an inducible model of this disorder.

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.

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 β-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.

Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

Alterations in the balance of cytoskeleton as well as energetic proteins are involved in the cardiac remodeling occurring in dilated cardiomyopathy (DCM). We used two-dimensional DIGE proteomics as a discovery approach to identify key molecular changes taking place in a temporally controlled model of DCM triggered by cardiomyocyte-specific serum response factor (SRF) knock-out in mice. We identified muscle creatine kinase (MCK) as the primary down-regulated protein followed by α-actin and α-tropomyosin down-regulation leading to a decrease of polymerized F-actin. The early response to these defects was an increase in the amount of desmin intermediate filaments and phosphorylation of the αB-crystallin chaperone. We found that αB-crystallin and desmin progressively lose their striated pattern and accumulate at the intercalated disk and the sarcolemma, respectively. We further show that desmin is a preferential target of advanced glycation end products (AGE) in mouse and human DCM. Inhibition of CK in cultured cardiomyocytes is sufficient to recapitulate both the actin depolymerization defect and the modification of desmin by AGE. Treatment with either cytochalasin D or glyoxal, a cellular AGE, indicated that both actin depolymerization and AGE contribute to desmin disorganization. Heat shock-induced phosphorylation of αB-crystallin provides a transient protection of desmin against glyoxal in a p38 MAPK-dependent manner. Our results show that the strong down-regulation of MCK activity contributes to F-actin instability and induces post-translational modification of αB-crystallin and desmin. Our results suggest that AGE may play an important role in DCM because they alter the organization of desmin filaments that normally support stress response and mitochondrial functions in cardiomyocytes.

An SRF/miR-1 axis regulates NCX1 and Annexin A5 protein levels in the normal and failing heart

AIMS The expression of the sodium/calcium exchanger NCX1 increases during cardiac hypertrophy and heart failure, playing an important role in Ca(2+) extrusion. This increase is presumed to result from stress signalling induced changes in the interplay between transcriptional and post-transcriptional regulations. We aimed to determine the impact of the SRF transcription factor known to regulate the NCX1 promoter and microRNA genes, on the expression of NCX1 mRNA and protein and annexin A5 (AnxA5), a Ca(2+)-binding protein interacting with NCX1 and increased during HF. METHODS AND RESULTS NCX1 mRNA was decreased while the protein was increased in the failing heart of the cardiomyocyte-restricted SRF knock-out mice (SRF(HKO)). The induction of NCX1 mRNA by the pro-hypertrophic drug phenylephrine observed in control mice was abolished in the SRF(HKO) though the protein was strongly increased. AnxA5 protein expression profile paralleled the expression of NCX1 protein in the SRF(HKO). MiR-1, a microRNA regulated by SRF, was decreased in the SRF(HKO) and repressed by phenylephrine. In vitro and in vivo manipulation of miR-1 levels and site-directed mutagenesis showed that NCX1 and AnxA5 mRNAs are targets of miR-1. AnxA5 overexpression slowed down Ca(2+) extrusion during caffeine application in adult rat cardiomyocytes. CONCLUSION Our study reveals the existence of a complex regulatory loop where SRF regulates the transcription of NCX1 and miR-1, which in turn functions as a rheostat limiting the translation of NCX1 and AnxA5 proteins. The decrease of miR-1 and increase of AnxA5 appear as important modulators of NCX1 expression and activity during heart failure.

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

SUMMARY 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.

Inactivation of Serum Response Factor Contributes To Decrease Vascular Muscular Tone and Arterial Stiffness in Mice

Rationale: Vascular smooth muscle (SM) cell phenotypic modulation plays an important role in arterial stiffening associated with aging. Serum response factor (SRF) is a major transcription factor regulating SM genes involved in maintenance of the contractile state of vascular SM cells. Objective: We investigated whether SRF and its target genes regulate intrinsic SM tone and thereby arterial stiffness. Methods and Results: The SRF gene was inactivated SM-specific knockout of SRF (SRFSMKO) specifically in vascular SM cells by injection of tamoxifen into adult transgenic mice. Fifteen days later, arterial pressure and carotid thickness were lower in SRFSMKO than in control mice. The carotid distensibility/pressure and elastic modulus/wall stress curves showed a greater arterial elasticity in SRFSMKO without modification in collagen/elastin ratio. In SRFSMKO, vasodilation was decreased in aorta and carotid arteries, whereas a decrease in contractile response was found in mesenteric arteries. By contrast, in mice with inducible SRF overexpression, the in vitro contractile response was significantly increased in all arteries. Without endothelium, the contraction was reduced in SRFSMKO compared with control aortic rings owing to impairment of the NO pathway. Contractile components (SM-actin and myosin light chain), regulators of the contractile response (myosin light chain kinase, myosin phosphatase target subunit 1, and protein kinase C–potentiated myosin phosphatase inhibitor) and integrins were reduced in SRFSMKO. Conclusions: SRF controls vasoconstriction in mesenteric arteries via vascular SM cell phenotypic modulation linked to changes in contractile protein gene expression. SRF-related decreases in vasomotor tone and cell-matrix attachment increase arterial elasticity in large arteries.

Posttranslational modifications of desmin and their implication in biological processes and pathologies.

Desmin, the muscle-specific intermediate filament, is involved in myofibrillar myopathies, dilated cardiomyopathy and muscle wasting. Desmin is the target of posttranslational modifications (PTMs) such as phosphorylation, ADP-ribosylation and ubiquitylation as well as nonenzymatic modifications such as glycation, oxidation and nitration. Several PTM target residues and their corresponding modifying enzymes have been discovered in human and nonhuman desmin. The major effect of phosphorylation and ADP-ribosylation is the disassembly of desmin filaments, while ubiquitylation of desmin leads to its degradation. The regulation of the desmin filament network by phosphorylation and ADP-ribosylation was found to be implicated in several major biological processes such as myogenesis, myoblast fusion, muscle contraction, muscle atrophy, cell division and possibly desmin interactions with its binding partners. Phosphorylation of desmin is also implicated in many forms of desmin-related myopathies (desminopathies). In this review, we summarize the findings on desmin PTMs and their implication in biological processes and pathologies, and discuss the current knowledge on the regulation of the desmin network by PTMs. We conclude that the desmin filament network can be seen as an intricate scaffold for muscle cell structure and biological processes and that its dynamics can be affected by PTMs. There are now precise tools to investigate PTMs and visualize cellular structures that have been underexploited in the study of desminopathies. Future studies should focus on these aspects.

Mosaic inactivation of the serum response factor gene in the myocardium induces focal lesions and heart failure

Regional alterations in ventricular mechanical functions are a primary determinant for the risk of myocardial injuries in various cardiomyopathies. The serum response factor (SRF) is a transcription factor regulating contractile and cytoskeletal genes and may play an important role in the remodelling of myocardium at the cellular level.