Rapamycin treatment unmasks a sex-specific pattern of scar expansion of the infarcted rat heart: The relationship between mTOR and KATP channel.

Inhibition of the mammalian target of rapamycin (mTOR) with the macrolide rapamycin or pharmacological suppression of KATP channel opening translated to scar expansion of the myocardial infarcted (MI) adult female rodent heart. The present study tested the hypotheses that rapamycin-mediated scar expansion was sex-specific and that mTOR signaling directly influenced KATP channel subunit expression/activity. Scar size was significantly larger in post-MI male rats as compared to the previous data reported in post-MI female rats. The reported scar expansion of rapamycin-treated post-MI female rats was not observed following the administration of the macrolide to post-MI male rats. Protein levels of the KATP channel subunits Kir6.2 and SUR2A and phosphorylation of the serine2448 residue of mTOR were similar in the normal heart of adult male and female rats. By contrast, greater tuberin inactivation characterized by the increased phosphorylation of the threonine1462 residue and reduced raptor protein levels were identified in the normal heart of adult female rats. Rapamycin pretreatment of phorbol 12,13-dibutyrate (PDBu)-treated neonatal rat ventricular cardiomyocytes (NNVMs) suppressed hypertrophy, inhibited p70S6K phosphorylation, and attenuated SUR2A protein upregulation. In the presence of low ATP levels, KATP channel activity detected in untreated NNVMs was significantly attenuated in PDBu-induced hypertrophied NNVMs via a rapamycin-independent pathway. Thus, rapamycin administration to post-MI rats unmasked a sex-specific pattern of scar expansion and mTOR signaling in PDBu-induced hypertrophied NNVMs significantly increased SUR2A protein levels. However, the biological advantage associated with SUR2A protein upregulation was partially offset by an mTOR-independent pathway that attenuated KATP channel activity in PDBu-induced hypertrophied NNVMs.

Regulation of PTP1B activation through disruption of redox-complex formation.

We have identified a molecular interaction between the reversibly oxidized form of protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ζ that regulates PTP1B activity. Destabilizing the transient interaction between 14-3-3ζ and PTP1B prevented PTP1B inactivation by reactive oxygen species and decreased epidermal growth factor receptor phosphorylation. Our data suggest that destabilizing the interaction between 14-3-3ζ and the reversibly oxidized and inactive form of PTP1B may establish a path to PTP1B activation in cells.

Filamentous nestin and nonmuscle myosin IIB are associated with a migratory phenotype in neonatal rat cardiomyocytes.

Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/ß mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.

SPECT imaging of pulmonary vascular disease in bleomycin-induced lung fibrosis using a vascular endothelium tracer.

BACKGROUND: Pulmonary hypertension (PH) complicating idiopathic pulmonary fibrosis (IPF) is associated to worse outcome. There is a great need for a non-invasive diagnostic modality to detect and evaluate the severity of pulmonary vascular disease (PVD). 99mTc-PulmoBind is a novel imaging agent that binds to the adrenomedullin (AM) receptor on the pulmonary microvascular endothelium. SPECT imaging employing the endothelial cell tracer 99mTc-PulmoBind was used to assess PVD associated with lung fibrosis. METHODS: Rats with selective right lung bleomycin-induced fibrosis were compared to control rats. SPECT imaging was performed after three weeks with 99mTc-PulmoBind and 99mTc-macroaggregates of albumin (MAA). PH and right ventricular (RV) function were assessed by echocardiography. Lung perfusion was evaluated by fluorescent microangiography. Lung AM receptor expression was measured by qPCR and by immunohistology. Relevance to human IPF was explored by measuring AM receptor expression in lung biopsies from IPF patients and healthy controls. RESULTS: The bleomycin group developed preferential right lung fibrosis with remodeling and reduced perfusion as assessed with fluorescent microangiography. These rats developed PH with RV hypertrophy and dysfunction. 99mTc-PulmoBind uptake was selectively reduced by 50% in the right lung and associated with reduced AM receptor expression, PH and RV hypertrophy. AM receptor was co-expressed with the endothelial cell protein CD31 in alveolar capillaries, and markedly reduced after bleomycin. Quantitative dynamic analysis of 99mTc-PulmoBind uptake in comparison to 99mTc-MAA revealed that the latter distributed only according to flow, with about 60% increased left lung uptake while left lung uptake of 99mTc-PulmoBind was not affected. Lung from human IPF patients showed important reduction in AM receptor expression closely associated with CD31. CONCLUSIONS: SPECT imaging with 99mTc-PulmoBind detects PVD and its severity in bleomycin-induced lung fibrosis. Reduced AM receptor expression in human IPF supports further clinical development of this imaging approach.

Structural dysregulation of the pulmonary autograft was associated with a greater density of p16INK4A-vascular smooth muscle cells.

The present study tested the hypothesis that a senescent phenotype of vascular smooth muscle cells (VSMCs) may represent the seminal event linked to maladaptive pulmonary autograft remodeling of a small number of patients that underwent the Ross procedure. The diameter of the pulmonary autograft (47±4 mm) of three male patients was significantly greater compared to the pulmonary artery (26±1 mm) excised from bicuspid aortic valve (BAV) patients. The pulmonary autograft was associated with a neointimal region and the adjacent medial region was significantly thinner compared to the pulmonary artery of BAV patients. Structural dysregulation was evident as elastin content of the medial region was significantly reduced in the pulmonary autograft compared to the pulmonary artery of BAV patients. By contrast, collagen content of the medial region of the pulmonary autograft and the pulmonary artery of BAV patients was not significantly different. Reduced medial elastin content of the pulmonary autograft was associated with increased protein levels of matrix metalloproteinase-9. The latter phenotype was not attributed to a robust inflammatory response as the percentage of Mac-2(+)-infiltrating monocytes/macrophages was similar between groups. A senescent phenotype was identified as protein levels of the cell cycle inhibitor p27kip1 were upregulated and the density of p16INK4A/non-muscle myosin IIB(+)-VSMCs was significantly greater in the pulmonary autograft compared to the pulmonary artery of BAV patients. Thus, senescent VSMCs may represent the predominant cellular source of increased matrix metalloproteinase-9 protein expression translating to maladaptive pulmonary autograft remodeling characterized by elastin degradation, medial thinning and neointimal formation.

The ascending aorta of male hypertensive bicuspid aortic valve patients preferentially associated with a cellular aneurysmal phenotype.

Male sex and hypertension represent risk factors in the progression of an aortic aneurysm. The present study examined the morphological/cellular phenotype of the ascending aorta (AA) of male and female patients diagnosed with a bicuspid aortic valve (BAV) to test the hypothesis that hypertension-induced remodeling of male BAV patients preferentially recapitulated the expression of a panel of proteins favoring aneurysm formation. The diameter of the AA of hypertensive male (35 ± 6 mm) and female (39 ± 5 mm) BAV patients was comparable to normotensive patients reflecting an early phase of vessel expansion. Morphological/structural remodeling of the medial region of the AA of male normotensive and hypertensive BAV patients were comparable. Protein levels of non-muscle myosin IIB, the cell cycle inhibitor p27kip1, tumor suppressor p53 and matrix metalloproteinase-2 and -9 were significantly upregulated in the AA of male hypertensive BAV patients. In female hypertensive BAV patients, collagen content was significantly increased whereas elastin content and medial width of the AA were similar to normotensive BAV patients. In the AA of female hypertensive BAV patients, matrix metalloproteinase-9 and p27kip1 protein levels were unchanged whereas p53 and matrix metalloproteinase-2 protein expression was significantly reduced. Nestin protein levels were diminished in the AA of male and female hypertensive BAV patients. Thus, sexual dimorphic remodeling of the AA was prevalent in hypertensive BAV patients. Moreover, during the early phase of vessel expansion, the AA of male hypertensive BAV patients was preferentially associated with the upregulation of a panel of proteins linked to progressive dilatation and potential aneurysm formation.

Protein tyrosine phosphatase 1B regulates miR-208b-argonaute 2 association and thyroid hormone responsiveness in cardiac hypertrophy.

Increased production of reactive oxygen species plays an essential role in the pathogenesis of several diseases, including cardiac hypertrophy. In our search to identify redox-sensitive targets that contribute to redox signaling, we found that protein tyrosine phosphatase 1B (PTP1B) was reversibly oxidized and inactivated in hearts undergoing hypertrophy. Cardiomyocyte-specific deletion of PTP1B in mice (PTP1B cKO mice) caused a hypertrophic phenotype that was exacerbated by pressure overload. Furthermore, we showed that argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, was a substrate of PTP1B in cardiomyocytes and in the heart. Our results revealed that phosphorylation at Tyr393 and inactivation of AGO2 in PTP1B cKO mice prevented miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1; also known as MED13) and contributed to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3) with propylthiouracil rescued pressure overload-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity is cardioprotective and that redox signaling is linked to thyroid hormone responsiveness and microRNA-mediated gene silencing in pathological hypertrophy.

Distinct Expression of Nonmuscle Myosin IIB in Pulmonary Arteries of Patients With Aortic Stenosis vs Insufficiency Undergoing a Ross Procedure.

BACKGROUND: Clinical studies have revealed a greater risk of pulmonary autograft dilation after the Ross procedure in patients with preoperative aortic insufficiency (AI). The present study examined whether the morphologic, biomechanical, and cellular properties of the pulmonary artery (PA) from patients with AI were phenotypically different compared with patients diagnosed with aortic stenosis (AS). METHODS: PA segments were harvested from patients undergoing the Ross procedure for AS (n = 16) and AI (n = 6). Preoperative aortic annulus was significantly larger (P < 0.05) in patients with AI (28.5 ± 1.8 mm) vs AS (22.8 ± 1.2 mm). Morphologic, biomechanical, and cellular phenotypes of the PA were analyzed. RESULTS: Collagen and elastin content in the media of the PA wall were similar in patients with AS and AI. Elastic modulus and energy loss of the PA were not significantly different between the groups. In the media of the PA, expression of a panel of vascular smooth muscle cell-specific proteins were similar in patients with AS and AI. In contrast, nonmuscle myosin IIB protein levels in the PA of AS patients were significantly higher compared with AI patients, and immunofluorescence identified staining in α-smooth muscle actin-positive vascular smooth muscle cells. CONCLUSIONS: Despite similar morphological and biomechanical properties, the disparate expression of nonmuscle myosin IIB protein distinguishes the PA of patients with AI from patients with AS. The biological role in vascular smooth muscle cells and the potential contribution of nonmuscle myosin IIB to pulmonary autograft dilation in a subset of AI patients after the Ross procedure remain to be determined.

p38α MAPK inhibition translates to cell cycle re-entry of neonatal rat ventricular cardiomyocytes and de novo nestin expression in response to thrombin and after apex resection.

The present study tested the hypothesis that p38α MAPK inhibition leads to cell cycle re-entry of neonatal ventricular cardiomyocytes (NNVMs) and de novo nestin expression in response to thrombin and after apex resection of the neonatal rat heart. Thrombin (1 U/ml) treatment of 1-day old NNVMs did not induce cell cycle re-entry or nestin expression. Acute exposure of NNVMs to thrombin increased p38α MAPK and HSP27 phosphorylation and p38α/ß MAPK inhibitor SB203580 abrogated HSP27 phosphorylation. Thrombin and SB203580 co-treatment of NNVMs led to bromodeoxyuridine incorporation and nestin expression. SB203580 (5 mg/kg) administration immediately after apex resection of 1-day old neonatal rat hearts and continued for two additional days shortened the fibrin clot length sealing the exposed left ventricular chamber. SB203580-treatment increased the density of troponin-T(+)-NNVMs that incorporated bromodeoxyuridine and expressed nuclear phosphohistone-3. Nestin(+)-NNVMs were selectively detected at the border of the fibrin clot and SB203580 potentiated the density that re-entered the cell cycle. These data suggest that the greater density of ventricular cardiomyocytes and nestin(+)-ventricular cardiomyocytes that re-entered the cell cycle after SB203580 treatment of the apex-resected neonatal rat heart during the acute phase of fibrin clot formation may be attributed in part to inhibition of thrombin-mediated p38α MAPK signalling.

N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties.

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of ß-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.

Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells.

Explaining naturalization and invasiveness: new insights from historical ornamental plant catalogs.

We identified plant attributes associated with naturalization and invasiveness using century-old ornamental plant catalogs from Québec (Canada). We tested the hypothesis that naturalization is determined by fewer factors than invasiveness, as the latter also requires dispersal, which introduces additional complexity. The approach we used took into account not only plant attributes as explanatory factors, but also propagule pressure, while accounting for phylogenetic relationships among species. Museum collections were used, in combination with scientific journal databases, to assess invasiveness. Particular attention was given to species that never escaped from gardens and thus represent cases of "failed" invasions. Naturalization in cold-temperate environments is determined by fewer factors than invasion, but only if phylogenetic links between species are taken into account, highlighting the importance of phylogenetic tools for analyzing species pools not resulting from a random selection of taxa. Hardiness is the main factor explaining naturalization in Québec. Invasion requires dispersal, as shown by three significant variables associated with the spread of diaspores in the invasiveness model (seed weight, hydrochory, number of seed dispersal modes). Plants that are not cold-hardy are likely to disappear from the market or nature, but the disappearance phenomenon is more complex, involving also seed dispersal abilities and propagule pressure. Factors contributing to naturalization or invasiveness may differ greatly between regions. Differences are due in part to the plant traits used in the models and the methodology. However, this study, conducted in a cold-temperate region, sheds new light on what is likely a context (climatic)-dependant phenomenon.