Dual level of interactions between calcineurin and PKC-epsilon in cardiomyocyte stretch.

OBJECTIVES: Myocardial stretch activates a number of interconnected pathways including the protein kinase C (PKC) pathway that in turn activates mitogen activated protein kinases (MAPK), leading to gene expression stimulation and ventricular hypertrophy. A role of calcineurin has also been shown during hypertrophy. The goal of our study was to look for a possible interconnection between PKC and calcineurin in myocardial stretch. METHODS: Neonatal rat cardiomyocytes were cultured for 5 days and a 15% stretch was applied. Expression of MAPK and PKC-epsilon was evaluated by Western blot analysis. The specific role of PKC-epsilon was evaluated by transfection of cardiomyocytes with a specific inhibitor peptide. Calcineurin and PKC-epsilon complex formation and co-localization were evaluated by co-immunoprecipitation and by immunolocalization. RESULTS: The PKC isoform involved in stretch-induced ERK and JNK activations was PKC-epsilon. We show here that calcineurin is also found to be involved in the stretch response and that calcineurin and PKC-epsilon co-operate at 2 levels during stretch. First, stretch-induced translocation of PKC-epsilon from the cytosolic to the membrane fraction was inhibited by calcineurin inhibitors, indicating that calcineurin was necessary for PKC-epsilon activation induced by stretch. A second level of interaction was the formation of a calcineurin-PKC-epsilon complex, which increased during stretch. Immunofluorescent studies indicated that, after stretch, calcineurin and PKC-epsilon were co-localized at the level of the perinuclear membrane. These results may have a major relevance in vivo since we also found similar PKC-epsilon-calcineurin complexes in the phase of thoracic aortic stenosis in rats during which heart failure develops. CONCLUSION: Calcineurin appears to be necessary for stretch-induced PKC-epsilon activation after which the phosphatase and the kinase are co-localized in a complex at the level of the perinuclear membrane where they may finely regulate the phosphorylation of their target proteins.

Chronic infusion of bradykinin delays the progression of heart failure and preserves vascular endothelium-mediated vasodilation in conscious dogs.

BACKGROUND: This study examined the effects of chronic bradykinin infusion on hemodynamics and myocardial and endothelial functions during the development of heart failure. METHODS AND RESULTS: Sixteen instrumented dogs were randomized to receive through the left atria either vehicle or bradykinin (1 microg/min) during ventricular pacing (250 bpm, 5 weeks). Hemodynamic and left ventricular (LV) parameters and the vasodilator responses to intravenous acetylcholine (0.3 to 3 microg/kg) and nitroglycerin (1 to 10 microg/kg) were examined in the control and after 3 and 5 weeks of pacing. The expression of endothelial NOS in femoral, carotid, and renal arteries was determined by Western blot analysis. After 3 weeks of pacing, changes in LV diastolic and systolic parameters were significantly lower in bradykinin-treated than vehicle-treated dogs (LV end-diastolic pressure, +10+/-3 versus +19+/-2 mm Hg; time constant of LV isovolumic relaxation, +11+/-2 versus +17+/-1 ms; LV wall thickening, -33+/-18% versus -75+/-9%; and cardiac output, -16+/-6% versus -32+/-6%; all P<0.05). Compared with vehicle-treated dogs, bradykinin-treated dogs had a reduced rightward shift of the diastolic LV pressure-diameter relation and a reduced diastolic LV wall stress. Similar trends were observed after 5 weeks. The vasodilator response to nitroglycerin was preserved in both groups. The response to acetylcholine was blunted in vehicle-treated but preserved in bradykinin-treated dogs. Vascular endothelial NOS expression decreased in vehicle-treated but was preserved in bradykinin-treated dogs. CONCLUSIONS: In conscious dogs, chronic bradykinin infusion delays the heart failure progression by preserving LV diastolic and systolic functions and by preserving vascular endothelial function.

Intracerebral administration of adeno-associated viral vector serotype rh.10 carrying human SGSH and SUMF1 cDNAs in children with mucopolysaccharidosis type IIIA disease: results of a phase I/II trial.

Mucopolysaccharidosis type IIIA is a severe degenerative disease caused by an autosomal recessive defect of a gene encoding a lysosomal heparan-N-sulfamidase, the N-sulfoglycosamine sulfohydrolase (SGSH), the catalytic site of which is activated by a sulfatase-modifying factor (SUMF1). Four children (Patients 1-3, aged between 5.5 and 6 years; Patient 4 aged 2 years 8 months) received intracerebral injections of an adeno-associated viral vector serotype rh.10-SGSH-IRES-SUMF1 vector in a phase I/II clinical trial. All children were able to walk, but their cognitive abilities were abnormal and had declined (Patients 1-3). Patients 1-3 presented with brain atrophy. The therapeutic vector was delivered in a frameless stereotaxic device, at a dose of 7.2×10(11) viral genomes/patient simultaneously via 12 needles as deposits of 60 µl over a period of 2 hr. The vector was delivered bilaterally to the white matter anterior, medial, and posterior to the basal ganglia. Immunosuppressive treatment (mycophenolate mofetil and tacrolimus) was initiated 15 days before surgery and maintained for 8 weeks (mycophenolate mofetil) or throughout follow-up (tacrolimus, with progressive dose reduction) to prevent elimination of transduced cells. Safety data collected from inclusion, during the neurosurgery period and over the year of follow-up, showed good tolerance, absence of adverse events related to the injected product, no increase in the number of infectious events, and no biological sign of toxicity related to immunosuppressive drugs. Efficacy analysis was necessarily preliminary in this phase I/II trial on four children, in the absence of validated surrogate markers. Brain atrophy evaluated by magnetic resonance imaging seemed to be stable in Patients 1 and 3 but tended to increase in Patients 2 and 4. Neuropsychological evaluations suggested a possible although moderate improvement in behavior, attention, and sleep in Patients 1-3. The youngest patient was the most likely to display neurocognitive benefit.

The EGF receptor activates ERK but not JNK Ras-dependently in basal conditions but ERK and JNK activation pathways are predominantly Ras-independent during cardiomyocyte stretch.

Myocardial stretch is a major determinant of ventricular hypertrophy, a physiological adaptational process that can be detrimental, leading to heart failure. Therapies aimed to limit the development of cardiac hypertrophy are thus currently evaluated. Among possible targets, the small G protein Ras and the epidermal growth factor receptor (EGFR) have been shown to be involved during stretch but their precise role in the activation of the major actors of hypertrophy, the mitogen activated protein kinases (MAPK) ERK and JNK is not well known. Our goal was thus was to evaluate precisely the activation pathways of ERK and JNK during stretch, with an emphasis on the role of the EGFR. For this purpose, neonatal rat cardiomyocytes in culture were stretched for different time durations. As measured by Western blot of their phosphorylated forms, ERK and JNK were activated by stretch. Ras inhibition decreased basal ERK phosphorylation but had no effect on stretch-induced ERK activation. Under basal conditions, EGFR activated ERK in a classical Ras-dependent manner. Upon stretch, EGFR transactivation activated ERK through both Ras-dependent and Ras-independent pathways. Interestingly, we also show that the Akt pathway participates in stretch-induced ERK activation with an involvement of EGFR. Unlike ERK, JNK activation is independent of either EGFR or PI3 kinase but dependent on other tyrosine kinases. In conclusion these data show different Ras-dependent and Ras-independent pathways in basal conditions and during stretch with a previously unrecognized role of Akt in the activation of ERK.

Structural analysis of the bacterial HPr kinase/phosphorylase V267F mutant gives insights into the allosteric regulation mechanism of this bifunctional enzyme.

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.

Dietary long-chain n-3 fatty acids modify blood and cardiac phospholipids and reduce protein kinase-C-delta and protein kinase-C-epsilon translocation.

The effects of an n-3 PUFA-enriched diet on cardiac cell membrane phospholipid fraction compositions and associated protein kinase-C (PKC) translocation modification have never been studied in higher mammals. This is of importance since membrane fatty acid composition has been shown to influence PKC signalling pathways. In the present study, we have tested whether the incorporation of n-3 PUFA in cardiac membrane phospholipids correlated with changes in the fatty acid composition of diacylglycerols (DAG) and led to a differential translocation of PKC isoforms. Two groups of five dogs were fed the standard diet supplemented with palm oil or fish oil for 8 weeks. Dogs fed a fish oil-enriched diet showed a preferential incorporation of EPA and, to a lesser extent, of DHA, at the expense of arachidonic acid, in the circulating TAG, plasma phospholipids, erythrocyte phospholipids and cardiomyocyte phospholipid fractions. Analysis of 1,2-DAG fatty acid composition also indicated a preferential enrichment of EPA compared with DHA. Associated with these results, a reduction in the expression of PKC-delta and PKC-epsilon isoforms in the particulate fractions was observed whereas no effect was seen for PKC-alpha and PKC-zeta. We conclude that a fish oil-enriched diet induces a modification in fatty acid composition of cardiac membrane phospholipids, associated with a differential translocation of PKC isoforms. These results can be explained by the production of structurally different DAG that may participate in some of the protective effects of n-3 PUFA against various chronic diseases.