Nonenzymatic lipid mediators, neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid.

Neuroprostanes are lipid mediators produced by nonenzymatic free radical peroxidation of docosahexaenoic acid (DHA). DHA is associated with a lower atherosclerosis risk, suggesting a beneficial role in cardiovascular diseases. The aim of this study was to investigate the influence of DHA peroxidation on its potentially antiarrhythmic properties (AAP) in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted mice. Calcium imaging and biochemical experiments indicate that cardiac arrhythmias induced by isoproterenol are associated with Ca(2+) leak from the sarcoplasmic reticulum after oxidation and phosphorylation of the type 2 ryanodine receptor (RyR2) leading to dissociation of the FKBP12.6/RyR2 complex. Both oxidized DHA and 4(RS)-4-F4t-NeuroP prevented cellular arrhythmias and posttranslational modifications of the RyR2 leading to a stabilized FKBP12.6/RyR2 complex. DHA per se did not have AAP. The AAP of 4(RS)-4-F4t-NeuroP was also observed in vivo. In this study, we challenged the paradigm that spontaneously formed oxygenated metabolites of lipids are undesirable as they are unconditionally toxic. This study reveals that the lipid mediator 4(RS)-4-F4t-neuroprostane derived from nonenzymatic peroxidation of docosahexaenoic acid can counteract such deleterious effects through cardiac antiarrhythmic properties. Our findings demonstrate 4(RS)-4-F4t-NeuroP as a mediator of the cardioprotective AAP of DHA. This discovery opens new perspectives for products of nonenzymatic oxidized ω3 polyunsaturated fatty acids as potent mediators in diseases that involve ryanodine complex destabilization such as ischemic events.

Resveratrol protects against protease inhibitor-induced reactive oxygen species production, reticulum stress and lipid raft perturbation.

OBJECTIVE: HIV protease inhibitors have been successfully used in highly active antiretroviral therapy of HIV-1 infection, but their benefits are compromised by a number of clinically important adverse side-effects. Several studies showed that protease inhibitors induce sarco/endoplasmic reticulum stress and overproduction of reactive oxygen species (ROS), but the hierarchy of these events was never established in protease inhibitor-treated cells. Our objective was to determine whether ROS production and sarco/endoplasmic reticulum stress were co-induced by protease inhibitors in human primary skeletal myotubes and whether antioxidant treatment with resveratrol could protect against protease inhibitor-induced cellular damages. DESIGN AND METHODS: We analyzed in cultures of primary human skeletal myotubes the effects of four protease inhibitors (atazanavir, lopinavir, ritonavir and saquinavir) on ROS production (by measuring the reduction of nitro blue tetrazolium and by fluorescence microscopy using CM-H2DCFDA), on sarco/endoplasmic reticulum stress induction (by immunofluorescence and electron microscopy) and on the expression and localization at lipid rafts of Caveolin 3 and Flotillin 1, two major components of lipid rafts (by immunoblotting and biochemical experiments). Cells were co-incubated with resveratrol to assess its protective effects. RESULTS: In myotubes, protease inhibitors increased ROS production, altered sarco/endoplasmic reticulum morphology, increased expression of C/EBP homologous protein, a sarco/endoplasmic reticulum stress marker, and decreased expression and localization at lipid rafts of Caveolin 3 and Flotillin 1. Importantly, these deleterious protease inhibitor effects were inhibited by the antioxidant resveratrol. CONCLUSION: Our findings demonstrate a protective effect of resveratrol against protease inhibitor-induced sarco/endoplasmic reticulum stress in human myotubes.

TC10 controls human myofibril organization and is activated by the sarcomeric RhoGEF obscurin.

The contractile activity of striated muscle depends on myofibrils that are highly ordered macromolecular complexes. The protein components of myofibrils are well characterized, but it remains largely unclear how signaling at the molecular level within the sarcomere and the control of assembly are coordinated. We show that the Rho GTPase TC10 appears during differentiation of human primary skeletal myoblasts and it is active in differentiated myotubes. We identify obscurin, a sarcomere-associated protein, as a specific activator of TC10. Indeed, TC10 binds directly to obscurin via its predicted RhoGEF motif. Importantly, we demonstrate that obscurin is a specific activator of TC10 but not the Rho GTPases Rac and Cdc42. Finally, we show that inhibition of TC10 activity by expression of a dominant-negative mutant or its knockdown by expression of specific shRNA block myofibril assembly. Our findings reveal a novel signaling pathway in human skeletal muscle that involves obscurin and the Rho GTPase TC10 and implicate this pathway in new sarcomere formation.

Exploring the FL-160-CRP gene family through sequence variability of the complement regulatory protein (CRP) expressed by the trypomastigote stage of Trypanosoma cruzi.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host-parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.