Moderate Exercise in Spontaneously Hypertensive Rats Is Unable to Activate the Expression of Genes Linked to Mitochondrial Dynamics and Biogenesis in Cardiomyocytes.

Hypertension (HTN) is a public health concern and a major preventable cause of cardiovascular disease (CVD). When uncontrolled, HTN may lead to adverse cardiac remodeling, left ventricular hypertrophy, and ultimately, heart failure. Regular aerobic exercise training exhibits blood pressure protective effects, improves myocardial function, and may reverse pathologic cardiac hypertrophy. These beneficial effects depend at least partially on improved mitochondrial function, decreased oxidative stress, endothelial dysfunction, and apoptotic cell death, which supports the general recommendation of moderate exercise in CVD patients. However, most of these mechanisms have been described on healthy individuals; the effect of moderate exercise on HTN subjects at a cellular level remain largely unknown. We hypothesized that hypertension in adult spontaneously hypertensive rats (SHRs) reduces the mitochondrial response to moderate exercise in the myocardium. Methods: Eight-month-old SHRs and their normotensive control-Wistar-Kyoto rats (WKYR)-were randomly assigned to moderate exercise on a treadmill five times per week with a running speed set at 10 m/min and 15° inclination. The duration of each session was 45 min with a relative intensity of 70-85% of the maximum O2 consumption for a total of 8 weeks. A control group of untrained animals was maintained in their cages with short sessions of 10 min at 10 m/min two times per week to maintain them accustomed to the treadmill. After completing the exercise protocol, we assessed maximum exercise capacity and echocardiographic parameters. Animals were euthanized, and heart and muscle tissue were harvested for protein determinations and gene expression analysis. Measurements were compared using a nonparametric ANOVA (Kruskal-Wallis), with post-hoc Dunn's test. Results: At baseline, SHR presented myocardial remodeling evidenced by left ventricular hypertrophy (interventricular septum 2.08 ± 0.07 vs. 1.62 ± 0.08 mm, p < 0.001), enlarged left atria (0.62 ± 0.1 mm vs. 0.52 ± 0.1, p = 0.04), and impaired diastolic function (E/A ratio 2.43 ± 0.1 vs. 1.56 ± 0.2) when compared to WKYR. Moderate exercise did not induce changes in ventricular remodeling but improved diastolic filling pattern (E/A ratio 2.43 ± 0.1 in untrained SHR vs. 1.89 ± 0.16 trained SHR, p < 0.01). Histological analysis revealed increased myocyte transversal section area, increased Myh7 (myosin heavy chain 7) expression, and collagen fiber accumulation in SHR-control hearts. While the exercise protocol did not modify cardiac size, there was a significant reduction of cardiomyocyte size in the SHR-exercise group. Conversely, titin expression increased only WYK-exercise animals but remained unchanged in the SHR-exercise group. Mitochondrial response to exercise also diverged between SHR and WYKR: while moderate exercise showed an apparent increase in mRNA levels of Ppargc1α, Opa1, Mfn2, Mff, and Drp1 in WYKR, mitochondrial dynamics proteins remained unchanged in response to exercise in SHR. This finding was further confirmed by decreased levels of MFN2 and OPA1 in SHR at baseline and increased OPA1 processing in response to exercise in heart. In summary, aerobic exercise improves diastolic parameters in SHR but fails to activate the cardiomyocyte mitochondrial adaptive response observed in healthy individuals. This finding may explain the discrepancies on the effect of exercise in clinical settings and evidence of the need to further refine our understanding of the molecular response to physical activity in HTN subjects.

A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

Bone integrity depends on a finely tuned balance between bone synthesis by osteoblasts and resorption by osteoclasts. The secretion capacity of mature osteoblasts requires strict control of proteostasis. Endoplasmic reticulum-associated degradation (ERAD) prevents the accumulation of unfolded ER proteins via dislocation to the cytosol and degradation by the proteasome. The ER membrane protein, homocysteine-inducible endoplasmic reticulum protein with ubiquitin-like domain 1 (HERPUD1), is a key component of the ERAD multiprotein complex which helps to stabilize the complex and facilitate the efficient degradation of unfolded proteins. HERPUD1 expression is strongly up-regulated by the unfolded protein response and cellular stress. The aim of the current study was to establish whether HERPUD1 and ERAD play roles in osteoblast differentiation and maturation. We evaluated preosteoblastic MC3T3-E1 cell and primary rat osteoblast differentiation by measuring calcium deposit levels, alkaline phosphatase activity, and runt-related transcription factor 2 and osterix expression. We found that ERAD and proteasomal degradation were activated and that HERPUD1 expression was increased as osteoblast differentiation progressed. The absence of HERPUD1 blocked osteoblast mineralization in vitro and significantly reduced alkaline phosphatase activity. In contrast, HERPUD1 overexpression activated the osteoblast differentiation program. Our results demonstrate that HERPUD1 and ERAD are important for the activation of the osteoblast maturation program and may be useful new targets for elucidating bone physiology.-Américo-Da-Silva, L., Diaz, J., Bustamante, M., Mancilla, G., Oyarzún, I., Verdejo, H. E., Quiroga, C. A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization.

Low Gene Dosage of Cdc42 Is Not Associated with Protein Dysfunction in Patients with Colorectal Cancer.

High incidence of Rho Cdc42-GTPase overexpression has been found in Colorectal Cancer (CRC) samples, suggesting its potential role in tumor development. However, no conclusive studies have shown the lack of mutations and/or copy number of Cdc42 gene in this type of samples. To understand mutation/deletion and copy number status of Cdc42 gene, CRC patients were evaluated for both parameters. More than Cdc42 mutants, single-nucleotide variants were found. Analysis of regions flanking the Cdc42 gene showed allelic imbalance; 58.7% were loss of heterozygosity (LOH) positive and 14.8% presented microsatellite instability. The highest LOH percentage was located between microsatellite markers D1S199 and D1S2674, where the Cdc42 gene is located. No association between gender, age, and tumor stage was found. LOH validation through gene dosage analysis showed most CRC patients with allelic imbalance also presented a low gene dosage of Cdc42, although equal amounts of Cdc42 mRNA were detected in all samples. Although changes in Cdc42 expression were not found in any condition, Cdc42 activation was different between high and normal gene dosage samples, but not between samples with normal and low copy number. Low dosage of Cdc42 was also not related to changes in methylation status at the Cdc42 promoter region. Results suggest that low copy of Cdc42 gene is not associated with Cdc42 protein dysfunction in CRC patients.

Molecular determinants of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) binding to transient receptor potential V1 (TRPV1) channels.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P2 interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.

K(+) channels: function-structural overview.

Potassium channels are particularly important in determining the shape and duration of the action potential, controlling the membrane potential, modulating hormone secretion, epithelial function and, in the case of those K(+) channels activated by Ca(2+), damping excitatory signals. The multiplicity of roles played by K(+) channels is only possible to their mammoth diversity that includes at present 70 K(+) channels encoding genes in mammals. Today, thanks to the use of cloning, mutagenesis, and the more recent structural studies using x-ray crystallography, we are in a unique position to understand the origins of the enormous diversity of this superfamily of ion channels, the roles they play in different cell types, and the relations that exist between structure and function. With the exception of two-pore K(+) channels that are dimers, voltage-dependent K(+) channels are tetrameric assemblies and share an extremely well conserved pore region, in which the ion-selectivity filter resides. In the present overview, we discuss in the function, localization, and the relations between function and structure of the five different subfamilies of K(+) channels: (a) inward rectifiers, Kir; (b) four transmembrane segments-2 pores, K2P; (c) voltage-gated, Kv; (d) the Slo family; and (e) Ca(2+)-activated SK family, SKCa.