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Aging is associated with a decline in cardiac function due to a decreased myocardial reserve. This adverse cardiac remodeling comprises of a variety of changes, including a reduction in mitochondrial function and a decline in the expression of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a central regulator of mitochondrial biogenesis and metabolic adaptation in the myocardium. To study the etiological involvement of PGC-1α in cardiac aging, we used mouse models mimicking the modest down- and upregulation of this coactivator in the old and the exercised heart, respectively. Youngmice with reduced cardiac expression of PGC-1α recapitulated part of the age-related impairment in mitochondrial gene expression, but otherwise did not aggravate the aging process. Inversely however, moderate overexpression of PGC-1α counteracts numerous key age-related remodeling changes, e.g., by improving blood pressure, age-associated apoptosis, and collagen accumulation, as well as in the expression of many, but not all cardiac genes involved in mitochondrial biogenesis, dynamics, metabolism, calcium handling and contractility. Thus, while the reduction of PGC-1α in the heart is insufficient to cause an aging phenotype, moderate overexpression reduces pathological remodeling of older hearts and could thereby contribute to the beneficial effects of exercise on cardiac function in aging. [ABSTRACT FROM AUTHOR]

Recent evidence supports that TNF-α, long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-α induced differentiation. The biological effects of TNF-α in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-α has growth-promoting effects in myotubes, and w& characterized the mechanisms leading to these effects. Treatment of C2C12 myotubes with TNF-α for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-α activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, p70S6K and 4E-BP1. TNF-α-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-α receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-α-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-α-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, p70S6K, and 4E-BP1. Rapamycin inhibited TNF-α-induced phosphorylation of the mTOR C1 target p70S6K without altering TNF-α-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-α enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Brugada syndrome (BS) is an inherited electrical cardiac disorder characterized by right bundle branch block pattern and ST segment elevation in leads V1 to V3 on surface electrocardiogram that can potentially lead to malignant ventricular tachycardia and sudden cardiac death. About 20% of patients have mutations in the only so far identified gene, SCN5A, which encodes the α-subunit of the human cardiac voltage-dependent sodium channel (hNav1.5). Fever has been shown to unmask or trigger the BS phenotype, but the associated molecular and the biophysical mechanisms are still poorly understood. We report on the identification and biophysical characterization of a novel heterozygous missense mutation in SCN5A, F1344S, in a 42-year-old male patient showing the BS phenotype leading to ventricular fibrillation during fever. Methods: The mutation was reproduced in vitro using site-directed mutagenesis and characterized using the patch clamp technique in the whole-cell configuration. Results: The biophysical characterization of the channels carrying the F1344S mutation revealed a 10mV mid-point shift of the G/V curve toward more positive voltages during activation. Raising the temperature to 40.5°C further shifted the mid-point activation by 18mV and significantly changed the slope factor in Nav1.5/F1344S mutant channels from −6.49 to −10.27mV. Conclusions: Our findings indicate for the first time that the shift in activation and change in the slope factor at a higher temperature mimicking fever could reduce sodium currents'' amplitude and trigger the manifestation of the BS phenotype. [Copyright &y& Elsevier]

Studies the biosynthesis of N-glycans in connective tissue of the snail, Lymnaea stagnalis. Glycoprotein activities; Minimal structure requirements; Prerequisite for the action of N-acetylglucosaminyltransferase.

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in β1-3 linkage to terminal GalNAc of GalNAcβ1- 4GlcNAc-R [R = β1-2Manα1-O(CH2)8COOMe, &β1-OMe, or α,β1-OH]. Using GalNAcβ1-4GlcNAcβ1-2Manα-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAcβ1-4GlcNAcβ1-2Manα1-O(CH2)8COOMe (Vmax 140 nmol · h-1 mg protein-1 Km 1.02 mM), GalNAcβ1-4GlcNAc (Vmax 105 nmol · h-1 · mg proteins-1 Km 0.99 mM), and GalNAcβ1 -4GlcNAcβ1 -OMe (Vmax 108 nmol ·h-1 · mg protein-1 Km 1.33 mM). The products formed from GalNAcβ1-4GlcNAcβ1-2Manα1-O(CH2)8COOMe and GalNAcβ1- 4G1cNAcβ1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz ¹H-NMR spectroscopy to be Galβ1 -3 GalNAcβ1 -4GlcNAcβ1 -2Manα1 -O(CH2)8 COO Me and Galβ1 -3GalNAcβ1 -4GlcNAcβ1 -OMe, respectively. The enzyme was inactive towards GlcNAc, GalNAcβ1 -3Galα1 -OMe, GalNAcα1-OC6H5, GalNAcα1 -ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP- Gal:GalNAcβ1-4GIcNAc-R β1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis. [ABSTRACT FROM AUTHOR]

Mechanistic target of rapamycin (mTOR) kinase operates in two functionally and structurally distinct multiprotein complexes mTORC1 and mTORC2. mTORC1 including its defining raptor protein is a critical mediator of myocardial growth. We induced cardiomyocyte specific raptor-KO (cKO) deletion to investigate a sex specific role of mTORC1 during cardiac adaptation in normotensive DOCA-salt mice. Untypical for this mild cardiac stress model, raptor-cKO females had sharper decrease in EF (51 vs. 26%) than males (50 vs. 32%) already at 3 weeks after start of DOCA-salt challenge. Male raptor-cKO mice mounted eccentric hypertrophic response upon DOCA-salt indicated by heart weight/tibia length ratios (6.7 to 8.0) as compared to their WT litters (6.6 to 7.6). In contrast female raptor-cKO (5.8 to 6.4) minimally differed from their litters (5.8 to 6.6). Severe dilative phenotype was detected in both sexes regardless of DOCA-salt challenge. In addition, we found tremendous diffuse left ventricular fibrosis which was also more prominent in raptor-cKO females. Collagen depositions in the LV visualized by Sirius red staining increased 10fold in males (1.0 to 10.6%) and 15fold in females (0.9 to 15.7%), respectively. Expression of raptor protein in the heart was diminished by 80% in raptor cKO mice of both sexes what lead to similar extent of decreased activity of direct mTORC1 downstream targets phospho-S6 ribosomal protein and phospho-4E-BP1Ser65 (68% reduction in males vs. 61% in females. Over-compensatory mTORC2 activation in cKO animals via phospho-AKTSer473 (12fold increase) and induction of anti-apoptotic signaling was similar in both sexes. However, raptor-cKO females failed to up-regulate expression of mitochondrial genes such as Mtnd1 (ND1), mt-Co1 (COX1) and Atp5i (ATP5k), stressing sex differences in mTORC1 dependent mitochondrial adaptation. In addition, transmission electron microscopy (TEM) points towards structural alterations of mitochondrial morphology.Intact mTORC1 function is important for myocardial adaptation in both sexes. More severe phenotype in females further implicates mTORC1 to be essential for intrinsic female cardio protection. [ABSTRACT FROM AUTHOR]