Patients experiencing statin-induced myalgia exhibit a unique program of skeletal muscle gene expression following statin re-challenge.

Statins, the 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitors, are widely prescribed for treatment of hypercholesterolemia. Although statins are generally well tolerated, up to ten percent of statin-treated patients experience myalgia symptoms, defined as muscle pain without elevated creatinine phosphokinase (CPK) levels. Myalgia is the most frequent reason for discontinuation of statin therapy.

Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI.

Role of PTEN in TNFα induced insulin resistance.

Aims/hypothesis: PTEN may play a reversible role in TNFα induced insulin resistance, which has been linked to obesity-associated insulin resistance (IR).

Methods: Western blots for PTEN and p-Akt were performed on H-411E liver cells incubated with insulin, TNFα, and in selected experiments VO-OHpic vanadium complex in the presence and absence of PTEN siRNA. Total PTEN was compared to β-actin loading control and p-Akt was compared to total Akt.

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA.

Pan-histone deacetylase inhibitors regulate signaling pathways involved in proliferative and pro-inflammatory mechanisms in H9c2 cells.

Background: We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA.

Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy.

We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles.

A critical role of Sp1 transcription factor in regulating gene expression in response to insulin and other hormones.

Specificity protein 1 (Sp1) belongs to a family of ubiquitously expressed, C(2)H(2)-type zinc finger-containing DNA binding proteins that activate or repress transcription of many genes in response to physiological and pathological stimuli. There is emerging evidence to indicate that in addition to functioning as 'housekeeping' transcription factors, members of Sp family may be key mediators of gene expression induced by insulin and other hormones. The founding member of the family, Sp1, by virtue of its multi-domain organization, potential for posttranslational modifications and interactions with numerous transcription factors, represents an ideal mediator of nuclear signaling in response to hormones.

Epigenetic regulation of cardiac muscle-specific genes in H9c2 cells by Interleukin-18 and histone deacetylase inhibitor m-carboxycinnamic acid bis-hydroxamide.

Interleukin-18 (IL-18) elicited a robust hypertrophy response in H9c2 cardiomyocytes as judged by their accelerated rates of protein synthesis and increased cell size. Evidently, IL-18 treatment also induced a cardiac hypertrophy-specific program of gene expression in H9c2 cardiomyocytes since they elicited enhanced expression of atrial naturetic factor (ANF), desmin, and skeletal alpha-actin genes accompanied by a canonical switch in the transcription of alpha- and beta-myosin heavy chain (MyHC) genes. Co-treatment of H9c2 cells with m-carboxycinnamic acid bis-hydroxamide (CBHA), an inhibitor of histone deacetylases, significantly blocked both morphological and molecular manifestations of IL-18-induced cardiac hypertrophy in vitro.

Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells.

O-glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A.

Proteome of H-411E (liver) cells exposed to insulin and tumor necrosis factor-alpha: analysis of proteins involved in insulin resistance.

Insulin resistance may be modeled in H-411E liver cells in tissue culture with the use of the cytokine tumor necrosis factor-alpha (TNF-alpha) and insulin. This tissue-culture model nicely mimics IR in human type 2 diabetes mellitus. After incubation of liver cells in tissue culture with INS alone, TNF-alpha alone, and TNF-alpha plus insulin, as well as a control sample, liver-cell extracts were separated on 2D polyacrylamide-gel electrophoresis on the basis of isoelectric point and molecular weight.

Insulin stimulates and diabetes inhibits O-linked N-acetylglucosamine transferase and O-glycosylation of Sp1.

Insulin stimulates both the biosynthesis of transcription factor Sp1 and its O-linked N-acetylglucosaminylation (O-GlcNAcylation), which promotes nuclear localization of Sp1 and its ability to transactivate calmodulin (CaM) gene transcription. To investigate this further, we incubated H-411E liver cells with insulin (10,000 microU/ml) and quantified the subcellular distribution of O-GlcNAc transferase (OGT) and O-GlcNAc-modified Sp1. We also examined the phosphorylation of Sp1 using both Western blot and incorporation of 32P into Sp1.

O-glycosylation of Sp1 and transcriptional regulation of the calmodulin gene by insulin and glucagon.

Both insulin and glucagon stimulate steady-state levels of Sp1 transcription factor, but only insulin stimulates transcription of the calmodulin (CaM) gene in liver. Because O-glycosylation of Sp1 by O-linked N-acetylglucosamine (O-GlcNAc) is thought to regulate its ability to activate transcription, we assayed the levels of Sp1 with anti-Sp1 and anti-O-GlcNAc antibodies in Western blots by use of extracts of H-411E liver cells treated with insulin (10,000 microU/ml) or glucagon (1.5 x 10(-5) M).

Paradoxical regulation of Sp1 transcription factor by glucagon.

Insulin is a potent regulator of Sp1 transcription factor. To examine if glucagon, which usually antagonizes insulin, regulates Sp1, we assessed the levels of Sp1 by Western blotting from H-411E cells exposed to glucagon with or without insulin. Glucagon alone (1.