BackgroundSkeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers.MethodsWe profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes.ResultsThe muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 × 10-8), post-translational modification (P < 9.7 × 10-5) and developmental (P < 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation.ConclusionsWe provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions.

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In vitro

In vertebrates, including the mammals, the chromosomal DNAs are modified by C methylation at a limited number of CpG dinucleotides, resulting in m 5 CpG, methylated at position 5 of the C residues. This methylation at CpG has been implicated in the regulation of a number of genetic activities during mammalian cell differentiation and embryo development. These activities include tissue-specific gene transcription, X chromosome inactivation, genomic imprinting, cellular defense against viral agents, and tumorigenesis (references 1, 2, 11, 13, 15, and 34 and references therein). The level of m 5 CpG in the vertebrate cells is presumably balanced by the combined actions of DNA cytosine (C-5) methyltransferases (CpG MTases) (2, 26) and DNA demethylases (3).It is generally accepted that regulation of the different cellular activities by DNA methylation is modulated mainly through the m 5 CpG residues. Indeed, m 5 CpG could be recognized by a specific class of proteins that consist of the methylated-DNA binding domain (MBD) (21). These "MBD proteins," in particular MeCP2 and MBD2/3, could preferentially bind to m 5 CpG-containing DNA, recruit histone deacetylase (HDAC)-containing complexes, and thus cause chromatin condensation in the vicinity of m 5 CpG (12,22,23,33). This is very likely one major, but not the only, scheme involved in the functioning of vertebrate DNA methylation.Unlike the vertebrates, several invertebrate species, including Drosophila melanogaster (27, 31), do not have apparent DNA methylation in their genomes. Nor has CpG MTase been reported to occur in these invertebrate animals. It was thus surprising to find mammalian CpG MTase-related proteins as well as MBD proteins expressed in Drosophila. A Drosophila protein, DmMTR1, is characteristically similar to the vertebrate maintenance CpG MTase, dnmt1, in several aspects (9). In particular, DmMTR1 molecules are located outside the nuclei during interphase of the syncytial Drosophila embryos, as is dnmt1 in the mouse blastocyst (19). However, DmMTR1 molecules appear to be rapidly transported into and then out of the nuclei again, as the embryos undergo the mitotic waves (9). Immunofluorescence data further indicate that DmMTR1 molecules "paint" the whole set of condensed Drosophila chromosomes throughout the mitotic phase, suggesting that it may play an essential role in the cell cycle-regulated condensation of the Drosophila chromosomes. In addition to DmMTR1, another Drosophila polypeptide, DmMT2, exhibiting high sequence homology to mammalian dnmt2, a relatively short homolog of dnmt1 but without detectable methylation activities (25), has been identified through a database search (9, 30). Expression of DmMT2 in Drosophila is developmentally regulated (9).Interestingly, several recent reports have also noted the identification of an MBD protein encoded by the Drosophila genome (9,30,33). Like the mammalian MBD (12,22,23,33), the Drosophila MBD protein, which was termed the "Drosophila MBD-like sequence" (33) or dMBD2/3 (30), interacts with HDAC in...

SK channels are upregulated in human patients and animal models of heart failure (HF). However, their activation mechanism and function in ventricular myocytes remain poorly understood. We aim to test the hypotheses that activation of SK channels in ventricular myocytes requires Ca(2+) release from sarcoplasmic reticulum (SR) and that SK currents contribute to reducing triggered activity. SK2 channels were overexpressed in adult rat ventricular myocytes using adenovirus gene transfer. Simultaneous patch clamp and confocal Ca(2+) imaging experiments in SK2-overexpressing cells demonstrated that depolarizations resulted in Ca(2+)-dependent outward currents sensitive to SK inhibitor apamin. SR Ca(2+) release induced by rapid application of 10 mM caffeine evoked repolarizing SK currents, whereas complete depletion of SR Ca(2+) content eliminated SK currents in response to depolarizations, despite intact Ca(2+) influx through L-type Ca(2+) channels. Furthermore, voltage-clamp experiments showed that SK channels can be activated by global spontaneous SR Ca(2+) release events Ca(2+) waves (SCWs). Current-clamp experiments revealed that SK overexpression reduces the amplitude of delayed afterdepolarizations (DADs) resulting from SCWs and shortens action potential duration. Immunolocalization studies showed that overexpressed SK channels are distributed both at external sarcolemmal membranes and along the Z-lines, resembling the distribution of endogenous SK channels. In summary, SR Ca(2+) release is both necessary and sufficient for the activation of SK channels in rat ventricular myocytes. SK currents contribute to repolarization during action potentials and attenuate DADs driven by SCWs. Thus SK upregulation in HF may have an anti-arrhythmic effect by reducing triggered activity.

These data suggest that enhancement of mSK channels in hypertrophic rat hearts protects from Ca2+-dependent arrhythmia and suggest that the protection is mediated via decreased mitochondrial ROS and subsequent decreased oxidation of reactive cysteines in RyR, which ultimately leads to stabilization of RyR-mediated Ca2+ release.

Key points Small‐conductance Ca 2+ ‐activated K + (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca 2+ ] in a biphasic manner. Submicromolar [Ca 2+ ] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca 2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function. PKA phosphorylation attenuates I SK rectification by reducing the Ca 2+ /voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca 2+ ] i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Abstract Small‐conductance Ca 2+ ‐activated K + (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs . Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L –1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L –1 ), which was eliminated by inhibition of PKA (1 μmol L –1 ). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L –1 ) evoked I SK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo ...

Background: A genetic variant within RNF207 is associated with a prolonged QT interval. QT interval prolongation may lead to arrhythmia, a risk factor for sudden cardiac death. Results: RNF207 regulates action potential duration and the repolarizing channel HERG. Conclusion: RNF207 is an important regulator of cardiac excitation. Significance: Understanding the composition and dynamics of membrane complexes is important in unraveling the mechanism of arrhythmias.