The Expression of microRNA and microRNA Clusters in the Aging Heart

Background: The microRNAs have been implicated in the process of cardiac development, cardiac hypertrophy, and heart failure. However, the impact of adult aging on cardiac expression of miRNA clusters, as well as both miRNA guide (miR) and passenger (miR*) strands has not been well established. Methods/Results: We explored the expression profile of both miR and miR * in the hearts of young adult versus old mice. We found that 65 miRNAs were differentially expressed in the old versus young adult hearts; approximately half of them were clustered miRNAs that were distributed in 11 miRNA clusters. Each miRNA cluster contained from 2 to as many as 71 miRNA genes. The majority of the clusters displayed similar expression, with most cluster members within a cluster being either increased or decreased together, suggesting that most clusters are likely to be regulated by a common signaling mechanism and that the combined expression of multiple miRNA genes in a cluster could pose an impact on a broad range of targets during aging. We also found age-related changes in the expression of miR*s. The expression of both miR and miR* correlated with that of pri-miRNA transcript over the time course from development and maturation through adult aging. Age-related changes in the expression of Ago1 and Ago2 proteins in the heart were also observed. Transfection assay revealed that both Ago1 and Ago2 synergistically induced miR-21 and miR-21 * when the mir-21 plasmid was co-transfected with either

Regulation of Serum Response Factor and Adiponectin by PPARγ Agonist Docosahexaenoic Acid

Recent studies indicate that significant health benefits involving the regulation of signaling proteins result from the consumption of omega-3 polyunsaturated fatty acids (ω-3 PUFAs). Serum response factor (SRF) is involved in transcriptional regulation of muscle growth and differentiation. SRF levels are increased in the aging heart muscle. It has not been examined whether SRF is made by adipocytes and whether SRF secretion by adipocytes is modulated by PPARγ agonist DHA. Adiponectin is made exclusively by adipocytes. We and others have previously reported that PUFAs such as DHA increase adiponectin levels and secretion in adipocytes. Here we show that DHA downregulates SRF with a simultaneous upregulation of adiponectin and that both these responses are reversible by PPARγ antagonist. Furthermore, there appears to be a direct relationship between DHA exposure and increased levels of membrane-associated high-density adiponectin, as well as lower levels of membrane-associated SRF. Thus, we find that the levels of SRF and adiponectin are inversely related in response to treatment with PPARγ agonist DHA. Decreased levels of SRF along with increase in membrane-associated adiponectin could in part mediate the health benefits of DHA

Regulation of cardiac microRNAs by serum response factor

Serum response factor (SRF) regulates certain microRNAs that play a role in cardiac and skeletal muscle development. However, the role of SRF in the regulation of microRNA expression and microRNA biogenesis in cardiac hypertrophy has not been well established. In this report, we employed two distinct transgenic mouse models to study the impact of SRF on cardiac microRNA expression and microRNA biogenesis. Cardiac-specific overexpression of SRF (SRF-Tg) led to altered expression of a number of microRNAs. Interestingly, downregulation of miR-1, miR-133a and upregulation of miR-21 occurred by 7 days of age in these mice, long before the onset of cardiac hypertrophy, suggesting that SRF overexpression impacted the expression of microRNAs which contribute to cardiac hypertrophy. Reducing cardiac SRF level using the antisense-SRF transgenic approach (Anti-SRF-Tg) resulted in the expression of miR-1, miR-133a and miR-21 in the opposite direction. Furthermore, we observed that SRF regulates microRNA biogenesis, specifically the transcription of pri-microRNA, thereby affecting the mature microRNA level. The mir-21 promoter sequence is conserved among mouse, rat and human; one SRF binding site was found to be in the mir-21 proximal promoter region of all three species. The mir-21 gene is regulated by SRF and its cofactors, including myocardin and p49/Strap. Our study demonstrates that the downregulation of miR-1, miR-133a, and upregulation of miR-21 can be reversed by one single upstream regulator, SRF. These results may help to develop novel therapeutic interventions targeting microRNA biogenesis

Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K(+) channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native I(K1) in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K(+) current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome

Modulation of the effects of class Ib antiarrhythmics on cardiac NaV1.5-encoded channels by accessory NaVβ subunits

Native myocardial voltage-gated sodium (NaV) channels function in macromolecular complexes comprising a pore-forming (α) subunit and multiple accessory proteins. Here, we investigated the impact of accessory NaVβ1 and NaVβ3 subunits on the functional effects of 2 well-known class Ib antiarrhythmics, lidocaine and ranolazine, on the predominant NaV channel α subunit, NaV1.5, expressed in the mammalian heart. We showed that both drugs stabilized the activated conformation of the voltage sensor of domain-III (DIII-VSD) in NaV1.5. In the presence of NaVβ1, the effect of lidocaine on the DIII-VSD was enhanced, whereas the effect of ranolazine was abolished. Mutating the main class Ib drug-binding site, F1760, affected but did not abolish the modulation of drug block by NaVβ1/β3. Recordings from adult mouse ventricular myocytes demonstrated that loss of Scn1b (NaVβ1) differentially affected the potencies of lidocaine and ranolazine. In vivo experiments revealed distinct ECG responses to i.p. injection of ranolazine or lidocaine in WT and Scn1b-null animals, suggesting that NaVβ1 modulated drug responses at the whole-heart level. In the human heart, we found that SCN1B transcript expression was 3 times higher in the atria than ventricles, differences that could, in combination with inherited or acquired cardiovascular disease, dramatically affect patient response to class Ib antiarrhythmic therapies

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background: Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase. Methodology/Principal Findings: To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage. Conclusions/Significance: This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and th

Pathogen reduction in human plasma using an ultrashort pulsed laser

Pathogen reduction is a viable approach to ensure the continued safety of the blood supply against emerging pathogens. However, the currently licensed pathogen reduction techniques are ineffective against non-enveloped viruses such as hepatitis A virus, and they introduce chemicals with concerns of side effects which prevent their widespread use. In this report, we demonstrate the inactivation of both enveloped and non-enveloped viruses in human plasma using a novel chemical-free method, a visible ultrashort pulsed laser. We found that laser treatment resulted in 2-log, 1-log, and 3-log reductions in human immunodeficiency virus, hepatitis A virus, and murine cytomegalovirus in human plasma, respectively. Laser-treated plasma showed ≥70% retention for most coagulation factors tested. Furthermore, laser treatment did not alter the structure of a model coagulation factor, fibrinogen. Ultrashort pulsed lasers are a promising new method for chemical-free, broad-spectrum pathogen reduction in human plasma