Elevated potassium outward currents in hyperoxia treated atrial cardiomyocytes.

Supplementation of 100% oxygen is a very common intervention in intensive care units (ICU) and critical care centers for patients with dysfunctional lung and lung disorders. Although there is advantage in delivering sufficient levels of oxygen, hyperoxia is reported to be directly associated with increasing in-hospital deaths. Our previous studies reported ventricular and electrical remodeling in hyperoxia treated mouse hearts, and in this article, for the first time, we are investigating the effects of hyperoxia on atrial electrophysiology using whole-cell patch-clamp electrophysiology experiments along with assessment of Kv1.5, Kv4.2, and KChIP2 transcripts and protein profiles using real-time quantitative RT-PCR and Western blotting. Our data showed that induction of hyperoxia for 3 days in mice showed larger outward potassium currents with shorter action potential durations (APD). This increase in current densities is due to significant increase in ultrarapid delayed rectifier outward K+ currents (IKur ) and rapidly activating, rapidly inactivating transient outward K+ current (Ito ) densities. We also observed a significant increase in both transcripts and protein levels of Kv1.5 and KChIP2 in hyperoxia treated atrial cardiomyocytes, whereas no significant change was observed in Kv4.2 transcripts or protein. The data presented here further support our previous findings that hyperoxia induces not only ventricular remodeling, but also atrial electrical remodeling.

Hyperoxia-induced cardiotoxicity and ventricular remodeling in type-II diabetes mice.

Hyperoxia, or supplemental oxygen, is regularly used in the clinical setting for critically ill patients in ICU. However, several recent studies have demonstrated the negative impact of this treatment in patients in critical care, including increased rates of lung and cardiac injury, as well as increased mortality. The purpose of this study was to determine the predisposition for arrhythmias and electrical remodeling in a type 2 diabetic mouse model (db/db), as a result of hyperoxia treatment. For this, db/db and their heterozygous controls were treated with hyperoxia (> 90% oxygen) or normoxia (normal air) for 72-h. Immediately following hyperoxia or normoxia treatments, mice underwent surface ECG. Excised left ventricles were used to assess ion channel expression, including for Kv1.4, Kv1.5, Kv4.2, and KChIP2. Serum cardiac markers were also measured, including cardiac troponin I and lactate dehydrogenase. Our results showed that db/db mice have increased sensitivity to arrhythmia. Normoxia-treated db/db mice displayed features of arrhythmia, including QTc and JT prolongation, as well as QRS prolongation. A significant increase in QRS prolongation was also observed in hyperoxia-treated db/db mice, when compared to hyperoxia-treated heterozygous control mice. Db/db mice were also shown to exhibit ion channel dysregulation, as demonstrated by down-regulation in Kv1.5, Kv4.2, and KChIP2 under hyperoxia conditions. From these results, we conclude that: (1) diabetic mice showed distinct pathophysiology, when compared to heterozygous controls, both in normoxia and hyperoxia conditions. (2) Diabetic mice were more susceptible to arrhythmia at normal air conditions; this effect was exacerbated at hyperoxia conditions. (3) Unlike in heterozygous controls, diabetic mice did not demonstrate cardiac hypertrophy as a result of hyperoxia. (4) Ion channel remodeling was also observed in db/db mice under hyperoxia condition similar to its heterozygous controls.

Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.

Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis.

Gradients of signalling and transcription factors govern many aspects of embryogenesis, highlighting the need for spatiotemporal control of regulatory protein levels. MicroRNAs are phylogenetically conserved small RNAs that regulate the translation of target messenger RNAs, providing a mechanism for protein dose regulation. Here we show that microRNA-1-1 (miR-1-1) and miR-1-2 are specifically expressed in cardiac and skeletal muscle precursor cells. We found that the miR-1 genes are direct transcriptional targets of muscle differentiation regulators including serum response factor, MyoD and Mef2. Correspondingly, excess miR-1 in the developing heart leads to a decreased pool of proliferating ventricular cardiomyocytes. Using a new algorithm for microRNA target identification that incorporates features of RNA structure and target accessibility, we show that Hand2, a transcription factor that promotes ventricular cardiomyocyte expansion, is a target of miR-1. This work suggests that miR-1 genes titrate the effects of critical cardiac regulatory proteins to control the balance between differentiation and proliferation during cardiogenesis.

Establishment of an arrhythmogenic right ventricular cardiomyopathy derived iPSC cell line (USFi004-A) carrying a heterozygous mutation in PKP2 (c.1799delA).

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an autosomal dominant inherited disease, with variable penetrance and expressivity. Currently, more than 14 different genetic loci have been reported for ARVC, the majority being desmosomal genes like Plakophilin-2 (PKP2). Here, we generated an iPSC cell line bearing a pathogenic heterozygous mutation in PKP2 (c.1799delA) from a patient affected by ARVC. Peripheral blood mononuclear cells were reprogrammed by Sendai virus vectors encoding KOS, KLF4, and c-MYC. Derived iPSCs expressed pluripotent markers, showed intact karyotype and demonstrated the ability to differentiate into three germ layers.

Distinct roles for the essential MYST family HAT Esa1p in transcriptional silencing.

Among acetyltransferases, the MYST family enzyme Esa1p is distinguished for its essential function and contribution to transcriptional activation and DNA double-stranded break repair. Here we report that Esa1p also plays a key role in silencing RNA polymerase II (Pol II)-transcribed genes at telomeres and within the ribosomal DNA (rDNA) of the nucleolus. These effects are mediated through Esa1p's HAT activity and correlate with changes within the nucleolus. Esa1p is enriched within the rDNA, as is the NAD-dependent protein deacetylase Sir2p, and the acetylation levels of key Esa1p histone targets are reduced in the rDNA in esa1 mutants. Although mutants of both ESA1 and SIR2 have enhanced rates of rDNA recombination, esa1 effects are more modest yet result in distinct structural changes of rDNA chromatin. Surprisingly, increased expression of ESA1 can bypass the requirement for Sir2p in rDNA silencing, suggesting that these two enzymes with seemingly opposing activities both contribute to achieve optimal nucleolar chromatin structure and function.

Premature MicroRNA-1 Expression Causes Hypoplasia of the Cardiac Ventricular Conduction System.

Mammalian cardiac Purkinje fibers (PFs) are specified from ventricular trabecular myocardium during mid-gestation and undergo limited proliferation before assuming their final form. MicroRNA-1 (miR-1), a negative regulator of proliferation, is normally expressed in the heart at low levels during the period of PF specification and outgrowth, but expression rises steeply after birth, when myocardial proliferation slows and postnatal cardiac maturation and growth commence. Here, we test whether premature up-regulation and overexpression of miR-1 during the period of PF morphogenesis influences PF development and function. Using a mouse model in which miR-1 is expressed under the control of the Myh6 promoter, we demonstrate that premature miR-1 expression leads to PF hypoplasia that persists into adulthood, and miR-1 TG mice exhibit delayed conduction through the ventricular myocardium beginning at neonatal stages. In addition, miR-1 transgenic embryos showed reduced proliferation within the trabecular myocardium and embryonic ventricular conduction system (VCS), a source of progenitor cells for the PF. This repression of proliferation may be mediated by direct translational inhibition by miR-1 of the cyclin dependent kinase Cdk6, a key regulator of embryonic myocardial proliferation. Our results suggest that altering the timing of miR-1 expression can regulate PF development, findings which have implications for our understanding of conduction system development and disease in humans.