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      Identification of optimal reference genes for transcriptomic analyses in normal and diseased human heart

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          Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.

          The effects of serum on the expression of four commonly used housekeeping genes were examined in serum-stimulated fibroblasts in order to validate the internal control genes for a quantitative RT-PCR assay. NIH 3T3 fibroblasts transfected with an inducible chimeric gene were serum-starved for 24 h and then induced with 15% serum for 8 h. Serum did not alter the amount of total RNA that was expressed in the cells, however, the amount of mRNA significantly increased over time with serum-stimulation. Both messenger and total RNA from each of the time points were reverse transcribed under two different conditions; one in which the reactions were normalized to contain equal amounts of RNA and another series of reactions that were not normalized to RNA content. The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for beta-actin, beta-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. The expression of beta-actin and GAPDH increased up to nine- and three-fold, respectively, under all conditions of reverse transcription (P 0.05). The expression of beta-2 microglobulin increased up to two-fold when assayed from cDNA synthesized from non-normalized mRNA, but was unaffected by serum when the reverse transcriptions were normalized to mRNA. beta-2 Microglobulin expression was found to be directly proportional to the amount of mRNA that was present in non-normalized reverse transcription reactions. Thus, beta-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while beta-actin and GAPDH are not. The internal control gene needs to be properly validated when designing quantitative gene expression studies.
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            Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart.

            The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion-channel functional expression. The present study addressed regionally defined differential ion-channel expression in the non-diseased human heart with a genomic approach. High-throughput real-time RT-PCR was used to quantify the expression patterns of 79 ion-channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non-diseased human donor hearts. Two-way non-directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left- versus right-sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cav alpha2 delta2, Nav beta1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression-portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na(+),K(+)-ATPase, NCX1, SERCA2, and the Ca(2+)-handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin-alpha. Nav1.5 and Nav beta1 were more strongly expressed in the endocardium. For selected genes, RT-PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion-channel subunit-gene expression in the non-diseased human heart. Our data point to significant regionally determined ion-channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion-channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion-current expression may be primarily transcriptional.
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              Parental atrial fibrillation as a risk factor for atrial fibrillation in offspring.

              Atrial fibrillation (AF) is the most common cardiac dysrhythmia in the United States. Whereas rare cases of familial AF have been reported, it is unknown if AF among unselected individuals is a heritable condition. To determine whether parental AF increases the risk for the development of offspring AF. Prospective cohort study (1983-2002) within the Framingham Heart Study, a population-based epidemiologic study. Participants were 2243 offspring (1165 women, 1078 men) at least 30 years of age and free of AF whose parents had both been evaluated in the original cohort. Development of new-onset AF in the offspring was prospectively examined in association with previously documented parental AF. Among 2243 offspring participants, 681 (30%) had at least 1 parent with documented AF; 70 offspring participants (23 women; mean age, 62 [range, 40-81] years) developed AF in follow-up. Compared with no parental AF, AF in at least 1 parent increased the risk of offspring AF (multivariable-adjusted odds ratio [OR], 1.85; 95% confidence interval [CI], 1.12-3.06; P =.02). These results were stronger when age was limited to younger than 75 years in both parents and offspring (multivariable-adjusted OR, 3.23; 95% CI, 1.87-5.58; P<.001) and when the sample was further limited to those without antecedent myocardial infarction, heart failure, or valve disease (multivariable-adjusted OR, 3.17; 95% CI, 1.71-5.86; P<.001). Parental AF increases the future risk for offspring AF, an observation supporting a genetic susceptibility to developing this dysrhythmia. Further research into the genetic factors predisposing to AF is warranted.
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                Author and article information

                Journal
                Cardiovascular Research
                Oxford University Press (OUP)
                0008-6363
                1755-3245
                February 2018
                February 01 2018
                September 22 2017
                February 2018
                February 01 2018
                September 22 2017
                : 114
                : 2
                : 247-258
                Article
                10.1093/cvr/cvx182
                29036603
                f000b098-9419-4f32-aa60-eb9a30747f77
                © 2017
                History

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