The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stressinducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease. Abbreviations ANKRD1, ankyrin repeat domain 1 protein; MARPs, muscle ankyrin repeat proteins; SRF, serum response factor; HF, heart failure; DHF, diastolic HF; LV/RV, left/right ventricular myocardium; LA/RA, left/right atrial

BackgroundMyocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and functional consequences of myocd upregulation in HF are still unclear.Methodology/Principal FindingsThe goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function.Conclusions/SignificanceThese data provide the first evidence that a moderate inhibition (e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.

Molecular predisposition of postnatal ventricular myocardium to chamber-dependent (concentric or eccentric) remodeling remains largely elusive. To this end, we compared gene expression in the left (LV) versus right ventricle (RV) in newborn piglets, using a differential display reverse transcription-PCR (DDRT-PCR) technique. Out of more than 5600 DDRT-PCR bands, a total of 153 bands were identified as being differentially displayed. Of these, 96 bands were enriched in the LV, whereas the remaining 57 bands were predominant in the RV. The transcripts, displaying over twofold LV-RV expression differences, were sequenced and identified by BLAST comparison to known mRNA sequences. Among the genes, whose expression was not previously recognized as being chamber-dependent, we identified a small cohort of key regulators of muscle cell growth/proliferation (MAP3K7IP2, MSTN, PHB2, APOBEC3F) and gene expression (PTPLAD1, JMJD1C, CEP290), which may be relevant to the chamber-dependent predisposition of ventricular myocardium to respond differentially to pressure (LV) and volume (RV) overloads after birth. In addition, our data demonstrate chamber-dependent alterations in expression of as yet uncharacterized novel genes, which may also be suitable candidates for association studies in animal models of LV/RV hypertrophy.

Background In the case of psoriatic patients, only a limited number of studies have related serum biological therapies and antidrug antibodies levels to clinical response. With respect to etanercept, the available evidence has not shown any relationship yet. Objective The aim of this study was to determine if there is any correlation among etanercept serum levels, the presence of anti-etanercept antibodies and clinical response to this treatment in psoriatic patients. Setting A 1500-bed hospital (A Coruña University Hospital Complex). Method A retrospective observational study in psoriatic patients treated with etanercept (50 mg once weekly) was carried out. Psoriasis Area and Severity Index scale and adverse reactions were recorded at the time of the extraction sample. The pharmacokinetic monitoring was evaluated at the previous time points by extracting peripheral blood samples before the dose administration. Etanercept and anti-etanercept antibodies concentrations were quantified by two sandwich-type ELISA immunoassays. The patients were classified into three groups (good, partial and nonresponders) in accordance with the treatment efficacy at the blood assessment moments. The Kruskall-Wallis test and Spearman correlation assay were used to assess the efficacy and incidence of adverse effects according to the etanercept concentration and anti-etanercept antibodies, considering p values of <0.05 as statistically significant. This statistical analysis was conducted using SPSS software (version19.0). Main outcome measures Etanercept and anti-etanercept antibodies trough serum levels and clinical response. Results 38 patients were included. 26 patients (68.4 %) were good, 5 (13.2 %) were partial and 7 (18.4 %) were non-responders. There was no significant difference with respect to etanercept levels: 2.7 μg/mL (range 0.7-5.6) versus 2.2 μg/mL (range 1.0-3.5) versus 1.73 μg/mL (range 0.1-2.3), respectively (p = 0.085). Nevertheless, a positive correlation between percentage decrease in the Psoriasis Area and Severity Index scale value with respect to the baseline value and etanercept concentration was found (p = 0.011). No anti-etanercept antibodies were detected; nor was there a significant difference in the incidence of adverse effects (p = 0.8523). Conclusions Our results showed a positive correlated between etanercept concentration and the percentage decrease in the Psoriasis Area and Severity Index scale value. The incidence of anti-etanercept antibodies in psoriatic patients was low.

Previous etanercept failure seems not influence the success and safety of adalimumab treatment in moderate to severe plaque psoriasis.