Interplay between cardiac transcription factors and non-coding RNAs in predisposing to atrial fibrillation

Review[Abstract] There is growing evidence that putative gene regulatory networks including cardio-enriched transcription factors, such as PITX2, TBX5, ZFHX3, and SHOX2, and their effector/target genes along with downstream non-coding RNAs can play a potentially important role in the process of adaptive and maladaptive atrial rhythm remodeling. In turn, expression of atrial fibrillation-associated transcription factors is under the control of upstream regulatory non-coding RNAs. This review broadly explores gene regulatory mechanisms associated with susceptibility to atrial fibrillation—with key examples from both animal models and patients—within the context of both cardiac transcription factors and non-coding RNAs. These two systems appear to have multiple levels of cross-regulation and act coordinately to achieve effective control of atrial rhythm effector gene expression. Perturbations of a dynamic expression balance between transcription factors and corresponding non-coding RNAs can provoke the development or promote the progression of atrial fibrillation. We also outline deficiencies in current models and discuss ongoing studies to clarify remaining mechanistic questions. An understanding of the function of transcription factors and non-coding RNAs in gene regulatory networks associated with atrial fibrillation risk will enable the development of innovative therapeutic strategies.Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; GRC 2013/06

Myocardial transcription factors in diastolic dysfunction: clues for model systems and disease

[Abstract] There are multiple intrinsic mechanisms for diastolic dysfunction ranging from molecular to structural derangements in ventricular myocardium. The molecular mechanisms regulating the progression from normal diastolic function to severe dysfunction still remain poorly understood. Recent studies suggest a potentially important role of core cardio-enriched transcription factors (TFs) in the control of cardiac diastolic function in health and disease through their ability to regulate the expression of target genes involved in the process of adaptive and maladaptive cardiac remodeling. The current relevant findings on the role of a variety of such TFs (TBX5, GATA-4/6, SRF, MYOCD, NRF2, and PITX2) in cardiac diastolic dysfunction and failure are updated, emphasizing their potential as promising targets for novel treatment strategies. In turn, the new animal models described here will be key tools in determining the underlying molecular mechanisms of disease. Since diastolic dysfunction is regulated by various TFs, which are also involved in cross talk with each other, there is a need for more in-depth research from a biomedical perspective in order to establish efficient therapeutic strategies.Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; GRC 2013/06

Expression of the Wilms tumor suppressor gene (Wt1) in a subpopulation of embryonic cardiomyocytes is required for cardiac development

The Wilms’ tumour gene, WT1, encodes a zinc-finger transcription factor involved in the development of several organs. WT1 is expressed during mammalian embryonic development in many tissues, including the urogenital system, spleen, spinal cord, diaphragm, coelomic epithelium and epicardium (Armstrong et al., 1993; Moore et al., 1999; Cano et al., 2016; Ariza et al., 2016; Carmona et al., 2016). Post-transcriptional modifications of the Wt1 pre-mRNA lead to the production of up to 24 different isoforms, which seem to serve distinct but overlapping cellular and developmental functions. We have checked if Wt1 is expressed by the embryonic myocardium. Using transgenic mice lines for lineage tracing (mWt1/IRES/GFPCre;ROSA26REYFP), we have detected a small population of cardiomyocytes from a Wt1-expressing cell lineage in early developmental stages (E8.5-E9.5). These cardiomyocytes were mainly located in the inflow tract, but some of them were observed in the ventricles. We confirmed Wt1 expression by RT-PCR in hearts before the attachment of proepicardial cells, when the cardiac tube is only constituted of myocardium and endocardium. We have also studied the mRNA levels of the four main isoforms of Wt1 in a reverse transcriptase-polymerase chain reaction (RT- PCR) assay. We have found differential expression of these Wt1 isoforms in the embryonic heart. Conditional deletion of Wt1 in cardiac Troponin T expressing cells caused severe damage in the developing heart, particularly muscular defects in the interventricular septum and free ventricular walls, as well as defective sinus venous formation. These embryos did not survive after birth. Likewise, conditional deletion of GATA4 in Wt1-expressing cells causes a similar phenotype in the myocardium, but also defects in the proepicardium, epicardium and subepicardial space, causing embryonic death around E11.5. Thus, we conclude that Wt1 is expressed in a subpopulation of early embryonic cardiomyocytes, and this expression seems to be essential for heart development.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry

[Abstract] We have developed a set of simple modifications of the green fluorescent protein (GFP)fragment reassembly assay in bacteria that permits: (i)fluorescent microscopy visualization of GFP reassembly only 1-2 h after induction of protein expression, thus approximating the detection of GFP reassembly to the real-time dynamics of protein complex formation in living cells; (ii) spectrofluorometric detection of reassembled GFP fluorescent signals directly in lysates from cell suspension thereby avoiding, in many cases, the need for tag-affinity isolation of protein complexes; and (iii) comparative quantification of signal intensity in numerous cell-sample lysates using a Bio-Rad IQ5 spectrofluorometric detection system (Bio-Rad Laboratories, Madrid, Spain). Collectively, the results demonstrate that the combination of microscopic and spectrofluorometric detection provides a time-saving and sensitive alternative to existing methods of fluorescence complementation analysis.Ministerio de Educación y Ciencia; SAF2004-01462Galicia. Consellería de Innovación, Industria e Comercio; PGIDIT04BTF161001P

El número en la escuela

El artículo “El Número en la Escuela”presenta avances de investigación realizados por estudiantes de la Maestría en Docencia de las Matemáticas de la Universidad Pedagógica Nacional como parte de sus respectivos trabajos de grado. El primero, referido a la formación de docentes de preescolar y primeros grados de educación primaria sobre Estructura Aditiva, expone la forma en que abordar dicha estructura contribuye al proceso de construcción del número Natural en los primeros grados de escolaridad. El segundo, basado en el modelo del profesor Carlo Federici, estudia la construcción de los números racionales como operadores sobre magnitudes, específicamente, sobre longitudes cuya representación son los segmentos. Y el tercero, permite reflexionar sobre el acercamiento al concepto de número real con estudiantes de Secundaria y Media

In vivo forced expression of myocardin in ventricular myocardium transiently impairs systolic performance in early neonatal pig heart

[Abstract] The aim of this study was to determine the effects of forced expression of myocd-A in the left ventricular (LV) myocardium on cardiac performance in early neonatal piglets. LV transfection with the gene for homeodomain only protein (hop), an antagonist of myocd-mediated activities, was also performed. Gene delivery was performed in 6-day-old piglets using a low-traumatic, catheter-based, video-assisted procedure developed by us for direct intra-myocardial injections of plasmid DNA into 3-4 target areas of the ventral LV free wall (LVFW). Two isoforms of porcine myocd were identified, cloned and characterized: the exon 11-lacking myocd-A and its larger exon 11-containig variant, myocd-B. In neonatal piglets, myocd-A seems to be a cardio-predominant isoform enriched in the LVFW/septum, whereas the myocd-B isoform is detected not only in the heart but also in various smooth muscle cell-containing tissues. Intramyocardial myocd-A gene delivery resulted in forced transgene expression in the target areas of the LVFW as compared to controls. On day 2 post-delivery, a marked decrease of LV-end systolic pressure values (an accepted marker for impaired LV function) was observed in myocd-A-transfected piglets as compared to hop-transfected and control groups. In addition, forced myocd-A expression in the LVFW caused abnormal ECG. A significant up-regulation of the gene for fetal-predominant muscle light chain 3F myosin was detected in myocd-A-transfected LVFWs harvested on day 2 post-delivery. Extended analysis on day 7 post-delivery revealed a drop decrease in myocd-A transgene expression in target LVFW regions which was correlated with normalization of the LV systolic parameters in experimented piglets.Ministerio de Educación y Ciencia; SAF2004-01462Galicia. Consellería de Innovación, Industria e Comercio; PGIDIT04BTF16001 PR

Identification of Candidate Genes Potentially Relevant to Chamber-Specific Remodeling in Postnatal Ventricular Myocardium

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

Exon-skipping brain natriuretic peptide variant is overexpressed in failing myocardium and attenuates brain natriuretic peptide production in vitro

Original research[Abstract] Brain natriuretic peptide/natriuretic peptide precursor B (NPPB) is one of the most studied genes in relation to heart failure (HF) conditions. However, it is still unclear as to whether alternative splicing could create NPPB mRNA variants, which may be expressed in normal and diseased myocardium. We aimed to identify and characterize a novel alternatively spliced variant of porcine and human NPPB resulting from exon 2 skipping (designated as ΔE2-NPPB). A variety of conventional molecular, biochemical and immunochemical methods were used to examine the expression and functional consequences of ΔE2-NPPB in vitro and in vivo. The pig ΔE2-NPPB mRNA is effectively translated into stable protein in cell-based assays but, in contrast to normally spliced NPPB, the ΔE2-NPPB protein is not secreted into the media. Co-transfection assays demonstrate that ΔE2-NPPB attenuates production and secretion of normally spliced NPPB, suggesting a negative feedback loop of NPPB signaling through generation of ΔE2-NPPB. The inhibitory effects of ΔE2-NPPB on the expression of NPPB are associated with sequence elements residing in exon 3 of ΔE2-NPPB. In piglets, ΔE2-NPPB gene expression is downregulated in both ventricles after birth, but it is markedly re-activated in the postnatal myocardium in experimental diastolic heart failure. In addition, we demonstrate that the exon-skipped NPPB variants are expressed in the postnatal and adult human myocardium and upregulated at end-stage HF due to dilated cardiomyopathy. Our work uncovers an important role of alternative exon skipping in the regulation of NPPB gene expression, thereby pinpointing a putative new mechanism for post-transcriptional regulation of NPPB production and secretion.Ministerio de Ciencia e Innovación; SAF2008- 00337Ministerio de Ciencia e Innovación; SAF2004-0146

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium

[Abstract] 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 stress-inducible 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.Ministerio de Ciencia e Innovación; SAF2004-01462Ministerio de Ciencia e Innovación; SAF2008-00337Galicia. Consellería de Economía e Industria; 08CSA008161P

La expresión miocárdica del gen supresor del tumor de Wilms (Wt1) es esencial para el desarrollo cardíaco y reaparece en cardiomiocitos adultos

El gen supresor del tumor de Wilms, Wt1, codifica un factor de transcripción del tipo “dedos de zinc” implicado en transcripción, regulación post-transcripcional e interacciones proteína-proteína. La función de este gen es indispensable para el desarrollo urogenital y está implicado también en el desarrollo embrionario de diversos órganos (hígado, bazo, adrenales); todos ellos incorporan durante su morfogénesis a una población mesenquimática derivada del epitelio celómico que los recubre y que es el tejido en el que Wt1 presenta su máxima expresión embrionaria. Durante el desarrollo cardiaco, Wt1 se expresa en el epicardio (epitelio celómico que recubre el corazón) y en las células mesenquimáticas derivadas del epicardio, pero su expresión no ha sido estudiada hasta ahora en el miocardio. En este trabajo hemos estudiado la expresión miocárdica de Wt1 utilizando dos líneas transgénicas de ratón, un knockin reportero (Wt1GFP/+) y un modelo de trazado de linaje (mWt1/IRES/GFPCre; ROSA26REYFP). Estos modelos animales nos han permitido identificar una población de cardiomiocitos que expresan Wt1 en estadios tempranos (desde E9 hasta E12) y cuyo linaje se encuentra sobre todo en los ventrículos y en el septo interventricular, dos regiones del corazón severamente afectadas por diversas enfermedades cardíacas adultas. De acuerdo con estos resultados, hemos detectado también la expresión de Wt1 en cardiomiocitos humanos adultos. Esta expresión disminuye significativamente en la insuficiencia cardiaca debida a isquemia, pero no sucede así en otras cardiomiopatías dilatadas no isquémicas. El análisis mediante citometría de flujo usando el reportero Wt1GFP/+ nos ha permitido cuantificar la población de cardiomiocitos Wt1+ en diferentes estadios y probar que esta población muestra los mayores niveles de expresión de VCAM de todo el corazón, identificando por lo tanto a una subpoblación singular de cardiomiocitos ventriculares. Un modelo in vitro de diferenciación hacia miocardio de células madre embrionarias de ratón ha mostrado también la breve expresión de Wt1 en una fracción de los cardiomiocitos obtenidos, lo que confirma que la expresión de Wt1 es un evento normal durante la diferenciación embrionaria de cardiomiocitos. Con el fin de comprobar si esta población de cardiomiocitos juega un papel determinado en la morfogénesis cardíaca, hemos realizado una deleción condicional de Wt1 usando la tecnología Cre/LoxP. Nuestros ratones mutantes (TnTCre;Wt1flox/flox) muestran defectos en el septo interventricular y paredes libres de ambos ventrículos. Estas anomalías suelen ser letales antes del parto, pero hemos detectado un número reducido de ratones mutantes adultos. En estos ratones el electrocardiograma muestra serias alteraciones que sugieren también defectos en el sistema de conducción. Todos estos resultados revelan que Wt1 puede desempeñar funciones hasta ahora desconocidas tanto en el desarrollo cardiaco como en la fisiología y la patofisiología del miocardio adulto. El estudio de estas funciones puede ser relevante desde el punto de vista traslacional ya que podrían ayudarnos a identificar dianas celulares o seleccionar subtipos de cardiomiocitos para el tratamiento de enfermedades cardiovasculares.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech