Functional Networks of Nucleocytoplasmic Transport-Related Genes Differentiate Ischemic and Dilated Cardiomyopathies. A New Therapeutic Opportunity

<div><p>Heart failure provokes alterations in the expression of nucleocytoplasmic transport-related genes. To elucidate the nucleocytoplasmic transport-linked functional network underlying the two major causes of heart failure, ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM), we examined global transcriptome profiles of left ventricular myocardium tissue samples from 31 patients (ICM, n = 10; DCM, n = 13) undergoing heart transplantation and control donors (CNT, n = 8) using RNA-Sequencing and GeneMANIA. Comparative profiling of ICM <i>versus</i> control and DCM <i>versus</i> control showed 1081 and 2440 differentially expressed genes, respectively (>1.29-fold; <i>P</i><0.05). GeneMANIA revealed differentially regulated functional networks specific to ICM and DCM. In comparison with CNT, differential expression was seen in 9 and 12 nucleocytoplasmic transport-related genes in ICM and DCM groups, respectively. <i>DDX3X</i>, <i>KPNA2</i>, and <i>PTK2B</i> were related to ICM, while <i>SMURF2</i>, <i>NUP153</i>, <i>IPO5</i>, <i>RANBP3</i>, <i>NOXA1</i>, and <i>RHOJ</i> were involved in DCM pathogenesis. Furthermore, the two pathologies shared 6 altered genes: <i>XPO1</i>, <i>ARL4, NFKB2</i>, <i>FHL3</i>, <i>RANBP2</i>, and <i>RHOU</i> showing an identical trend in expression in both ICM and DCM. Notably, the core of the derived functional networks composed of nucleocytoplasmic transport-related genes (<i>XPO1</i>, <i>RANBP2</i>, <i>NUP153</i>, <i>IPO5</i>, <i>KPNA2</i>, and <i>RANBP3</i>) branched into several pathways with downregulated genes. Moreover, we identified genes whose expression levels correlated with left ventricular mass index and left ventricular function parameters in HF patients. Collectively, our study provides a clear distinction between the two pathologies at the transcriptome level and opens up new possibilities to search for appropriate therapeutic targets for ICM and DCM.</p></div

Functional network for ICM (A) and DCM (B).

<p>Analysis was based on protein-protein interaction (dark blue lines) and pathway (light blue lines) databases with the nucleocytoplasmic transport-related genes (>1.29-fold; <i>P</i><0.05). GeneMANIA retrieved known and predicted interactions between these genes and added extra genes (small grey circles) that are strongly connected to query genes (large circles). Red indicates upregulation, green indicates downregulation and black indicates no differences in the trend expression between the two comparisons (ICM <i>vs</i>. CNT and DCM <i>vs</i>. CNT).</p

Clinical characteristics of patients according to HF etiology.

<p>Data are showed as the mean value ± SD. ICM, ischemic cardiomyopathy; DCM, dilated cardiomyopathy; NYHA, New York Heart Association; BMI, body mass index; EF, ejection fraction; FS, fractional shortening; LVESD, left ventricular end-systolic diameter; LVEDD, left ventricular end-diastolic diameter; LV mass, left ventricular mass; LVMI, left ventricular mass index.</p><p>*<i>P</i><0.05 significantly different between ICM and DCM patients.</p

Correlations between gene expression and echocardiographic parameters (<i>P</i><0.05).

<p>Correlations between gene expression and echocardiographic parameters (<i>P</i><0.05).</p

New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos

<p>Novel next-generation sequencing procedures have rapidly emerged into the preimplantation genetic screening framework. This work presents the design and validation of a new low-coverage whole-genome sequencing assay for aneuploidy detection in single blastomeres and trophectodermal samples from preimplantation embryos. The validation ensures analytical sensitivity, specificity, robustness, precision, limit of detection, resolution, and reproducibility. Specific parameters to measure the performance are defined, and the results are compared with a standardized array-based method to stablish the concordance. From the single cell genomics point of view, the main novelties are the length of reads of the libraries (150 nucleotides) together with a paired-end strategy and the design of an original algorithm and copy number viewer. A total of 129 samples were included in six experimental runs using a MiSeq Illumina platform. Samples included: single amniocytes, single blastomeres (cleavage-stage embryos), trophectoderm samples (blastocyst), and diluted DNA. Sensitivity and specificity were calculated per chromosome yielding 96% and 99%, respectively. The percentage of concordant samples was 98.2% and all of the aneuploid samples were confirmed. In conclusion, the validation yields highly reliable and reproducible results, representing an accurate and cost-effective strategy for the routine detection of aneuploidy in human embryos.</p

Protein expression levels of COL8A1, COL16A1, TGF-β1, and MMP2 in dilated cardiomyopathy.

<p>Bar graph comparing protein levels of non-fibrillar collagens COL8A1 and COL16A1 (A), and TGF-β1 and MMP2 (B) levels in dilated hearts (grey bars) <i>vs</i>. controls (dark bars). For western blot analyses 28 LV samples from DCM patients were used. The values from the controls were set to 1. Bars display fold change (FC) ± standard error of the mean (SEM). Results were considered statistically significant at *<i>P</i> < 0.05 <i>vs</i>. CNT.</p

Altered expression levels of collagen genes in dilated hearts.

<p>(A) Bar graph comparing mRNA expression levels of collagen genes in dilated hearts (grey bars) <i>vs</i>. controls (dark bars). (B) Principal Component Analysis based on non-fibrillar collagen fold change values, shows a clear differentiation of the DCM and CNT groups. For RNA-seq analyses 13 LV samples from DCM patients were used. The values from the controls were set to 1. Bars display fold change (FC) ± standard error of the mean (SEM). Results were considered statistically significant at *<i>P</i> < 0.05 and **<i>P</i> < 0.01 <i>vs</i>. CNT.</p

Relationship between collagen mRNA expression levels and remodeling parameters of DCM patients.

<p>Left ventricular mass index (LVMI) vs. <i>COL8A1</i> mRNA levels. Arbitrary units (au).</p

Expressed mRNA levels of molecular markers of proliferation, hypertrophy, apoptosis, fibrosis and inflammation in DCM patients.

<p>Expressed mRNA levels of molecular markers of proliferation, hypertrophy, apoptosis, fibrosis and inflammation in DCM patients.</p

Relationships between the differentially expressed non-fibrillar collagens COL8A1 and COL16A1, and proliferation, apoptosis, and fibrosis molecular markers at mRNA and protein levels in DCM patients.

<p>Relationships between the differentially expressed non-fibrillar collagens COL8A1 and COL16A1, and proliferation, apoptosis, and fibrosis molecular markers at mRNA and protein levels in DCM patients.</p