Transcriptomics-Based Network Medicine Approach Identifies Metformin as a Repurposable Drug for Atrial Fibrillation

Effective drugs for atrial fibrillation (AF) are lacking, resulting in significant morbidity and mortality. This study demonstrates that network proximity analysis of differentially expressed genes from atrial tissue to drug tar-gets can help prioritize repurposed drugs for AF. Using enrichment analysis of drug-gene signatures and functional testing in human inducible pluripotent stem cell (iPSC)-derived atrial-like cardiomyocytes, we identify metformin as a top repurposed drug candidate for AF. Using the active compactor, a new design analysis of large-scale longitudinal electronic health record (EHR) data, we determine that metformin use is significantly associated with a reduced risk of AF (odds ratio = 0.48, 95%, confidence interval [CI] 0.36- 0.64, p \u3c 0.001) compared with standard treatments for diabetes. This study utilizes network medicine meth-odologies to identify repurposed drugs for AF treatment and identifies metformin as a candidate drug

PANCR, the PITX2 Adjacent Noncoding RNA, Is Expressed in Human Left Atria and Regulates PITX2c Expression

Background-Genome-wide studies reveal that genetic variants at chromosome 4q25 constitute the strongest locus associated with atrial fibrillation, the most frequent arrhythmia. However, the mechanisms underlying this association are unknown. Our goal is to find and characterize left atrial-expressed transcripts in the chromosome 4q25 atrial fibrillation risk locus that may play a role in atrial fibrillation pathogenesis. Methods and Results-RNA sequencing performed on human left/right pairs identified an intergenic long noncoding RNA adjacent to the PITX2 gene, which we have named PANCR (PITX2 adjacent noncoding RNA). In a human tissue screen, PANCR was expressed specifically in the left atria and eye and in no other chambers of the heart. The levels of PANCR and PITX2c RNAs were highly correlated in 233 human left atrial appendage samples. PANCR levels were not associated with either atrial rhythm status or the genotypes of the chromosome 4q25 atrial fibrillation risk variants. Both PANCR and PITX2c RNAs were induced early during differentiation of human embryonic stem cells into cardiomyocytes. Because long noncoding RNAs often control gene expression, we performed siRNA-mediated knockdown of PANCR, and this treatment repressed PITX2c expression and mimicked the effects of PITX2c knockdown on global mRNA and miRNA expression. Cell fractionation studies demonstrate that PANCR is primarily localized in the cytoplasm. Conclusions-PANCR and PITX2c are coordinately expressed early during cardiomyocyte differentiation from stem cells. PANCR knockdown decreased PITX2c expression in differentiated cardiomyocytes, altering the transcriptome in a manner similar to PITX2c knockdown

PANCR, the PITX2

BACKGROUND: Genome-wide studies reveal that genetic variants at chromosome 4q25 constitute the strongest locus associated with atrial fibrillation (AF), the most frequent arrhythmia. However, the mechanisms underlying this association are unknown. Our goal is to find and characterize left atrial expressed transcripts in the chromosome 4q25 AF risk locus that may play a role in AF pathogenesis. METHODS AND RESULTS: RNA sequencing performed on human left/right pairs identified an intergenic long noncoding RNA (lncRNA) adjacent to the PITX2 gene, which we have named PANCR (PITX2 adjacent noncoding RNA). In a human tissue screen, PANCR was expressed specifically in the left atria and eye, and in no other chambers of the heart. The levels of PANCR and PITX2c RNAs were highly correlated in 233 human left atrial appendage samples. PANCR levels were not associated with either atrial rhythm status or the genotypes of the chromosome 4q25 AF risk variants. Both PANCR and PITX2c RNAs were induced early during differentiation of human embryonic stem cells into cardiomyocytes. Since lncRNAs often control gene expression, we performed siRNA-mediated knockdown of PANCR; and, this treatment repressed PITX2c expression and mimicked the effects of PITX2c knockdown on global mRNA and miRNA expression. Cell fractionation studies demonstrate that PANCR is primarily localized in the cytoplasm. CONCLUSIONS: PANCR and PITX2c are coordinately expressed early during cardiomyocyte differentiation from stem cells. PANCR knockdown decreased PITX2c expression in differentiated cardiomyocytes, altering the transcriptome in a manner similar to PITX2c knockdown

Atrial Fibrillation associated chromosome 4q25 variants are not associated with PITX2c expression in human adult left atrial appendages.

Atrial Fibrillation (AF), the most common sustained arrhythmia, has a strong genetic component, but the mechanism by which common genetic variants lead to increased AF susceptibility is unknown. Genome-wide association studies (GWAS) have identified that the single nucleotide polymorphisms (SNPs) most strongly associated with AF are located on chromosome 4q25 in an intergenic region distal to the PITX2 gene. Our objective was to determine whether the AF-associated SNPs on chromosome 4q25 were associated with PITX2c expression in adult human left atrial appendages. Analysis of a lone AF GWAS identified four independent AF risk SNPs at chromosome 4q25. Human adult left atrial appendage tissue was obtained from 239 subjects of European Ancestry and used for SNP analysis of genomic DNA and determination of PITX2c RNA expression levels by quantitative PCR. Subjects were divided into three groups based on their history of AF and pre-operative rhythm. AF rhythm subjects had higher PITX2c expression than those with history of AF but in sinus rhythm. PITX2c expression was not associated with the AF risk SNPs in human adult left atrial appendages in all subjects combined or in each of the three subgroups. However, we identified seven SNPs modestly associated with PITX2c expression located in the introns of the ENPEP gene, ∼54 kb proximal to PITX2. PITX2c expression in human adult left atrial appendages is not associated with the chromosome 4q25 AF risk SNPs; thus, the mechanism by which these SNPs are associated with AF remains enigmatic

4q25 SNPs Independently Associated with AF.

<p>Results from logistic regression fits of the following 2 models.</p><p>Marginal Model for each SNP: History of lone AF using as covariates Sex +4 principal components of genetic sharing.</p><p>Full Model for each SNP: History of lone AF using as covariates Sex +4 principal components of genetic sharing+other 3 SNPs shown on table.</p

Left atrial appendage surgical and donor patient characteristics.

*<p>Median (interquartile range).</p>&<p>Not including donors, for which this information not available.</p>#<p>p-value by chi-square test.</p>$<p>p-value by Kruskal Wallis nonparametric ANOVA.</p>##<p>p-value by chi-square comparing only AF/SR and AF/AF groups.</p

Adjusted and unadjusted expression of <i>PITX2c</i> in human left atrial appendages in AF controls.

<p>Log2 <i>PITX2c</i> expression, normalized to <i>ACTC1</i>, in the 16 donor and 24 surgical No AF samples uncorrected (A), or after correction for age and sex (B). There was no significant difference in <i>PITX2c</i> expression between these groups by non-parametric Mann-Whitney t-test. Individual values are shown along with median and interquartile range.</p