Targeted myocardial gene expression in failing hearts by RNA sequencing

Abstract

BackgroundMyocardial recovery with left ventricular assist device (LVAD) therapy is highly variable and difficult to predict. Next generation ribonucleic acid (RNA) sequencing is an innovative, rapid, and quantitative approach to gene expression profiling in small amounts of tissue. Our primary goal was to identify baseline transcriptional profiles in non-ischemic cardiomyopathies that predict myocardial recovery in response to LVAD therapy. We also sought to verify transcriptional differences between failing and non-failing human hearts.MethodsRNA was isolated from failing (n = 16) and non-failing (n = 8) human hearts. RNA from each patient was reverse transcribed and quantitatively sequenced on the personal genome machine (PGM) sequencer (Ion torrent) for 95 heart failure candidate genes. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser Suite™. Differential expression analyses were conducted by empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes between failing and non-failing groups, and between responder and non-responder groups respectively. Targeted cardiac gene messenger RNA (mRNA) expression was analyzed in proportion to the total number of reads. Gene expression profiles from the PGM sequencer were validated by performing RNA sequencing (RNAseq) with the Illumina Hiseq2500 sequencing system.ResultsThe failing sample population was 75% male with an average age of 50 and a left ventricular ejection fraction (LVEF) of 16%. Myosin light chain kinase (MYLK) and interleukin (IL)-6 genes expression were significantly higher in LVAD responders compared to non-responders. Thirty-six cardiac genes were expressed differentially between failing and non-failing hearts (23 decreased, 13 elevated). MYLK, Beta-1 adrenergic receptor (ADRB1) and myosin heavy chain (MYH)-6 expression were among those significantly decreased in failing hearts compared to non-failing hearts. Natriuretic peptide B (NPPB) and IL-6 were significantly elevated. Targeted gene expression profiles obtained from the Ion torrent PGM sequencer were consistent with those obtained from Illumina HiSeq2500 sequencing system.ConclusionsHeart failure is associated with a network of transcriptional changes involving contractile proteins, metabolism, adrenergic receptors, protein phosphorylation, and signaling factors. Myocardial MYLK and IL-6 expression are positively correlated with ejection fraction (EF) response to LVAD placement. Targeted RNA sequencing of myocardial gene expression can be utilized to predict responders to LVAD therapy and to better characterize transcriptional changes in human heart failure.

Novel myocardial markers GADD45G and NDUFS5 identified by RNA-sequencing predicts left ventricular reverse remodeling in advanced non-ischemic heart failure: a retrospective cohort study

“…First, we identified markers for predicting LVRR by comprehensive transcriptome analysis of single samples obtained prior to treatment. Some studies showed associations between certain mRNAs or miRNAs and myocardial recovery or subsequent VAD removal after LVAD implantation [13,14,44]. However, these studies analyzed only limited number of Fig.…”

“…Dhar et al performed RNA-seq for the cardiac samples obtained from the patients with non-ischemic advanced heart failure requiring LVAD implantation, and showed that the expressions of myosin light chain kinase and interleukin-6 genes were significantly higher in the LVAD responders as compared to the non-responders. In this study, however, only 95 genes associated with heart failure were examined [44]. RNAseq in the present study performed a comprehensive transcriptome analysis, thereby demonstrating previously unknown, novel mechanisms of LVRR.…”

“…First, this study was carried out at a single institution with a small number of samples. Previous studies examining myocardial samples from severely failing hearts could collect only around 10 samples for each treatment group [14,20,44]. These facts probably reflect the small population of patients whose heart failure status are severe enough to assess pathological specimen of the heart.…”

Transcriptional and Free Radical Responses to LVAD Therapy

“…An additional 16 patients had blood drawn at the time of LVAD implant and post-LVAD for measurement of free radicals. Our RNA library construction, targeted DNA template and sequencing were performed, as we previously described [4]. The study targeted 140 genes were previously identi ed by quantitate RT PCR, gene arrays and next generation sequencing has been associated with heart failure [4,5,[10][11][12][13][14][15][16].…”

“…While these devices provide immediate improvement in cardiac output and quality of life, signi cant problems remain such as death due to infections, ongoing heart failure, strokes, and device malfunctions [3]. Furthermore, recovery of heart function and device explant is rare with many patients deteriorating over time [4].…”