Dyssynchrony and resynchronization in heart failure - Effects on regional and global gene expression in a murine pacemaker model

Cardiac resynchronization therapy (CRT) improves prognosis for patients with heart failure with reduced ejection fraction and dyssynchrony due to left bundle branch block (LBBB) or right ventricular pacing (RVP). On a functional level, CRT reverses the heterogeneity in timing of contractions between different left ventricular wall segments and conveys a reversal of dyssynchrony-induced cardiac remodelling. The mechanisms behind the positive effects of resynchronization are unknown. However, a canine model of dyssynchrony and resynchronization has shown improved inotropy by modulation of calcium response and reversed cardiomyocyte hypertrophy.1 A heterogeneity in gene expression between early and late activated left ventricular wall segments has also been described in dogs with LBBB, and this pattern was ameliorated with resynchronization.2 We have recently described a pacemaker fitted for mice, that allows for in vivo studies of dyssynchrony induced by RVP.3 In this study, we describe regional and global gene expression changes induced by dyssynchrony and reversed by resynchronization. The mouse heart tissue used in this study was provided from a previously published experiment where a detailed study protocol can be found.3 Heart failure was induced through ischaemia/reperfusion and a pacemaker implanted. Mice were divided into three groups: synchronous heart failure (SHF) with sinus rhythm for 4 weeks (n = 10), dyssynchronous heart failure (DHF) by right ventricular pacing for 4 weeks (n = 10), and resynchronized heart failure (RHF) by right ventricular pacing for 2 weeks followed by 2 weeks in sinus rhythm (n = 9). Mice were sacrificed at 4 weeks and the septum (early activated) and lateral wall (late activated) used for RNA sequencing in this study. RNA was isolated from 3 mg of heart muscle. Trueseq Poly-A enriched libraries (Illumina) were prepared and single-end sequenced using Nextseq 75 cycles. Differential gene expression analysis was performed on the mapped read counts and a false discovery rate (FDR) of <1% was considered significant. We found 14 361 genes expressed in the tissue. Regional differences in the left ventricular gene expression were determined by comparing the late-activated lateral wall and the early-activated septum within each group (Figure 1A–C). Expression of 1923 genes (13%) differed between late and early activated wall segments in DHF. There were no differences in gene expression between the late- and early-activated wall segments in SHF or RHF. Hence, dyssynchrony induces a marked heterogeneity in gene expression within the left ventricle that is not observed with resynchronization. (A–C) Volcano plots describing the regional (intracardiac) heterogeneity in gene expression of the different groups. The −Log10 of the P-value is given on the y-axis (false detection rate <1%) and Log2 of fold change on the x-axis. A significant P-value of <0.05, after correction for false detection rate <1%, is indicated by a grey horizontal line. The dots correspond to one gene detected by RNA sequencing. Red dots correspond to genes with a significantly changed expression. (D) A VENN diagram of the genes with globally changed expression levels (between-group comparisons). The overlap is shown in the middle and the two genes with the highest fold change are given in the box to the right. (E) The 69 genes that changed globally in all comparisons between dyssynchronous heart failure and synchronous heart failure or resynchronized heart failure are listed. Upregulated genes to the left and downregulated to the right. Cdca3, cell division cycle-associated protein 3; DHF, dyssynchronous heart failure; E, early-activated septum; FDR, false detection rate; Fignl1, fidgetin-like protein 1; L, late-activated left ventricular later wall segment; RHF, resynchronized heart failure; SHF, synchronous heart failure. Next, we wanted to find out whether there were global changes in gene expression induced by dyssynchrony and reversed with resynchronization. We compared gene expression in the same left ventricular segments between the three groups. DHF-Late differed from SHF-Late in the expression of 837 genes (6%) and from RHF-Late in the expression of 305 genes (2%). When SHF-Late was compared to RHF-Late no significant difference in gene expression was found. The same pattern appeared when gene expression in the early activated septal segment was compared between groups. DHF-Early compared to SHF-Early had 1400 genes (10%) with different expression and DHF-Early compared to RHF-Early 768 genes (5%). SHF-Early compared to RHF-Early had similar gene expression. Hence, dyssynchrony induces gene expression changes in both early- and late-activated left ventricular segments that are not found with resynchronization. From the globally changed genes, we identified 69 genes that changes expression in all between-group comparisons (Figure 1D and E). Two genes were upregulated > 2-fold; Cell division cycle-associated protein 3 (Cdca3) and Fidgetin-like protein 1 (Fignl1), both connected to the cell cycle. The global change in gene expression induced by dyssynchrony and reversed with resynchronization described in this study is novel and may constitute a platform for further studies of its biological relevance. That the biological changes with dyssynchrony and resynchronization are not only localized to the late-activated lateral left ventricular segment, even though this part of the ventricle is subjected to the most obvious remodelling and stress, is a notion supported by earlier studies of protein expression in dogs4 and a smaller study in humans with endomyocardial biopsies from the septum.5 The regional heterogeneity in gene expression with dyssynchrony and its resolution with resynchronization align with earlier studies in a canine model which increases the translational importance of the mouse-pacemaker model. The lack of gene expression heterogeneity in RHF and SHF, which was observed in DHF indicate that this gene expression pattern may play a role in DHF-associated remodelling and may be reversed when synchronized contractions are reinstituted. Among the 69 genes involved in all global changes Cdca3 and Fignl1 had a fold change >2. These are genes associated with the cell cycle, however, cardiomyocytes regenerate at a very low rate after the antenatal period. Increased mitotic activity could stem from supportive cells like fibroblasts or smooth muscle cells. Another possibility is that the cardiomyocyte hypertrophy observed in DHF activates genes associated with the cell cycle. We used a heart failure, mouse-pacemaker model to demonstrate that long-term dyssynchronous activation and contraction of the failing heart induce changes in gene expression, which are both regional and global. Importantly, resynchronization was not associated with these gene expression changes. This study demonstrates the biological relevance of the model and facilitates further studies of cellular and molecular processes with dyssynchrony and resynchronization in an animal model in which genetic manipulation is feasible. Dr. Marcus Ståhlberg is a specialist and associate professor of cardiology at Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden. He has been trained at Karolinska Institutet, Stockholm, Sweden (MD and PhD) and Johns Hopkins University, Baltimore, Maryland, USA (Postdoc) where he developed the mouse pacemaker model used in this study. Clinical and research expertise include arrhythmias, heart failure, CEID-implantation and cardiovascular dysautonomia. We thank Dr David A. Kass for providing materials for the study. The Swedish Research Council (2014-352); and The Swedish Heart Lung Foundation (20170093, 20180500). Data from this study will be made available upon request to the corresponding author.