Heart failure-associated changes in RNA splicing of sarcomere genes

Abstract

Background: Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts.

Methods and results: We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy.

Conclusions: Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

Figure 1. Systematic differences of RNA splicing in ICM compared to control

Genes were scored for likelihood of alternative splicing (FIRMA score). Two way clustering of samples and genes with significantly different FIRMA scores (FDR < 0.01) separated samples by diagnosis, with one misassignment. Heat map is color coded by FIRMA score (blue-red, −1.9 to +1.9 standard deviations from the mean). Genes with significantly different gene level expression (P < 0.05) were excluded.

Fig. 2. Differences in Alternative Splicing between ICM and control

a–b. Examples of exon array data suggestive of differential alternative splicing between ICM and control. Expression values are in log2 scale. Black arrows indicate direction of transcription. n=15. c. Structure and representative RTPCR of major and minor isoforms in control and ICM heart samples. Blue boxes indicate RefSeq exons. Red triangles indicate probeset with differential signal detected by exon array. Primers are indicated as arrows beneath gene models. EF, exonic forward. ER, exonic reverse. TF, intronic forward. TR, intronic reverse. d. Percentage of minor variant by RTPCR. n=15. *, P < 0.05 by Welch's t-test. NS, not significant.

Figure 3. Disease-associated differences in splicing of sarcomere genes in diseased compared to control myocardium

a. Minor variant fraction of TNNI3 and MYH7-1 were measured by RTPCR in control, ICM, DCM, and AS samples (5 per group). b. Minor variant fraction of TNNT2, MYH7-2, and FLNC sarcomere genes were measured by RTPCR in control, ICM, DCM, and AS samples (10 per group). c. LVAD mechanical unloading did not normalize splicing of the four sarcomere genes in heart failure. n=4. NS, not significant. *, P < 0.05, using Welch's t-test with Sidak correction.

Figure 4. Lower RNA Splicing Efficiency in Heart Failure

Expression of 5 introns and one exon for 4 genes was measured by qRTPCR. The intron/exon ratio was expressed relative to control (defined as 1). a. Comparison of all measured intron/exon ratios between control and ICM. b. Comparison of measured intron/exon ratios between control (blue) and ICM (red), analyzed by gene. c. Individual analysis of each intron/exon ratio. Black lines indicate the group mean. Each point represents the intron/exon ratio from a single sample, measured in duplicate. Controls lacking RT showed no detectable signal. P value indicates Welch's t-test between ICM and control.

Fig 5. Analysis of sarcomere gene exon-exon junctions by capillary electrophoresis of RTPCR products

a. TNNT2 transcript and alternative splicing variant due to differential exon 7 5' splice acceptor use. b. Detection of variation of exon-exon junctions by capillary electrophoresis. A standard curve was generated by mixing known proportions of cloned wild-type and c.203_205del TNNT2. The peak area ratios from capillary electrophoresis analysis were linearly related to the input ratio. c. Representative chromatograms of control and ICM samples analyzed using the capillary electrophoresis assay. No significant difference in the proportion of c203_205del variant was detected between groups (n=15).

Figure 6. Altered RNA Splicing Distinctly Clusters Control and Disease Samples

MYH7-2, TNNT2, and FLNC splicing ratios measured in 60 samples were visualized by principal component analysis. The control samples were tightly clustered together and clearly distinguished from the disease samples. Arrow indicates sample prone to misclassification as control.