The splicing factor hnRNPL demonstrates conserved myocardial regulation across species and is altered in heart failure

Abstract

Heart failure (HF) is highly prevalent. Mechanisms underlying HF remain incompletely understood. Splicing factors (SF), which control pre-mRNA alternative splicing, regulate cardiac structure and function. This study investigated regulation of the splicing factor heterogeneous nuclear ribonucleoprotein-L (hnRNPL) in the failing heart. hnRNPL protein increased in left ventricular tissue from mice with transaortic constriction-induced HF and from HF patients. In left ventricular tissue, hnRNPL was detected predominantly in nuclei. Knockdown of the hnRNPL homolog Smooth in Drosophila induced cardiomyopathy. Computational analysis of predicted mouse and human hnRNPL binding sites suggested hnRNPL-mediated alternative splicing of tropomyosin, which was confirmed in C2C12 myoblasts. These findings identify hnRNPL as a sensor of cardiac dysfunction and suggest that disturbances of hnRNPL affect alternative splicing in HF.

Keywords: cardiomyopathy; heart failure; hnRNPL; mRNA splicing; splicing factors.

Figure 1.. hnRNPL is expressed across species in mammalian left ventricle and upregulated in cardiomyopathy.

A. hnRNPL expression in left ventricle tissue from mice subjected to control (sham) surgery, or with heart failure induced by: mild left ventricular pressure overload by transaortic constriction (mild TAC); severe TAC; or doxorubicin. Left ventricular tissue from dogs with or without myxomatous mitral valve-induced heart failure is also shown. B. Immunoblot of hnRNPL from human left ventricular tissue of nonfailing donors (NF), hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) patients. C. Densitometry of hnRNPL expression normalized to GAPDH. Each dot represents individual patient sample, and error bars represent ± standard error of the mean. ADU, arbitrary densitometric units. *, p<0.05 by 1-way ANOVA Dunnett’s multiple comparison. §, p<0.05 by global 1-way ANOVA.

Figure 2.. hnRNPL is expressed in the nucleus in LV tissue, and increases in expression after experimental LV pressure overload.

A. Nuclear labeling with DAPI or immunocytochemistry using anti-hnRNPL antibody and AF488-conjugated secondary antibody, in LV tissue from 12-week-old mice subjected to 4-week sham or transaortic constriction (TAC) surgery. White arrow denotes hnRNPL positive nucleus. Scale bars represent 50 pixels. B. Immunoblot for hnRNPL expression in LV tissue from male mice subjected to TAC for 7 days. C. Quantitation of hnRNPL protein expression normalized to loading control (Ponceau stain). Each dot represents protein sample from individual mouse. Error bars represent ± standard error of the mean. ADU, arbitrary densitometric units. *, p<0.05 by Student’s 2-tailed unpaired T test.

Figure 3.. Knockdowns of the hnRNPL homolog gene, smooth (sm), induce dilated cardiomyopathy phenotype and early mortality in Drosophila.

Heart function of whole-body sm knockdown and control flies was assessed using Optical Coherence Tomography (OCT). A. Representative OCT imaging of WT control and sm knockdown flies. B. End Diastolic Dimension, C. End Systolic Dimension and D. Fractional Shortening in sm knockdown flies backcrossed into two separate genetic backgrounds (Mut 1 and Mut 2). Each dot represents data from individual organism. Error bars represent ± standard error of the mean. *, p<0.01 and **, p<0.0001 vs knockdown groups by 1-way ANOVA with Dunnett’s multiple comparisons test.

Figure 4.. hnRNPL mediates alternative splicing of tropomyosin transcripts in striated muscle.

A. Expression and splicing change in Tpm1 in C2C12 myoblasts transfected with scrambled shRNA (Scr) or Hnrnpl shRNA (KD), viewed in IGV (v.2.10.2), shown reading right-to-left. The boxed exon marked with * represents the final exon for Tpm1 transcript variants 1, 5, and 13, dubbed Tpm1.1, located at chr9:55,935,172–66,935,449 (GRCm39). The boxed exon marked with ** represents the final exon for Tpm1 transcript variants 2, 6, 7, 8, and 9, dubbed Tpm1.2, located at chr9:66,929,875–66,930,723 (GRCm39). B. qPCR data of Tpm1 from RNA isolated from C2C12 myoblasts, using a common Tpm1 probe and probes specific for the final exons of interest, comprising Tpm1.1 and Tpm1.2 splicing pools as in A. Error bars represent ± standard error of the mean. ***, p<0.0001 by Student’s 2-tailed unpaired T test. N=3 biological replicates, each with 3 technical replicates, per shRNA. C. Nanopore RNA sequencing statistics for Tpm1. D. Left: The different terminal exons of the mouse Tpm1 gene (Ensembl Gene ENSMUSG00000032366), on the reverse strand of chromosome 9; the transcript is shown reading from left to right. The final exon includes 2 variants with different coding sequence regions (dark green) and UTRs (light green). “Score” denotes log-odds scores from a position specific scoring matrix for binding sites, constructed using experimentally validated human binding sequences. Right: The last two exons of the human TPM1 gene (Ensembl Gene ENSG00000140416, GRCh38) on the forward strand of chromosome 15.