The MEF2 transcriptional target DMPK induces loss of sarcomere structure and cardiomyopathy

Aims The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling. Methods Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified and results myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity. Conclusion Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure

The RNA-binding protein Rbm38 is dispensable during pressure overload-induced cardiac remodeling in mice

The importance of tightly controlled alternative pre-mRNA splicing in the heart is emerging. The RNA binding protein Rbm24 has recently been identified as a pivotal cardiac splice factor, which governs sarcomerogenesis in the heart by controlling the expression of alternative protein isoforms. Rbm38, a homolog of Rbm24, has also been implicated in RNA processes such as RNA splicing, RNA stability and RNA translation, but its function in the heart is currently unknown. Here, we investigated the role of Rbm38 in the healthy and diseased adult mouse heart. In contrast to the heart- and skeletal muscle-enriched protein Rbm24, Rbm38 appears to be more broadly expressed. We generated somatic Rbm38 -/- mice and show that global loss of Rbm38 results in hematopoietic defects. Specifically, Rbm38 -/- mice were anemic and displayed enlarged spleens with extramedullary hematopoiesis, as has been shown earlier. The hearts of Rbm38 -/- mice were mildly hypertrophic, but cardiac function was not affected. Furthermore, Rbm38 deficiency did not affect cardiac remodeling (i.e. hypertrophy, LV dilation and fibrosis) or performance (i.e. fractional shortening) after pressure-overload induced by transverse aorta constriction. To further investigate molecular consequences of Rbm38 deficiency, we examined previously identified RNA stability, splicing, and translational targets of Rbm38. We found that stability targets p21 and HuR, splicing targets Mef2d and Fgfr2, and translation target p53 were not altered, suggesting that these Rbm38 targets are tissue-specific or that Rbm38 deficiency may be counteracted by a redundancy mechanism. In this regard, we found a trend towards increased Rbm24 protein expression in Rbm38 -/- hearts. Overall, we conclude that Rbm38 is critical in hematopoiesis, but does not play a critical role in the healthy and diseased heart

AAV9-mediated Rbm24 overexpression induces fibrosis in the mouse heart

The RNA-binding protein Rbm24 has recently been identified as a pivotal splicing factor in the developing heart. Loss of Rbm24 in mice disrupts cardiac development by governing a large number of muscle-specific splicing events. Since Rbm24 knockout mice are embryonically lethal, the role of Rbm24 in the adult heart remained unexplored. Here, we used adeno-associated viruses (AAV9) to investigate the effect of increased Rbm24 levels in adult mouse heart. Using high-resolution microarrays, we found 893 differentially expressed genes and 1102 differential splicing events in 714 genes in hearts overexpressing Rbm24. We found splicing differences in cardiac genes, such as PDZ and Lim domain 5, Phospholamban, and Titin, but did not find splicing differences in previously identified embryonic splicing targets of Rbm24, such as skNAC, αNAC, and Coro6. Gene ontology enrichment analysis demonstrated increased expression of extracellular matrix (ECM)-related and immune response genes. Moreover, we found increased expression of Tgfβ-signaling genes, suggesting enhanced Tgfβ-signaling in these hearts. Ultimately, this increased activation of cardiac fibroblasts, as evidenced by robust expression of Periostin in the heart, and induced extensive cardiac fibrosis. These results indicate that Rbm24 may function as a regulator of cardiac fibrosis, potentially through the regulation of TgfβR1 and TgfβR2 expression

Rbm24 is upregulated in Rbm38 -/- hearts.

<p>A. Rbm24 mRNA levels were measured by qPCR. B. Western blot of Rbm24 in wildtype versus Rbm38 -/- hearts. Statistical significance was tested by a 1-way ANOVA with LSD posthoc-testing.</p

Rbm38 is ubiquitously expressed.

<p>A. Sequence identity of mouse Rbm24 and Rbm38. RRM represents the RNA-recognition motif, including the submotifs RNP1 and RNP2. B. qPCR of Rbm24 and Rbm38 in mouse tissues. Left ventricular (LV) heart tissue is highlighted in red. Values are corrected for the geometric mean of the following reference genes: Gapdh, Hprt, Pgk1, Rpl32, and Tbp. C. qPCR of Rbm38 in sham-operated and TAC-operated wildtype mice (C57/Bl6) 12 weeks after surgery. Hypertrophied mice were TAC-operated mice with an ejection fraction > 35%, failing mice were TAC-operated mice with an ejection fraction < 20%. D. qPCR of Rbm38 in neonatal rat cardiomyocytes (NRCM) and neonatal rat fibroblasts (NRF). Significance was tested with a 2-tailed Student’s t-test, * indicates p < 0.05 versus sham.</p

Nur77 protects against adverse cardiac remodelling by limiting neuropeptide Y signalling in the sympathoadrenal-cardiac axis

Aims: Cardiac remodelling and heart failure are promoted by persistent sympathetic activity. We recently reported that nuclear receptor Nur77 may protect against sympathetic agonist-induced cardiac remodelling in mice. The sympathetic co-transmitter neuropeptide Y (NPY) is co-released with catecholamines and is a known cardiac modulator and predictor of heart failure mortality. Recently, transcriptome analyses revealed NPY as a putative target of Nur77. In this study, we assess whether Nur77 modulates adverse cardiac remodelling via NPY signalling. Methods and results: Nur77 represses NPY expression in the PC12 adrenal chromaffin cell line. Accordingly, NPY levels are higher in adrenal gland, plasma, and heart from Nur77-KO compared to wild-type mice. Conditioned medium from Nur77-silenced chromaffin cells and serum from Nur77-KO mice induce marked hypertrophy in cultured neonatal rat cardiomyocytes, which is inhibited by the NPY type 1 receptor (NPY1R) antagonist BIBO3304. In cardiomyocytes from Nur77-KO mice, intracellular Ca2+ is increased partially via the NPY1R. This is independent from elevated circulating NPY since cardiomyocyte-specific Nur77-deficient mice (CM-KO) do not have elevated circulating NPY, but do exhibit BIBO3304-sensitive, increased cardiomyocyte intracellular Ca2+. In vivo, this translates to NPY1R antagonism attenuating cardiac calcineurin activity and isoproterenol-induced cardiomyocyte hypertrophy and fibrosis in full-body Nur77-KO mice, but not in CM-KO mice. Conclusions: The cardioprotective action of Nur77 can be ascribed to both inhibition of circulating NPY levels and to cardiomyocyte-specific modulation of NPY-NPY1R signalling. These results reveal the underlying mechanism of Nur77 as a promising modifier gene in heart failure

Rbm38 knockout mice have hematopoietic defects.

<p>A. RT-PCR of Rbm38 mRNA transcript in wildtype and Rbm38 -/- hearts. Δex3-4 represents the transcript lacking exon 3 and 4. B. qPCR of wildtype Rbm38 mRNA with primers designed within exon 4 and 5. C. Representative photograph of a wildtype and Rbm38 -/- spleen of 15–18 weeks old mice. D. Spleen weight/body weight ratio of 15–18 weeks old wildtype (n = 7) and Rbm38 -/- mice (n = 3). E. Representative images of H&E staining on section of wildtype and Rbm38 -/- spleens. Note the increased number of nucleated red blood cells in the splenic red pulp indicative of extramedullary hematopoiesis (EMH). F. Hemoglobin levels in the blood of wildtype (n = 7) and Rbm38 -/- mice (n = 5). Significance was tested with a 2-tailed Student’s t-test, * indicates p < 0.05 versus wildtype.</p