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Spatiotemporal regulation of subcellular protein kinase A (PKA) activity for precise substrate phosphorylation is essential for cellular responses to hormonal stimulation. Ryanodine receptor 2 (RyR2) and (sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) represent two critical targets of β adrenoceptor (βAR) signaling on the sarcoplasmic reticulum membrane for cardiac excitation and contraction coupling. Using novel biosensors, we show that cardiac β1AR signals to both RyR2 and SERCA2a nanodomains in cardiomyocytes from mice, rats, and rabbits, whereas the β2AR signaling is restricted from these nanodomains. Phosphodiesterase 4 (PDE4) and PDE3 control the baseline PKA activity and prevent β2AR signaling from reaching the RyR2 and SERCA2a nanodomains. Moreover, blocking inhibitory G protein allows β2AR signaling to the RyR2 but not the SERCA2a nanodomains. This study provides evidence for the differential roles of inhibitory G protein and PDEs in controlling the adrenergic subtype signaling at the RyR2 and SERCA2a nanodomains in cardiomyocytes.

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.

Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.

Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20 (RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-Akt-mTOR Kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels, while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.

Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mecha- nisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed.

Obesity is a major public health problem worldwide. In the United States, one-third of women of reproductive age are obese. Human studies show that maternal obesity (MO) predisposes offspring to cardiovascular disease. However, the underlying mechanisms remain unclear. Given the similarities between pregnancy in sheep and humans, we studied sheep to examine the impact of MO on fetal cardiomyocyte contractility at term. We observed that MO impaired cardiomyocyte contractility by reducing peak shortening and shortening/relengthening velocity, prolonging time to relengthening. MO disrupted Ca(2+) homeostasis in fetal cardiomyocytes, increasing intracellular Ca(2+) and inducing cellular Ca(2+) insensitivity. The Ca(2+)-release channel was impaired, but Ca(2+) uptake was unaffected by MO. The upstream kinases that phosphorylate the Ca(2+)-release channel—ryanodine receptor-2, PKA, and calmodulin-dependent protein kinase II—were activated in MO fetuses. Contractile dysfunction was associated with an increased ratio of myosin heavy chain (MHC)-β to MHC-α and upregulated cardiac troponin (cTn)-T and tropomyosin, as well as cTn-I phosphorylation. In summary, this is the first characterization of the effects of MO on fetal cardiomyocyte contractility. Our findings indicate that MO impairs fetal cardiomyocyte contractility through altered intracellular Ca(2+) handling, overloading fetal cardiomyocyte intracellular Ca(2+) and aberrant myofilament protein composition. These mechanisms may contribute to developmental programming by MO of offspring cardiac function and predisposition to later life cardiovascular disease in the offspring.—Wang, Q., Zhu, C., Sun, M., Maimaiti, R., Ford, S. P., Nathanielsz, P. W., Ren, J., Guo, W. Maternal obesity impairs fetal cardiomyocyte contractile function in sheep.

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.

RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR, we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting, MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.

Dear Editor, Diastolic heart failure (DHF) and systolic heart failure (SHF) are two main subsets of chronic heart failure that are commonly encountered in clinical practice (Chatterjee and Massie, 2007). Although there have been considerable advances in the treatment of SHF, the molecular and biochemical mechanisms mediating the structural remodeling and principal functional derangement in both SHF and DHF remain unclear. In particular, effective therapeutic regimens are still lacking for DHF; thus, it is imperative to better understand the molecular and biochemical mechanisms of DHF in an effort to seek novel therapeutic avenues for patients suffering from DHF. DHF is manifested by stiffer ventricular walls, while SHF has overly compliant ventricular walls. Therefore, altering ventricular wall stiffness will be a potential therapeutic strategy for both DHF and SHF. Recently, increasing attention has been paid by researchers and cardiologists to titin in regard to developing an alternative therapy for both DHF and SHF. Titin, a giant sarcomeric protein, is a key determinant of myocardial passive tension and is largely responsible for the diastolic properties of the heart (LeWinter and Granzier, 2014). Two major classes of titin are co-expressed in mammalian cardiac muscles, namely the smaller N2B isoform (3.0 MDa) and the larger N2BA isoform (3.2–3.7 MDa). The larger N2BA isoform is more compliant and develops lower passive tension, while the N2B isoform is stiffer and develops higher passive tension. At varying ratios of N2BA to N2B isoforms, the sarcomeres develop an intermediate level of passive tension affecting myofibrillar extensibility and passive force generation and altering the stiffness of the cardiac walls (Cazorla et al., 2000; Freiburg et al., 2000). Therefore, the manipulation of their ratios has been considered a therapy for reducing pathological diastolic stiffness or increasing pathological systolic stiffness. However, the mechanisms regulating titin isoform transition remain elusive. Recently, RNA binding motif 20 (RBM20), a splicing factor, has been identified as a major regulator of titin isoform transition (Guo et al., 2012), while the thyroid hormone-triiodothyronine (T3) has also been reported to regulate titin isoform transition (Wu et al., 2007; Kruger et al., 2008). Nevertheless, prior to this report, the relationship between RBM20 and T3 in the regulation of titin isoform transition remains unknown. The present study was designed to evaluate the role of RBM20 in T3-regulated titin isoform transition, and the molecular signaling mechanisms linking T3 and RBM20. There are indications that thyroid gland maturation and titin isoform transition occur in the fetus at approximately the same time (Polk, 1995; Kruger et al., 2006). Indeed, in the presence of RBM20, T3 treatment in primary cultures of neonatal rat cardiomyocytes (CMs) increased the smaller N2B isoform expression (Kruger et al., 2008). However, it is unknown whether T3 can still regulate titin isoform transition in the absence of RBM20. Hence, we examined the influence of T3 on titin isoform transition in the absence of RBM20. In homozygous RBM20 knockout rats (Rbm20−/−), only the largest N2BA isoform (∼3.9 MDa) is present (Guo et al., 2010), and in heterozygous RBM20 knockout rats (Rbm20+/−), the intermediate sized titin N2BA isoforms (3.2–3.7 MDa) are co-expressed with the smaller N2B isoform, while, in adult wild type rats (Rbm20+/+), the small, stiffer N2B isoform predominates (Guo et al., 2012). This makes the RBM20 deficient rat model a unique tool for this study. We isolated and cultured neonatal ventricular cardiomyocytes (NVCMs) of Rbm20+/+, Rbm20+/−, and Rbm20−/− rats. With the addition of T3 in a serum-starved medium, the ratios of N2B to N2BA isoforms increased significantly, from ∼23% to ∼59% of total titin isoforms (N2B+N2BA) in Rbm20+/+ rats (Figure 1A) and from ∼6% to ∼23% of total titin isoforms in Rbm20+/− group (Figure 1B). However, increased N2B expression did not occur in Rbm20−/− NVCMs with T3 supplementation. Virtually no N2B was expressed in either treated or untreated Rbm20−/− NVCMs (Figure 1C). These results suggest that RBM20 plays an indispensable role in the regulation of titin isoform transition triggered by T3. Figure 1 Effect of T3 on titin isoform transition in RBM20 deficient NVCMs and rats. (A, B, D, and E) N2B-titin isoform increased with T3 treatment in primary cultures of Rbm20+/+ and Rbm20+/− NVCMs and rats when compared with control without T3 treatment. ... In order to further confirm whether T3-regulated titin isoform transition is RBM20-dependent, we performed in vivo assays. We treated the Rbm20+/+, Rbm20+/−, and Rbm20−/− rats independently with T3 and propylthiouracil (PTU, a drug that inhibits the secretion of thyroid hormone). After a 90-day treatment with subcutaneously implanted T3 pellets in Rbm20+/+, Rbm20+/−, and Rbm20−/− rats, we harvested the hearts, and observed titin isoform transitions by resolving titin bands with a 1% SDS-agarose gel. The N2B isoform was significantly increased in Rbm20+/+ and Rbm20+/− rat hearts (Figure 1D), with increases of N2B isoform from ∼84% to 92% of total titin in Rbm20+/+ rats, and from ∼16% to 36% of total titin in Rbm20+/− rats (Figure 1E). No N2B isoform was expressed in Rbm20−/− rat hearts under T3 treatment when compared with control groups that were implanted with placebo (Figure 1F). An equal number of age-matched rats were used for the PTU treatment by feeding a diet containing 0.15% PTU. After 3 months of treatment, with low thyroid status, the N2BA isoform was increased and the N2B isoform was decreased from ∼87% to 80% of total titin in Rbm20+/+ rats (Figure 1G). The N2B isoform was nearly undetectable with PTU treatment in Rbm20+/− rats (Figure 1H). However, no changes were observed in Rbm20−/− rats treated with PTU when compared with control groups without PTU treatment (Figure 1I). These data further confirm that RBM20 is an essential factor for thyroid hormone-regulated titin isoform transition. Next, we examined the mechanisms linking RBM20 and T3 in the regulation of titin isoform transition. Western blot analysis using anti-RBM20 antibody indicated two bands (∼135 and 145 kDa) in control primary cultures of Rbm20+/+ and Rbm20+/− NVCMs (Figure 1J and K). With supplementation of T3, the intensity of the 145 kDa band increased in primary cultures of Rbm20+/+ and Rbm20+/− NVCMs when compared with the control without T3, while supplementation of LY 294002 (LY, a PI3K inhibitor) with or without T3 produced slightly decreased intensity of the 145 kDa band, and a significant reduction in the intensity of the 135 kDa band (Figure 1J and K). These results suggest that RBM20 is likely posttranslationally modified. If this is the case, the posttranslational modification of RBM20 could be phosphorylation, given that the phosphorylation of serine–arginine (SR) proteins (Guo et al., 2012) is essential for pre-mRNA splicing (Prasad et al., 1999). A previous study reported that T3 regulates titin isoform transition via the PI3K/Akt pathway (Kruger et al., 2008). Our results also demonstrated that T3 supplementation increased AKT activation and the N2B isoform, while LY supplementation decreased AKT activation and the N2B isoform (Supplementary Figures S1A–C and S2A–C) in primary cultures. In in vivo assays with rats treated with T3 and PTU, we found that the activation and phosphorylation of Akt were increased with T3 treatment, and deceased with PTU treatment (Supplementary Figures S3A–C and S4A–C). These results suggest that the phosphorylation of RBM20 is crucial for titin isoform transition triggered by T3. The mechanism could be that first T3 activates Akt and then Akt phosphorylates RBM20, and finally the phosphorylated RBM20 regulates titin isoform transition. Band density analysis also demonstrated that total RBM20 expression was increased with T3 supplementation when compared with control without T3 supplementation in the medium. Additionally, LY supplementation reduced the total amount of RBM20 when compared with control without T3 or LY supplementation (Figure 1J and K). These results imply that T3-regulated titin isoform transition could also activate the mTOR pathway and increase gene expression of RBM20. The relative expression of Rbm20 mRNA was also validated by real-time PCR, and the results revealed that the mRNA level increased with T3 supplementation while it decreased with LY supplementation (Supplementary Figure S5A and B). Therefore, we conclude that titin isoform transition triggered by T3 is linked to RBM20 via the PI3K/Akt/mTOR signaling pathway, phosphorylating RBM20 and/or increasing gene expression of RBM20 (Supplementary Figure S6). Taken together, these findings present significant clinical implications for patients with SHF or DHF. Our data suggest that T3 treatment in SHF may increase the stiffness of ventricular walls through upregulating the stiffer N2B isoform. In particular, patients with DHF can be benefited from the treatment to decrease the stiffer N2B isoform. Overall, our data should strengthen the understanding of the regulatory mechanism of titin isoform transition, which might help to decipher the mechanisms of developmental or diseased titin isoform transition, and further, they provide new insights for improving diastolic and systolic dysfunction. [Supplementary material is available at Journal of Molecular Cell Biology online. We thank Mark D. Hanna for expert technical assistance and laboratory management. We are grateful to the support of Agricultural Experiment Station at University of Wyoming and the Regional Hatch Project (NC1184). This work was supported by a pilot grant from NIH-P20-GM-103432 and Startup Funds from the University of Wyoming.]