Cardiac tissue-specific repression of CELF activity disrupts alternative splicing and causes cardiomyopathy

Members of the CELF family of RNA binding proteins have been implicated in alternative splicing regulation in developing heart. Transgenic mice that express a nuclear dominant-negative CELF protein specifically in the heart (MHC-CELFDelta) develop cardiac hypertrophy and dilated cardiomyopathy with defects in alternative splicing beginning as early as 3 weeks after birth. MHC-CELFDelta mice exhibit extensive cardiac fibrosis, severe cardiac dysfunction, and premature death. Interestingly, the penetrance of the phenotype is greater in females than in males despite similar levels of dominant-negative expression, suggesting that there is sex-specific modulation of splicing activity. The cardiac defects in MHC-CELFdelta mice are directly attributable to reduced levels of CELF activity, as crossing these mice with mice overexpressing CUG-BP1, a wild-type CELF protein, rescues defects in alternative splicing, the severity and incidence of cardiac hypertrophy, and survival. We conclude that CELF protein activity is required for normal alternative splicing in the heart in vivo and that normal CELF-mediated alternative splicing regulation is in turn required for normal cardiac function.     

Reactivation of Fetal Splicing Programs in Diabetic Hearts Is Mediated by Protein Kinase C Signaling

Diabetic cardiomyopathy is one of the complications of diabetes that eventually leads to heart failure and death. Aberrant activation of PKC signaling contributes to diabetic cardiomyopathy by mechanisms that are poorly understood. Previous reports indicate that PKC is implicated in alternative splicing regulation. Therefore, we wanted to test whether PKC activation in diabetic hearts induces alternative splicing abnormalities. Here, using RNA sequencing we identified a set of 22 alternative splicing events that undergo a developmental switch in splicing, and we confirmed that splicing reverts to an embryonic pattern in adult diabetic hearts. This network of genes has important functions in RNA metabolism and in developmental processes such as differentiation. Importantly, PKC isozymes α/β control alternative splicing of these genes via phosphorylation and up-regulation of the RNA-binding proteins CELF1 and Rbfox2. Using a mutant of CELF1, we show that phosphorylation of CELF1 by PKC is necessary for regulation of splicing events altered in diabetes. In summary, our studies indicate that activation of PKCα/β in diabetic hearts contributes to the genome-wide splicing changes through phosphorylation and up-regulation of CELF1/Rbfox2 proteins. These findings provide a basis for PKC-mediated cardiac pathogenesis under diabetic conditions.     

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.     

The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing

Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.     

Cell Cycle Re-Entry and Mitochondrial Defects in Myc-Mediated Hypertrophic Cardiomyopathy and Heart Failure

While considerable evidence supports the causal relationship between increases in c-Myc (Myc) and cardiomyopathy as a part of a “fetal re-expression” pattern, the functional role of Myc in mechanisms of cardiomyopathy remains unclear. To address this, we developed a bitransgenic mouse that inducibly expresses Myc under the control of the cardiomyocyte-specific MHC promoter. In adult mice the induction of Myc expression in cardiomyocytes in the heart led to the development of severe hypertrophic cardiomyopathy followed by ventricular dysfunction and ultimately death from congestive heart failure. Mechanistically, following Myc activation, cell cycle markers and other indices of DNA replication were significantly increased suggesting that cell cycle-related events might be a primary mechanism of cardiac dysfunction. Furthermore, pathological alterations at the cellular level included alterations in mitochondrial function with dysregulation of mitochondrial biogenesis and defects in electron transport chain complexes I and III. These data are consistent with the known role of Myc in several different pathways including cell cycle activation, mitochondrial proliferation, and apoptosis, and indicate that Myc activation in cardiomyocytes is an important regulator of downstream pathological sequelae. Moreover, our findings indicate that the induction of Myc in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure, and that sustained induction of Myc, leading to cell cycle re-entry in adult cardiomyocytes, represents a maladaptive response for the mature heart.     

Involvement of autophagy in cardiac remodeling in transgenic mice with cardiac specific over-expression of human programmed cell death 5

Programmed cell death 5 (PDCD5) is a cytosolic protein suppressing growth of multiple types of cancer cells through activating p53. We hypothesized that PDCD5 plays an essential role in cardiac remodeling and function. PDCD5 was significantly up-regulated in the hearts from mice subjected to angiotensin II treatment or transverse aortic constriction. Thus, we generated transgenic mice over-expressing human PDCD5 under the control of alpha myosin heavy chain promoter to examine the role of PDCD5 in cardiac remodeling. Transgenic founder died spontaneously displayed enlarged heart. The high PDCD5 over-expressing line (10-fold) showed reduced survival rate, increase in heart weight normalized to body weight. Real-Time RT-PCR analysis revealed fetal gene program was up-regulated. Echocardiography and histopathological examination showed characteristics of dilated cardiomyopathy and heart failure in transgenic mice. Western blot and immunohistochemistry analysis showed autophagy was dramatically increased in transgenic mice as compared to WT littermates control mice, while apoptosis remained unchanged. The enhanced autophagy in high over-expressing line was associated with significant increase in p53 activity and its downstream target damage-regulated autophagy modulator expression. The low over-expressing line (3.5-fold) appeared normal, but was more susceptible to angiotensin II-induced cardiac hypertrophy. This study is the first providing evidence that PDCD5 plays an important role in cardiac remodeling.     

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a β-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca²⁺ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased β myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.     

Serial Examination of an Inducible and Reversible Dilated Cardiomyopathy in Individual Adult Drosophila

Recent work has demonstrated that Drosophila can be used as a model of dilated cardiomyopathy, defined as an enlarged cardiac chamber at end-diastole when the heart is fully relaxed and having an impaired systolic function when the heart is fully contracted. Gene mutations that cause cardiac dysfunction in adult Drosophila can result from abnormalities in cardiac development or alterations in post-developmental heart function. To clarify the contribution of transgene expression to post-developmental cardiac abnormalities, we applied strategies to examine the temporal and spacial effects of transgene expression on cardiac function. We engineered transgenic Drosophila based on the well-characterized temperature-sensitive Gal80 protein in the context of the bipartite Gal4/UAS transgenic expression system in Drosophila employing the cardiac specific driver, tinCΔ4-Gal4. Then, we developed a strategy using optical coherence tomography to serially measure cardiac function in the individual flies over time course of several days. As a proof of concept we examined the effects of the expression of a human mutant delta-sarcoglycan associated with familial heart failure and observed a reversible, post-developmental dilated cardiomyopathy in Drosophila. Our results show that the unique imaging strategy based on the non-destructive, non-invasive properties of optical coherence tomography can be applied to serially examine cardiac function in individual adult flies. Furthermore, the induction and reversal of cardiac transgene expression can be investigated in adult flies thereby providing insight into the post-developmental effects of transgene expression.     

ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo

Members of the CUG-BP and ETR-3 like factor (CELF) protein family bind within conserved intronic elements (called MSEs) flanking the cardiac troponin T (cTNT) alternative exon 5 and promote exon inclusion in vivo and in vitro. Here we use a comparative deletion analysis of two family members (ETR-3 and CELF4) to identify separate domains required for RNA binding and splicing activity in vivo. CELF proteins contain two adjacent RNA binding domains (RRM1 and RRM2) near the N-terminus and one RRM (RRM3) near the C-terminus, which are separated by a 160–230 residue divergent domain of unknown function. Either RRM1 or RRM2 of CELF4 are necessary and sufficient for binding MSE RNA and RRM2 plus an additional 66 amino acids of the divergent domain are as effective as full-length protein in activating MSE-dependent splicing in vivo. Non-overlapping N- and C-terminal regions of ETR-3 containing either RRM1 and RRM2 or RRM3 plus segments of the adjacent divergent domain activate MSE-dependent exon inclusion demonstrating an unusual functional redundancy of the N- and C-termini of the protein. These results identify specific regions of ETR-3 and CELF4 that are likely targets of protein–protein interactions required for splicing activation.     

BaxΔ2 Is a Novel Bax Isoform Unique to Microsatellite Unstable Tumors

The pro-death Bcl-2 family protein and tumor suppressor Bax is frequently mutated in tumors with microsatellite instability (MSI). The mutation often results in a “Bax negative” phenotype and therefore is generally thought to be beneficial to the development of the tumor. Here, we report the identification of a novel Bax isoform, BaxΔ2, which is unique to microsatellite unstable tumors. BaxΔ2 is generated by a unique combination of a microsatellite deletion in Bax exon 3 and alternative splicing of Bax exon 2. Consistently, BaxΔ2 is only detected in MSI cell lines and primary tumors. BaxΔ2 is a potent cell death inducer but does not directly target mitochondria. In addition, BaxΔ2 sensitizes certain MSI tumor cells to a subset of chemotherapeutic agents, such as adriamycin. Thus, our data provide evidence that mutation and alternative splicing of tumor suppressors such as Bax are not always beneficial to tumor development but can be detrimental instead.     

Establishment of a Novel Murine Model of Ischemic Cardiomyopathy with Multiple Diffuse Coronary Lesions

Objectives

Atherosclerotic lesions of the coronary arteries are the pathological basis for myocardial infarction and ischemic cardiomyopathy. Progression of heart failure after myocardial infarction is associated with cardiac remodeling, which has been studied by means of coronary ligation in mice. However, this ligation model requires excellent techniques. Recently, a new murine model, HypoE mouse was reported to exhibit atherogenic Paigen diet-induced coronary atherosclerosis and myocardial infarction; however, the HypoE mice died too early to make possible investigation of cardiac remodeling. Therefore, we aimed to modify the HypoE mouse model to establish a novel model for ischemic cardiomyopathy caused by atherosclerotic lesions, which the ligation model does not exhibit.

Methods and Results

In our study, the sustained Paigen diet for the HypoE mice was shortened to 7 or 10 days, allowing the mice to survive longer. The 7-day Paigen diet intervention starting when the mice were 8 weeks old was adequate to permit the mice to survive myocardial infarction. Our murine model, called the “modified HypoE mouse”, was maintained until 8 weeks, with a median survival period of 36 days, after the dietary intervention (male, n = 222). Echocardiography demonstrated that the fractional shortening 2 weeks after the Paigen diet (n = 14) significantly decreased compared with that just before the Paigen diet (n = 6) (31.4±11.9% vs. 54.4±2.6%, respectively, P<0.01). Coronary angiography revealed multiple diffuse lesions. Cardiac remodeling and fibrosis were identified by serial analyses of cardiac morphological features and mRNA expression levels in tissue factors such as MMP-2, MMP-9, TIMP-1, collagen-1, and TGF-β.

Conclusion

Modified HypoE mice are a suitable model for ischemic cardiomyopathy with multiple diffuse lesions and may be considered as a novel and convenient model for investigations of cardiac remodeling on a highly atherogenic background.