Extracellular vesicles from differentiated stem cells contain novel proangiogenic miRNAs and induce angiogenic responses at low doses.

Extracellular vesicles (EVs) released from healthy endothelial cells (ECs) have shown potential for promoting angiogenesis, but their therapeutic efficacy remains poorly understood. We have previously shown that transplantation of a human embryonic stem cell-derived endothelial cell product (hESC-ECP), promotes new vessel formation in acute ischemic disease in mice, likely via paracrine mechanism(s). Here, we demonstrated that EVs from hESC-ECPs (hESC-eEVs) significantly increased EC tube formation and wound closure in vitro at ultralow doses, whereas higher doses were ineffective. More important, EVs isolated from the mesodermal stage of the differentiation (hESC-mEVs) had no effect. Small RNA sequencing revealed that hESC-eEVs have a unique transcriptomic profile and are enriched in known proangiogenic microRNAs (miRNAs, miRs). Moreover, an in silico analysis identified three novel hESC-eEV-miRNAs with potential proangiogenic function. Differential expression analysis suggested that two of those, miR-4496 and miR-4691-5p, are highly enriched in hESC-eEVs. Overexpression of miR-4496 or miR-4691-5p resulted in increased EC tube formation and wound closure in vitro, validating the novel proangiogenic function of these miRNAs. In summary, we demonstrated that hESC-eEVs are potent inducers of EC angiogenic response at ultralow doses and contain a unique EV-associated miRNA repertoire, including miR-4496 and miR-4691-5p, with novel proangiogenic function.

Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

Cardiac circRNAs arise mainly from constitutive exons rather than alternatively spliced exons.

Circular RNAs (circRNAs) are a relatively new class of RNA molecules, and knowledge about their biogenesis and function is still in its infancy. It was recently shown that alternative splicing underlies the formation of circular RNAs (circRNA) arising from the Titin (TTN) gene. Since the main mechanism by which circRNAs are formed is still unclear, we hypothesized that alternative splicing, and in particular exon skipping, is a major driver of circRNA production. We performed RNA sequencing on human and mouse hearts, mapped alternative splicing events, and overlaid these with expressed circRNAs at exon-level resolution. In addition, we performed RNA sequencing on hearts of Rbm20 KO mice to address how important Rbm20-mediated alternative splicing is in the production of cardiac circRNAs. In human and mouse hearts, we show that cardiac circRNAs are mostly (∼90%) produced from constitutive exons and less (∼10%) from alternatively spliced exons. In Rbm20 KO hearts, we identified 38 differentially expressed circRNAs of which 12 were produced from the Ttn gene. Even though Ttn appeared the most prominent target of Rbm20 for circularization, we also detected Rbm20-dependent circRNAs arising from other genes including Fan1, Stk39, Xdh, Bcl2l13, and Sorbs1 Interestingly, only Ttn circRNAs seemed to arise from Rbm20-mediated skipped exons. In conclusion, cardiac circRNAs are mostly derived from constitutive exons, suggesting that these circRNAs are generated at the expense of their linear counterpart and that circRNA production impacts the accumulation of the linear mRNA.

RBM20 Mutations Induce an Arrhythmogenic Dilated Cardiomyopathy Related to Disturbed Calcium Handling.

BACKGROUND: Mutations in RBM20 (RNA-binding motif protein 20) cause a clinically aggressive form of dilated cardiomyopathy, with an increased risk of malignant ventricular arrhythmias. RBM20 is a splicing factor that targets multiple pivotal cardiac genes, such as Titin (TTN) and CAMK2D (calcium/calmodulin-dependent kinase II delta). Aberrant TTN splicing is thought to be the main determinant of RBM20-induced dilated cardiomyopathy, but is not likely to explain the increased risk of arrhythmias. Here, we investigated the extent to which RBM20 mutation carriers have an increased risk of arrhythmias and explore the underlying molecular mechanism. METHODS: We compared clinical characteristics of RBM20 and TTN mutation carriers and used our previously generated Rbm20 knockout (KO) mice to investigate downstream effects of Rbm20-dependent splicing. Cellular electrophysiology and Ca2+ measurements were performed on isolated cardiomyocytes from Rbm20 KO mice to determine the intracellular consequences of reduced Rbm20 levels. RESULTS: Sustained ventricular arrhythmias were more frequent in human RBM20 mutation carriers than in TTN mutation carriers (44% versus 5%, respectively, P=0.006). Splicing events that affected Ca2+- and ion-handling genes were enriched in Rbm20 KO mice, most notably in the genes CamkIIδ and RyR2. Aberrant splicing of CamkIIδ in Rbm20 KO mice resulted in a remarkable shift of CamkIIδ toward the δ-A isoform that is known to activate the L-type Ca2+ current ( ICa,L). In line with this, we found an increased ICa,L, intracellular Ca2+ overload and increased sarcoplasmic reticulum Ca2+ content in Rbm20 KO myocytes. In addition, not only complete loss of Rbm20, but also heterozygous loss of Rbm20 increased spontaneous sarcoplasmic reticulum Ca2+ releases, which could be attenuated by treatment with the ICa,L antagonist verapamil. CONCLUSIONS: We show that loss of Rbm20 disturbs Ca2+ handling and leads to more proarrhythmic Ca2+ releases from the sarcoplasmic reticulum. Patients that carry a pathogenic RBM20 mutation have more ventricular arrhythmias despite a similar left ventricular function, in comparison with patients with a TTN mutation. Our experimental data suggest that RBM20 mutation carriers may benefit from treatment with an ICa,L blocker to reduce their arrhythmia burden.

RBM20 Regulates Circular RNA Production From the Titin Gene.

RATIONALE: RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules. OBJECTIVE: To establish the role of RBM20 in the formation of circRNAs in the heart. METHODS AND RESULTS: Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. CONCLUSIONS: We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs.

Insights into alternative splicing of sarcomeric genes in the heart.

Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial layer in gene expression, greatly expanding protein diversity and governing complex biological processes in the cardiomyocyte. At the core of cardiac contraction, the physical properties of the sarcomere are carefully orchestrated through alternative splicing to fit the varying demands on the heart. By the recent discovery of RBM20 and RBM24, two major heart and skeletal muscle-restricted splicing factors, it became evident that alternative splicing events in the heart occur in regulated networks rather than in isolated events. Analysis of knockout mice of these splice factors has shed light on the importance of these fundamental processes in the heart. In this review, we discuss recent advances in our understanding of the role and regulation of alternative splicing in the developing and diseased heart, specifically within the sarcomere. Through various examples (titin, myomesin, troponin T, tropomyosin and LDB3) we illustrate how alternative splicing regulates the functional properties of the sarcomere. Finally, we evaluate opportunities and obstacles to modulate alternative splicing in therapeutic approaches for cardiac disease.

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins.

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components lamin A and lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representative LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of lamin C protein was reduced by the expected approximately 50%. Clearly decreased lamin A and lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of lamin A protein were more reduced suggesting an effect on expression of lamin A from the wild type allele. PCR analysis of the ratio of lamin A to lamin C transcripts showed unchanged relative amounts of lamin A transcript suggesting that the effect on the wild type allele was operative at the protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that heterozygosity for the nonsense mutation causes NMD degradation of the mutant transcripts blocking expression of the truncated mutant protein and an additional trans effect on lamin A protein levels expressed from the wild type allele. We discuss the possibility that skewing of the lamin A to lamin C ratio may contribute to ensuing processes that destabilize cardiomyocytes and trigger cardiomyopathy.

Non-coding RNAs to treat vascular smooth muscle cell dysfunction.

Vascular smooth muscle cell (vSMC) dysfunction is a critical contributor to cardiovascular diseases, including atherosclerosis, restenosis and vein graft failure. Recent advances have unveiled a fascinating range of non-coding RNAs (ncRNAs) that play a pivotal role in regulating vSMC function. This review aims to provide an in-depth analysis of the mechanisms underlying vSMC dysfunction and the therapeutic potential of various ncRNAs in mitigating this dysfunction, either preventing or reversing it. We explore the intricate interplay of microRNAs, long-non-coding RNAs and circular RNAs, shedding light on their roles in regulating key signalling pathways associated with vSMC dysfunction. We also discuss the prospects and challenges associated with developing ncRNA-based therapies for this prevalent type of cardiovascular pathology.

COVID-19 plasma induces subcellular remodelling within the pulmonary microvascular endothelium.

BACKGROUND: COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect multiple organ systems, including the pulmonary vasculature. Endothelial cells (ECs) are thought to play a key role in the propagation of COVID-19, however, our understanding of the exact scale of dysregulation sustained by the pulmonary microvasculature (pMV) remains incomplete. Here we aim to identify transcriptional, phenotypic, and functional changes within the pMV induced by COVID-19. METHODS AND RESULTS: Human pulmonary microvascular endothelial cells (HPMVEC) treated with plasma acquired from patients hospitalised with severe COVID-19 were compared to HPMVEC treated with plasma from patients hospitalised without COVID-19 but with other severe illnesses. Exposure to COVID-19 plasma caused a significant functional decline in HPMVECs as seen by a decrease in both cell viability via the WST-1 cell-proliferation assay and cell-to-cell barrier function as measured by electric cell-substrate impedance sensing. High-content imaging using a Cell Painting image-based assay further quantified morphological variations within sub-cellular organelles to show phenotypic changes in the whole endothelial cell, nucleus, mitochondria, plasma membrane and nucleolus morphology. RNA-sequencing of HPMVECs treated with COVID-19 plasma suggests the observed phenotype may, in part, be regulated by genes such as SMAD7, BCOR, SFMBT1, IFIT5 and ZNF566 which are involved in transcriptional regulation, protein monoubiquitination and TGF-ß signalling. CONCLUSION AND IMPACT: During COVID-19, the pMV undergoes significant remodelling, which is evident based on the functional, phenotypic, and transcriptional changes seen following exposure to COVID-19 plasma. The observed morphological variation may be responsible for downstream complications, such as a decline in overall cellular function and cell-to-cell barrier integrity. Moreover, genes identified through bulk RNA sequencing may contribute to our understanding of the observed phenotype and assist in developing strategies that can inform the rescue of the dysregulated endothelium.

CHAP is a newly identified Z-disc protein essential for heart and skeletal muscle function.

In recent years, the perception of Z-disc function has changed from a passive anchor for myofilaments that allows transmission of force, to a dynamic multicomplex structure, capable of sensing and transducing extracellular signals. Here, we describe a new Z-disc protein, which we named CHAP (cytoskeletal heart-enriched actin-associated protein), expressed in differentiating heart and skeletal muscle in vitro and in vivo. Interestingly, in addition to its sarcomeric localization, CHAP was also able to translocate to the nucleus. CHAP was associated with filamentous actin in the cytoplasm and the nucleus when expressed ectopically in vitro, but in rat neonatal cardiomyocytes, CHAP disrupted the subcellular localization of alpha-actinin, another Z-disc protein. More importantly, knockdown of CHAP in zebrafish resulted in aberrant cardiac and skeletal muscle development and function. These findings suggest that CHAP is a critical component of the sarcomere with an important role in muscle development.

Mapping the developing human cardiac endothelium at single-cell resolution identifies MECOM as a regulator of arteriovenous gene expression.

AIMS: Coronary vasculature formation is a critical event during cardiac development, essential for heart function throughout perinatal and adult life. However, current understanding of coronary vascular development has largely been derived from transgenic mouse models. The aim of this study was to characterize the transcriptome of the human foetal cardiac endothelium using single-cell RNA sequencing (scRNA-seq) to provide critical new insights into the cellular heterogeneity and transcriptional dynamics that underpin endothelial specification within the vasculature of the developing heart. METHODS AND RESULTS: We acquired scRNA-seq data of over 10 000 foetal cardiac endothelial cells (ECs), revealing divergent EC subtypes including endocardial, capillary, venous, arterial, and lymphatic populations. Gene regulatory network analyses predicted roles for SMAD1 and MECOM in determining the identity of capillary and arterial populations, respectively. Trajectory inference analysis suggested an endocardial contribution to the coronary vasculature and subsequent arterialization of capillary endothelium accompanied by increasing MECOM expression. Comparative analysis of equivalent data from murine cardiac development demonstrated that transcriptional signatures defining endothelial subpopulations are largely conserved between human and mouse. Comprehensive characterization of the transcriptional response to MECOM knockdown in human embryonic stem cell-derived EC (hESC-EC) demonstrated an increase in the expression of non-arterial markers, including those enriched in venous EC. CONCLUSIONS: scRNA-seq of the human foetal cardiac endothelium identified distinct EC populations. A predicted endocardial contribution to the developing coronary vasculature was identified, as well as subsequent arterial specification of capillary EC. Loss of MECOM in hESC-EC increased expression of non-arterial markers, suggesting a role in maintaining arterial EC identity.

Human embryonic stem cell-derived endothelial cell product injection attenuates cardiac remodeling in myocardial infarction.

Background: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium. Aim: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy. Materials and methods: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice (n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand-receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts. Results: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling. Conclusion: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction.

CAZIP, a novel protein expressed in the developing heart and nervous system.

Recently, we have performed a whole genome micro-array analysis on human embryonic stem cells differentiating toward cardiomyocytes, which resulted in the identification of novel genes that were highly up-regulated during differentiation. Here, we describe one of these novel genes annotated as KIAA0774. The predicted protein contains a leucine-zipper domain at the C-terminus and has at least two isoforms (358 and 1354 amino acids). Whole-mount in situ hybridization confirmed that the mRNA of both the mouse and chicken orthologs of KIAA0774 is expressed during early cardiac development. Hence, we named this protein CAZIP (cardiac zipper protein). Later during embryonic development, Cazip was also expressed in parts of the nervous system. Northern blot and real-time polymerase chain reaction analysis showed that Cazip is expressed in heart and brain in adult mice. These results suggest a role for CAZIP in development and function of the heart and nervous system in vertebrates.

Extracellular Vesicle miRNAs in the Promotion of Cardiac Neovascularisation.

Cardiovascular disease (CVD) is the leading cause of mortality worldwide claiming almost 17. 9 million deaths annually. A primary cause is atherosclerosis within the coronary arteries, which restricts blood flow to the heart muscle resulting in myocardial infarction (MI) and cardiac cell death. Despite substantial progress in the management of coronary heart disease (CHD), there is still a significant number of patients developing chronic heart failure post-MI. Recent research has been focused on promoting neovascularisation post-MI with the ultimate goal being to reduce the extent of injury and improve function in the failing myocardium. Cardiac cell transplantation studies in pre-clinical models have shown improvement in cardiac function; nonetheless, poor retention of the cells has indicated a paracrine mechanism for the observed improvement. Cell communication in a paracrine manner is controlled by various mechanisms, including extracellular vesicles (EVs). EVs have emerged as novel regulators of intercellular communication, by transferring molecules able to influence molecular pathways in the recipient cell. Several studies have demonstrated the ability of EVs to stimulate angiogenesis by transferring microRNA (miRNA, miR) molecules to endothelial cells (ECs). In this review, we describe the process of neovascularisation and current developments in modulating neovascularisation in the heart using miRNAs and EV-bound miRNAs. Furthermore, we critically evaluate methods used in cell culture, EV isolation and administration.

The clinical outcome of LMNA missense mutations can be associated with the amount of mutated protein in the nuclear envelope.

AIMS: Lamin A/C mutations are generally believed to be associated with a severe prognosis. The aim of this study was to investigate disease expression in three affected families carrying different LMNA missense mutations. Furthermore, the potential molecular disease mechanisms of the mutations were investigated in fibroblasts obtained from mutation carriers. METHODS AND RESULTS: A LMNA-p.Arg216Cys missense mutation was identified in a large family with 36 mutation carriers. Disease expression was unusual with a late onset and a favourable prognosis. Two smaller families with severe disease expression were shown to carry a LMNA-p.Arg471Cys and LMNA-p.Arg471His mutation, respectively. LMNA gene and protein expression was investigated in eight different mutation carriers by quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and protein mass spectrometry. The results showed that all mutation carriers incorporated mutated lamin protein into the nuclear envelope. Interestingly, the ratio of mutated to wild-type protein was only 30:70 in LMNA-p.Arg216Cys carriers with a favourable prognosis while LMNA-p.Arg471Cys and LMNA-p.Arg471His carriers with a more severe outcome expressed significantly more of the mutated protein by a ratio of 50:50. CONCLUSION: The clinical findings indicated that some LMNA mutations may be associated with a favourable prognosis and a low risk of sudden death. Protein expression studies suggested that a severe outcome was associated with the expression of high amounts of mutated protein. These findings may prove to be helpful in counselling and risk assessment of LMNA families.

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 and results: Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified 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.

Z-disc protein CHAPb induces cardiomyopathy and contractile dysfunction in the postnatal heart.

AIMS: The Z-disc is a crucial structure of the sarcomere and is implicated in mechanosensation/transduction. Dysregulation of Z-disc proteins often result in cardiomyopathy. We have previously shown that the Z-disc protein Cytoskeletal Heart-enriched Actin-associated Protein (CHAP) is essential for cardiac and skeletal muscle development. Furthermore, the CHAP gene has been associated with atrial fibrillation in humans. Here, we studied the misregulated expression of CHAP isoforms in heart disease. METHODS AND RESULTS: Mice that underwent transverse aortic constriction and calcineurin transgenic (Tg) mice, both models of experimental heart failure, displayed a significant increase in cardiac expression of fetal isoform CHAPb. To investigate whether increased expression of CHAPb postnatally is sufficient to induce cardiomyopathy, we generated CHAPb Tg mice under the control of the cardiac-specific αMHC promoter. CHAPb Tg mice displayed cardiac hypertrophy, interstitial fibrosis and enlargement of the left atrium at three months, which was more pronounced at the age of six months. Hypertrophy and fibrosis were confirmed by evidence of activation of the hypertrophic gene program (Nppa, Nppb, Myh7) and increased collagen expression, respectively. Connexin40 and 43 were downregulated in the left atrium, which was associated with delayed atrioventricular conduction. Tg hearts displayed both systolic and diastolic dysfunction partly caused by impaired sarcomere function evident from a reduced force generating capacity of single cardiomyocytes. This co-incided with activation of the actin signalling pathway leading to the formation of stress fibers. CONCLUSION: This study demonstrated that the fetal isoform CHAPb initiates progression towards cardiac hypertrophy, which is accompanied by delayed atrioventricular conduction and diastolic dysfunction. Moreover, CHAP may be a novel therapeutic target or candidate gene for screening in cardiomyopathies and atrial fibrillation.