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The IgCAM coxsackie–adenovirus receptor (CAR) is essential for embryonic heart development and electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analyzed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wild type and CAR knockout (KO) embryos. Surprisingly, in the absence of the CAR, cultured cardiomyocytes showed increased frequencies of beating and calcium cycling. Increased beatings of heart organ cultures were also induced by the application of reagents that bind to the extracellular region of the CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed size increases but decreased in the total numbers of membrane-localized Cx43 clusters. This was accompanied by improved cell–cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that the CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions.

Tissue dissociation, a crucial step in single-cell sample preparation, can alter the transcriptional state of a sample through the intrinsic cellular stress response. Here we demonstrate a general approach for measuring transcriptional response during sample preparation. In our method, transcripts made during dissociation are labeled for later identification upon sequencing. We found general as well as cell-type-specific dissociation response programs in zebrafish larvae, and we observed sample-to-sample variation in the dissociation response of mouse cardiomyocytes despite well-controlled experimental conditions. Finally, we showed that dissociation of the mouse hippocampus can lead to the artificial activation of microglia. In summary, our approach facilitates experimental optimization of dissociation procedures as well as computational removal of transcriptional perturbation response.

Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm(20S637A)) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.

The article looks at the outcome of the two legal proceedings in the Schüth and IR cases. In both cases employees of the Catholic Church – a choirmaster and organist in a Catholic parish and a trained physician working as Head of the Internal Medicine Department of a Catholic hospital - were dismissed because of the violation of the Basic Regulations on Employment Relationships in the Service of the Church. In the Schüth case Article 8 of the European Convention on Human Rights (ECHR), which protects the right to private and family life, had been violated. In the IR case the Charter of Fundamental Rights of the European Union and the Directive establishing a general framework for equal treatment in employment and occupation were applicable. The dismissal in IR was held to be unequal treatment in employment. But the outcome of both cases was very different. We find that Union law and in particular Article 47 of the Charter of Fundamental Rights of the European Union makes all the difference. In the Schüth case, the employment relationship was terminated and the claimant’s only consolation was a claim for damages from the State. In the IR case, on the other hand, the termination was declared invalid and the employment relationship continued, i.e. the head physician did not lose his job. The comparison of the cases demonstrates that European law, backed by Article 47 of the Charter of Fundamental Rights of the European Union, has not only permeated procedural law, it has also led to an increase in judicial reviews of substantive law which in the application of Union law is a far cry from a mere plausibility review.

Heart failure with preserved ejection fraction (HFpEF) is prevalent and deadly, but so far, there is no targeted therapy. A main contributor to the disease is impaired ventricular filling, which we improved with antisense oligonucleotides (ASOs) targeting the cardiac splice factor RBM20. In adult mice with increased wall stiffness, weekly application of ASOs over 2 months increased expression of compliant titin isoforms and improved cardiac function as determined by echocardiography and conductance catheter. RNA sequencing confirmed RBM20-dependent isoform changes and served as a sensitive indicator of potential side effects, largely limited to genes related to the immune response. We validated our approach in human engineered heart tissue, showing down-regulation of RBM20 to less than 50% within 3 weeks of treatment with ASOs, resulting in adapted relaxation kinetics in the absence of cardiac pathology. Our data suggest anti-RBM20 ASOs as powerful cardiac splicing regulators for the causal treatment of human HFpEF.

Introduction: The versatility of the human genome is increased by the process of alternative mRNA splicing. Impaired splicing of the cardiac transcriptome is involved in the pathophysiology of heart failure. Especially, mutations in cardiac-specific splicing factors such as RBM20 cause severe forms of cardiomyopathy. Aim: We aimed to identify novel cardiomyopathy-associated splicing factors in the human heart using a score of myocardial tissue specificity including 53 human tissues and disease-associated expression changes in hearts of dilated cardiomyopathy (DCM) patients and controls. Methods & Results: We found the splicing factor Slm2 to be significantly upregulated on the mRNA and protein level in the human myocardium of patients diagnosed with DCM (1.88 and 5.3-fold, both Pin vivo resulted in DCM in zebrafish and sarcomere irregularity and altered calcium cycling in human cardiomyocytes. Sequencing of RNAs bound to Slm2 isolated from diseased human hearts, revealed the interaction of Slm2 with important mRNA transcripts encoding for sarcomere constituents, such as troponin I, troponin T, tropomyosin and titin. In the failing human myocardium, Slm2 bound to an alternative titin mRNA variant including retained introns. In detail, Slm2 interacted with exons of the titin mRNA encoding for the PEVK region, which mediates flexibility and contractile properties of the heart muscle. Using a splice reporter assay, which contains parts of titin’s PEVK region, we found Slm2 mediating the splicing process of intron retention. Conclusion: Slm2 is a novel splicing factor in the diseased human myocardium, maintaining cardiomyocyte integrity and function by binding and splicing essential sarcomere constituents including titin.

Purpose Atrial fibrillation (AF) is the most common sustained arrhythmia. It carries a large healthcare burden and is associated with serious complications. The arrhythmia has a substantial genetic component and is associated with several structural genes, including the gene TTN. A recent large genome-wide association study on AF found an association to RBM20. The RBM20 gene is a splicing factor targeting TTN, RYR2 and CAMK2D among other cardiac genes. Using Next-Generation Sequencing and data derived from the UK Biobank, we aimed to reveal the role of RBM20 in AF. Methods and results We examined the burden of rare (Minor allele frequency (MAF) To examine the effect of rare missense RBM20 variants in the splicing of TTN, we screened an in-house cohort of 531 Scandinavian early-onset AF patients using targeted sequencing. We filtered for rare (MAF20) variants and identified nine missense variants and three novel LOF variants in RBM20. To evaluate the effect of these RBM20 variants, we constructed a series of human RBM20 single nucleotide base exchange mutants. The splicing activity of the variants was measured with RT-qPCR on HEK293 cells transfected with a TTN241–3 splicing reporter. Four of these variants resulted in a significantly altered splicing activity in TTN, with the largest effect observed for LOF variants. In order to examine the biological effect of RBM20 variants on structural changes in atrial tissue, we used a Norwegian Brown rat animal model with loss of RBM20. In this model, Transmission Electron Microscopy revealed altered sarcomere and mitochondrial structure in its atrial cardiomyocytes. Furthermore, nanopore RNA sequencing of atrial tissue from the aforementioned animal model indicated altered expression in several key cardiac genes, including TTN and PITX2. Conclusion Rare RBM20 LOF variants are significantly enriched in AF cases, seen in a large population of 175,000 individuals. We demonstrated that the effect of LOF RBM20 on alternative TTN splicing can be detected on an individual level in patients with AF. Studies using an animal model indicates that LOF in RBM20 may affect atrial function through altered expression of several genes in the atria, and may cause structural changes in the atrial cardiomyocytes. This suggests that RBM20 may be involved in AF pathogenesis mediated through an atrial cardiomyopathy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Novo Nordisk Foundation Pre-Graduate Scholarships (NNF18OC0053094)The Hallas Møller Emerging Investigator grant (Novo Nordisk Foundation (NNF17OC0031204))

Splice regulators play an essential role in the transcriptomic diversity of all eukaryotic cell types and organ systems. Recent evidence suggests a contribution of splice-regulatory networks in many diseases, such as cardiomyopathies. Adaptive splice regulators, such as RNA-binding motif protein 20 (RBM20) determine the physiological mRNA landscape formation, and rare variants in the RBM20 gene explain up to 6% of genetic dilated cardiomyopathy (DCM) cases. With ample knowledge from RBM20-deficient mice, rats, swine and induced pluripotent stem cells (iPSCs), the downstream targets and quantitative effects on splicing are now well-defined and the prerequisites for corrective therapeutic approaches are set. This review article highlights some of the recent advances in the field, ranging from aspects of granule formation to 3D genome architectures underlying RBM20-related cardiomyopathy. Promising therapeutic strategies are presented and put into context with the pathophysiological characteristics of RBM20-related diseases.

SummaryRNA-binding proteins (RBPs) are major regulators of gene expression at the post-transcriptional level. While many posttranslational modification sites in RBPs have been identified, little is known about how these modifications regulate RBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Applying qRIC to HEK293T cells quantified pull-down efficiencies of over 300 RBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Over hundred phosphorylation events increased or decreased pull-down efficiency compared to the unmodified RBPs and thus have regulatory potential. Our data captures known regulatory phosphorylation sites in ELAVL1, SF3B1 and UPF1 and identifies new potentially regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localization, association with cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify functional posttranslational modification sites in RBPs.HighlightsqRIC globally quantifies the fraction of RNA-binding proteins pulled down with mRNACombining qRIC with phosphoproteomics identifies sites that affect RNA bindingPhosphorylation sites in RBM20 regulate its function in splicing

Background: Titin is a giant elastic protein that spans the half-sarcomere from Z-disk to M-band. It acts as a molecular spring and mechanosensor and has been linked to striated muscle disease. The pathways that govern titin-dependent cardiac growth and contribute to disease are diverse and difficult to dissect. Methods: To study titin deficiency versus dysfunction, the authors generated and compared striated muscle specific knockouts (KOs) with progressive postnatal loss of the complete titin protein by removing exon 2 (E2-KO) or an M-band truncation that eliminates proper sarcomeric integration, but retains all other functional domains (M-band exon 1/2 [M1/2]-KO). The authors evaluated cardiac function, cardiomyocyte mechanics, and the molecular basis of the phenotype. Results: Skeletal muscle atrophy with reduced strength, severe sarcomere disassembly, and lethality from 2 weeks of age were shared between the models. Cardiac phenotypes differed considerably: loss of titin leads to dilated cardiomyopathy with combined systolic and diastolic dysfunction—the absence of M-band titin to cardiac atrophy and preserved function. The elastic properties of M1/2-KO cardiomyocytes are maintained, while passive stiffness is reduced in the E2-KO. In both KOs, we find an increased stress response and increased expression of proteins linked to titin-based mechanotransduction (CryAB, ANKRD1, muscle LIM protein, FHLs, p42, Camk2d, p62, and Nbr1). Among them, FHL2 and the M-band signaling proteins p62 and Nbr1 are exclusively upregulated in the E2-KO, suggesting a role in the differential pathology of titin truncation versus deficiency of the full-length protein. The differential stress response is consistent with truncated titin contributing to the mechanical properties in M1/2-KOs, while low titin levels in E2-KOs lead to reduced titin-based stiffness and increased strain on the remaining titin molecules. Conclusions: Progressive depletion of titin leads to sarcomere disassembly and atrophy in striated muscle. In the complete knockout, remaining titin molecules experience increased strain, resulting in mechanically induced trophic signaling and eventually dilated cardiomyopathy. The truncated titin in M1/2-KO helps maintain the passive properties and thus reduces mechanically induced signaling. Together, these findings contribute to the molecular understanding of why titin mutations differentially affect cardiac growth and have implications for genotype-phenotype relations that support a personalized medicine approach to the diverse titinopathies.

Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and nonphosphorylated forms of mRBPs. Almost 200 phosphorylation events affected pull-down efficiency compared with the unmodified mRBPs and thus have regulatory potential. Our data capture known regulatory phosphorylation sites in ELAVL1, SF3B1, and UPF1 and identify potential regulatory sites. Follow-up experiments on the splicing regulator RBM20 revealed multiple phosphorylation sites in the C-terminal disordered region affecting nucleocytoplasmic localization, association with cytoplasmic ribonucleoprotein granules, and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.

Rationale: CAV3 (caveolin3) variants associated with arrhythmogenic cardiomyopathy and muscular dystrophy can disrupt post-Golgi surface trafficking. As CAV1 (caveolin1) was recently identified in cardiomyocytes, we hypothesize that conserved isoform-specific protein/protein interactions orchestrate unique cardiomyocyte microdomain functions. To analyze the CAV1 versus CAV3 interactome, we employed unbiased live-cell proximity proteomic, isoform-specific affinity, and complexome profiling mass spectrometry techniques. We demonstrate the physiological relevance and loss-of-function mechanism of a novel CAV3 interactor in gene-edited human induced pluripotent stem cell cardiomyocytes. Objective: To identify differential CAV1 versus CAV3 protein interactions and to define the molecular basis of cardiac CAV3 loss-of-function. Methods and Results: Combining stable isotope labeling with proximity proteomics, we applied mass spectrometry to screen for putative CAV3 interactors in living cardiomyocytes. Isoform-specific affinity proteomic and co-immunoprecipitation experiments confirmed the monocarboxylate transporter McT1 (monocarboxylate transporter type 1) versus aquaporin1, respectively, as CAV3 or CAV1 specific interactors in cardiomyocytes. Superresolution stimulated emission depletion microscopy showed distinct CAV1 versus CAV3 cluster distributions in cardiomyocyte transverse tubules. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9 nuclease)-mediated CAV3 knockout uncovered a stabilizing role for McT1 surface expression, proton-coupled lactate shuttling, increased late Na + currents, and early afterdepolarizations in human induced pluripotent stem cell-derived cardiomyocytes. Complexome profiling confirmed that McT1 and the Na,K-ATPase form labile protein assemblies with the multimeric CAV3 complex. Conclusions: Combining the strengths of proximity and affinity proteomics, we identified isoform-specific CAV1 versus CAV3 binding partners in cardiomyocytes. McT1 represents a novel class of metabolically relevant CAV3-specific interactors close to mitochondria in cardiomyocyte transverse tubules. CAV3 knockout uncovered a previously unknown role for functional stabilization of McT1 in the surface membrane of human cardiomyocytes. Strikingly, CAV3 deficient cardiomyocytes exhibit action potential prolongation and instability, reproducing human reentry arrhythmias in silico. Given that lactate is a major substrate for stress adaption both in the healthy and the diseased human heart, future studies of conserved McT1/CAV3 interactions may provide rationales to target this muscle-specific assembly function therapeutically.

RNA-binding proteins (RBPs) are major regulators of gene expression at the post-transcriptional level. While many posttranslational modification sites in RBPs have been identified, little is known about how these modifications regulate RBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of cellular RBPs pulled down with polyadenylated mRNAs. Applying qRIC to HEK293T cells quantified pull-down efficiencies of over 300 RBPs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and non-phosphorylated forms of RBPs. Over hundred phosphorylation events increased or decreased pull-down efficiency compared to the unmodified RBPs and thus have regulatory potential. Our data captures known regulatory phosphorylation sites in ELAVL1, SF3B1 and UPF1 and identifies new potentially regulatory sites. Follow-up experiments on the cardiac splicing regulator RBM20 revealed that multiple phosphorylation sites in the C-terminal disordered region affect nucleo-cytoplasmic localization, association with cytosolic RNA granules and alternative splicing. Together, we show that qRIC is a scalable method to identify functional posttranslational modification sites in RBPs.

Alternative mRNA splicing is a fundamental process to increase the versatility of the genome. In humans, cardiac mRNA splicing is involved in the pathophysiology of heart failure. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) cause severe forms of cardiomyopathy. To identify novel cardiomyopathy-associated splicing factors, RNA-seq and tissue-enrichment analyses were performed, which identified up-regulated expression of Sam68-Like mammalian protein 2 (SLM2) in the left ventricle of dilated cardiomyopathy (DCM) patients. In the human heart, SLM2 binds to important transcripts of sarcomere constituents, such as those encoding myosin light chain 2 (MYL2), troponin I3 (TNNI3), troponin T2 (TNNT2), tropomyosin 1/2 (TPM1/2), and titin (TTN). Mechanistically, SLM2 mediates intron retention, prevents exon exclusion, and thereby mediates alternative splicing of the mRNA regions encoding the variable proline-, glutamate-, valine-, and lysine-rich (PEVK) domain and another part of the I-band region of titin. In summary, SLM2 is a novel cardiac splicing regulator with essential functions for maintaining cardiomyocyte integrity by binding to and processing the mRNAs of essential cardiac constituents such as titin.

Molecular and cellular mechanisms of mutations of splicing factors in heart function are not well understood. The splicing of precursor mRNA is dependent on an essential group of splicing factors containing serine-arginine (SR) domain(s) that are critical for protein-RNA and protein-protein interaction in the spliceosome assembly. Phosphorylation of SR domains plays a key role in splicing control and the distribution of splicing factors in the cell. RNA binding motif 20 (RBM20) is a splicing factor predominantly expressed in muscle tissues with the highest expression level in the heart. However, its phosphorylation status is completely unknown up-to-date. In this study, we identified sixteen amino acid residues that are phosphorylated by middle-down mass spectrometry. Four of them are located in the SR domain, and two out of the four residues, S638 and S640, play an essential role in splicing control and facilitate RBM20 shuttling from the nucleus to the cytoplasm. Re-localization of RBM20 promotes protein aggregation in the cytoplasm. We have also verified that SR-protein kinases (SRPKs), cdc2-like kinases (CLKs) and protein kinase B (PKB or AKT) phosphorylate S638 and S640. Mutations of S638A and S640G reduce RBM20 phosphorylation and disrupt the splicing. Taken together, we determine the phosphorylation status of RBM20 and provide the first evidence that phosphorylation on SR domain is crucial for pre-mRNA splicing and protein trafficking. Our findings reveal a new role of RBM20 via protein shuttling in cardiac function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Introduction: Published work has shown that RNA binding motif 20 (RBM20) can improve ventricular stiffening through altering titin sizes in HFpEF, a syndrome with no effective therapeutic options. Reducing RBM20 activity to treat diastolic dysfunction is promising, but it may cause side effects due to its multiple targets except for titin. RBM20 is one of the serine-arginine (SR) proteins of which the phosphorylation plays a critical role in gene splicing. Here we investigate the phosphorylation status of RBM20 and its potential role in diastolic dysfunction. Methods: Insect cell expression system was utilizedfor RBM20 expression. Purified RBM20 was subjected to the middle-down mass spectrometry (MS) analysis. Identified phosphorylation sites were mutated for in-vitro splicing and dual luciferase splice reporter assay through co-transfection with titin mini-gene. In-vitro kinase and co-IP assays were performed to identify the kinases that phosphorylate RBM20. Since phosphorylation is also important for translocation of RNA binding proteins, the confocal microscope was applied to estimate RBM20 trafficking. Results: Sixteen phosphorylation sites with four of them on SR domain of RBM20 were identified with MS analysis. In-vitro splicing and dual luciferase reporter assays revealed that two phosphorylation sites on SR domain play a major role in titin mini-gene splicing. In-vitro kinase assay indicated that kinases SRPK, CLK, and AKT can all phosphorylate RBM20, and co-IP confirmed the interaction between kinases and RBM20. Co-transfection of these kinases with RBM20 and titin mini-gene revealed that splicing pattern was different from co-transfection of mutated RBM20. Interestingly, two-phosphorylation sites mutation on SR domain facilitated RBM20 trafficking from the nucleus to the cytoplasm and thus, form RNA granules, suggesting the new role of RBM20 phosphorylation in diastolic dysfunction. In-vivo studies with mutation knock-in mouse model will be warranted in near future. Conclusion: These results suggest that RBM20 phosphorylation plays an important role in titin splicing, which is a potential target to treat diastolic dysfunction. RBM20 trafficking could be another new pathway to treat HFpEF.

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

SUMMARY Recent advances in induced pluripotent stem cell (iPSC) technology and directed differentiation of iPSCs into cardiomyocytes (iPSC-CMs) make it possible to model genetic heart disease in vitro. We apply CRISPR/Cas9 genome editing technology to introduce three RBM20 mutations in iPSCs and differentiate them into iPSC-CMs to establish an in vitro model of RBM20 mutant dilated cardiomyopathy (DCM). In iPSC-CMs harboring a known causal RBM20 variant, the splicing of RBM20 target genes, calcium handling, and contractility are impaired consistent with the disease manifestation in patients. A variant (Pro633Leu) identified by exome sequencing of patient genomes displays the same disease phenotypes, thus establishing this variant as disease causing. We find that all-trans retinoic acid upregulates RBM20 expression and reverts the splicing, calcium handling, and contractility defects in iPSC-CMs with different causal RBM20 mutations. These results suggest that pharmacological upregulation of RBM20 expression is a promising therapeutic strategy for DCM patients with a heterozygous mutation in RBM20., Graphical Abstract, In Brief Briganti et al. use iPSC and CRISPR/Cas9 to create a model of RBM20-deficient dilated cardiomyopathy (DCM) that recapitulates mRNA splicing and contractile defects of the disease. They evaluate pharmacological upregulation of RBM20 as a therapeutic strategy. All-trans retinoic acid upregulates RBM20 expression and ameliorates the in vitro hallmarks of disease.

This protocol provides a walk through for complex processing of fresh human cardiac tissue followed by tissue dissociation for single cell RNA sequencing and nuclei isolation for single nuclei RNA sequencing. The protocol can be used for various regions of the heart muscle, including the ventricles, atria and septum. Each section looks slightly different and will contain different amount of tissue, meaning that the number of applications and the amount of stored tissue will vary. The protocol is also applicable to animal cardiac tissue with minor modifications adjusting for size and amount of the tissue.

Heart failure with preserved ejection fraction (HFpEF) is a syndrome characterized by increased diastolic stiffness, for which effective therapies are lacking. Slater et al. show that metformin lowers titin-based passive stiffness in an HFpEF mouse model and may therefore be of therapeutic benefit., Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome characterized by a preserved ejection fraction but increased diastolic stiffness and abnormalities of filling. Although the prevalence of HFpEF is high and continues to rise, no effective therapies exist; however, the diabetic drug metformin has been associated with improved diastolic function in diabetic patients. Here we determine the therapeutic potential of metformin for improving diastolic function in a mouse model with HFpEF-like symptoms. We combine transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) supplementation to obtain a mouse model with increased diastolic stiffness and exercise intolerance. Echocardiography and pressure–volume analysis reveal that providing metformin to TAC/DOCA mice improves diastolic function in the left ventricular (LV) chamber. Muscle mechanics show that metformin lowers passive stiffness of the LV wall muscle. Concomitant with this improvement in diastolic function, metformin-treated TAC/DOCA mice also demonstrate preserved exercise capacity. No metformin effects are seen in sham operated mice. Extraction experiments on skinned ventricular muscle strips show that the metformin-induced reduction of passive stiffness in TAC/DOCA mice is due to an increase in titin compliance. Using phospho-site-specific antibodies, we assay the phosphorylation of titin’s PEVK and N2B spring elements. Metformin-treated mice have unaltered PEVK phosphorylation but increased phosphorylation of PKA sites in the N2B element, a change which has previously been shown to lower titin’s stiffness. Consistent with this result, experiments with a mouse model deficient in the N2B element reveal that the beneficial effect of metformin on LV chamber and muscle stiffness requires the presence of the N2B element. We conclude that metformin offers therapeutic benefit during HFpEF by lowering titin-based passive stiffness.

Aims Heart failure with preserved ejection fraction (HFpEF) and pathological cardiac aging share a complex pathophysiology, including extracellular matrix remodelling (EMR). Protease-activated receptor 2 (PAR2) deficiency is associated with EMR. The roles of PAR1 and PAR2 have not been studied in HFpEF, age-dependent cardiac fibrosis, or diastolic dysfunction (DD). Methods and results Evaluation of endomyocardial biopsies from patients with HFpEF (n = 14) revealed that a reduced cardiac PAR2 expression was associated with aggravated DD and increased myocardial fibrosis (r = −0.7336, P = 0.0028). In line, 1-year-old PAR2-knockout (PAR2ko) mice suffered from DD with preserved systolic function, associated with an increased age-dependent α-smooth muscle actin expression, collagen deposition (1.7-fold increase, P = 0.0003), lysyl oxidase activity, collagen cross-linking (2.2-fold increase, P = 0.0008), endothelial activation, and inflammation. In the absence of PAR2, the receptor-regulating protein caveolin-1 was down-regulated, contributing to an augmented profibrotic PAR1 and transforming growth factor beta (TGF-β)-dependent signalling. This enhanced TGF-β/PAR1 signalling caused N-proteinase (ADAMTS3) and C-proteinase (BMP1)-related increased collagen I production from cardiac fibroblasts (CFs). PAR2 overexpression in PAR2ko CFs reversed these effects. The treatment with the PAR1 antagonist, vorapaxar, reduced cardiac fibrosis by 44% (P = 0.03) and reduced inflammation in a metabolic disease model (apolipoprotein E-ko mice). Patients with HFpEF with upstream PAR inhibition via FXa inhibitors (n = 40) also exhibited reduced circulating markers of fibrosis and DD compared with patients treated with vitamin K antagonists (n = 20). Conclusions Protease-activated receptor 2 is an important regulator of profibrotic PAR1 and TGF-β signalling in the heart. Modulation of the FXa/FIIa-PAR1/PAR2/TGF-β-axis might be a promising therapeutic approach to reduce HFpEF.

The heart senses and responds to changes in mechanical loading conditions by initiating signaling events to produce structural and functional remodeling through a process known as mechanotransduction. Within cardiac myocytes, the giant protein titin has been implicated as a key player in the conversion of mechanical stimuli into downstream signaling events as part of the adaptive responses of mechanotransduction. The N2B element within the extensible I-band of titin has been proposed to be a stretch sensor and signaling hot spot important to titin’s role as a mechanosensor. To investigate the role of the N2B element in mechanotransduction, N2B knockout (N2BKO) mice were subjected to cardiac pressure overload by transverse aortic constriction (TAC) or volume overload by aortocaval fistula (ACF). Following 4 weeks TAC, WT mice respond with 37% LV hypertrophy (4.0 ± 0.1 mg/gBW for TAC vs 2.9 ± 0.1 mg/gBW for sham) compared to 51% (3.8 ± 0.1 mg/gBW for TAC vs 2.5 ± 0.1 mg/gBW for sham) in N2BKO. In contrast, N2BKO mice demonstrated a severe lack of a hypertrophic response following 1-week ACF with 26% (4.0 ± 0.1 mg/gBW for ACF vs 3.1 ± 0.1 mg/gBW for sham) LV hypertrophy in WT compared to -2% (2.8 ± 0.1 mg/gBW for ACF vs 2.8 ± 0.1 mg/gBW for sham) in N2BKO. Additionally, while there was no incidence of mortality in WT mice subjected to ACF, approximately 36% (5 of 14) of N2BKO mice died within 1 week following ACF. Western blot analysis of canonical hypertrophy signaling kinases revealed no differences between WT and N2BKO mice at baseline or 1 week post-ACF. Four-and-a half LIM domains 1 and 2 (FHL1 and FHL2) have previously been shown to localize to the N2B element and FHL1 has been linked to mechanotransduction in TAC. In N2BKO, FHL1 protein is expressed at levels comparable to WT in both TAC and ACF; however, FHL2 protein levels are reduced by ~98% compared to WT (1.00 ± 0.26 for WT vs 0.02 ± 0.01% for N2BKO). These data indicate that while the N2B element is not required for mechanotransduction in pressure overload of the LV, it is critical for mechanosensing and the adaptive hypertrophy response during volume overload. FHL2 is an attractive candidate as a link between titin’s N2B element and the hypertrophy response following ACF and further work will critical examine its involvement.

International audience; Mutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients.

PAR-CLIP (photoactivatable ribonucleoside–enhanced crosslinking and immunoprecipitation) facilitates the identification and mapping of protein/RNA interactions. So far, it has been limited to select cell-lines as it requires efficient 4SU uptake. To increase transcriptome complexity and thus identify additional RNA-protein interaction sites we fused HEK 293 T-Rex cells (HEK293-Y) that express the RNA binding protein YBX1 with PC12 cells expressing eGFP (PC12-eGFP). The resulting hybrids enable PAR-CLIP on a neuronally expanded transcriptome (Fusion-CLIP) and serve as a proof of principle. The fusion cells express both parental marker genes YBX1 and eGFP and the expanded transcriptome contains human and rat transcripts. PAR-CLIP of fused cells versus the parental HEK293-Y identified 768 novel RNA targets of YBX1. We were able to trace the origin of the majority of the short PAR-CLIP reads as they differentially mapped to the human and rat genome. Furthermore, Fusion-CLIP expanded the CAUC RNA binding motif of YBX1 to UCUUUNNCAUC. The fusion of HEK293-Y and PC12-eGFP cells resulted in cells with a diverse genome expressing human and rat transcripts that enabled the identification of novel YBX1 substrates. The technique allows the expansion of the HEK 293 transcriptome and makes PAR-CLIP available to fusion cells of diverse origin.

RNA-binding motif protein 20 (RBM20) is a cardiac splice regulator that adapts cardiac filling via its diverse substrates—including the sarcomeric protein titin. The molecular basis and regulation of RBM20-dependent exon exclusion are largely unknown. In tissue culture experiments, we show that the combination of RNA recognition motif (RRM) and C-terminus is necessary and sufficient for RBM20 activity, indicating an important function of the ZnF2 domain in splicing repression. Using splice reporter and in vitro binding assays targeting titin exons 241–243, we identified a minimal genomic segment that is necessary for RBM20-mediated splicing repression of the alternative exon. Here, RBM20 binds the cluster containing most RBM20 binding motifs through its RRM domain and represses the upstream and downstream introns. For subsequent exon exclusion, specific regions upstream, downstream and within the alternative exon 242 are required. Regulation of exon exclusion involves PTB4 as a novel titin splice regulator, which counteracts RBM20 repressor activity in HEK293 cells. Together, these mechanistic insights into the regulation and action of RBM20 and PTB4 provide a basis for the future development of RBM20 modulators that adapt titin elasticity in cardiac disease.

Safe anaerobic metabolism Naked mole-rats live in large colonies deep underground in hypoxic conditions. Park et al. found that these animals fuel anaerobic glycolysis with fructose by a rewired pathway that avoids tissue damage (see the Perspective by Storz and McClelland). These results provide insight into the adaptations that this strange social rodent has to make for life underground. They also have implications for medical practice, particularly for understanding how to protect tissues from hypoxia. Science , this issue p. 307 ; see also p. 248

The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail links CAR to the cytoskeleton and intracellular signaling cascades. In the heart, CAR is crucial for embryonic development, electrophysiology, and coxsackievirus B infection. Noncardiac functions are less well understood, in part due to the lack of suitable animal models. Here, we generated a transgenic mouse that rescued the otherwise embryonic-lethal CAR knockout (KO) phenotype by expressing chicken CAR exclusively in the heart. Using this rescue model, we addressed interspecies differences in coxsackievirus uptake and noncardiac functions of CAR. Survival of the noncardiac CAR KO (ncKO) mouse indicates an essential role for CAR in the developing heart but not in other tissues. In adult animals, cardiac activity was normal, suggesting that chicken CAR can replace the physiological functions of mouse CAR in the cardiomyocyte. However, chicken CAR did not mediate virus entry in vivo , so that hearts expressing chicken instead of mouse CAR were protected from infection and myocarditis. Comparison of sequence homology and modeling of the D1 domain indicate differences between mammalian and chicken CAR that relate to the sites important for virus binding but not those involved in homodimerization. Thus, CAR-directed anticoxsackievirus therapy with only minor adverse effects in noncardiac tissue could be further improved by selectively targeting the virus-host interaction while maintaining cardiac function. IMPORTANCE Coxsackievirus B3 (CVB3) is one of the most common human pathogens causing myocarditis. Its receptor, the coxsackievirus and adenovirus receptor (CAR), not only mediates virus uptake but also relates to cytoskeletal organization and intracellular signaling. Animals without CAR die prenatally with major cardiac malformations. In the adult heart, CAR is important for virus entry and electrical conduction, but its nonmuscle functions are largely unknown. Here, we show that chicken CAR expression exclusively in the heart can rescue the otherwise embryonic-lethal CAR knockout phenotype but does not support CVB3 infection of adult cardiomyocytes. Our findings have implications for the evolution of virus-host versus physiological interactions involving CAR and could help to improve future coxsackievirus-directed therapies inhibiting virus replication while maintaining CAR's cellular functions.

The cellular basis of the Frank–Starling “Law of the Heart” is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension–pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.

Aims: In this study, we aimed to clinically and genetically characterize LVNC patients and investigate the prevalence of variants in known and novel LVNC disease genes. Introduction: Left ventricular non-compaction cardiomyopathy (LVNC) is an increasingly recognized cause of heart failure, arrhythmia, thromboembolism, and sudden cardiac death. We sought here to dissect its genetic causes, phenotypic presentation and outcome. Methods and results: In our registry with follow-up of in the median 61 months, we analysed 95 LVNC patients (68 unrelated index patients and 27 affected relatives; definite familial LVNC = 23.5%) by cardiac phenotyping, molecular biomarkers and exome sequencing. Cardiovascular events were significantly more frequent in LVNC patients compared with an age-matched group of patients with non-ischaemic dilated cardiomyopathy (hazard ratio = 2.481, P = 0.002). Stringent genetic classification according to ACMG guidelines revealed that TTN, LMNA, and MYBPC3 are the most prevalent disease genes (13 patients are carrying a pathogenic truncating TTN variant, odds ratio = 40.7, Confidence interval = 21.6-76.6, P