Blood taken immediately after fatal resuscitation attempts yields higher quality DNA for genetic studies as compared to autopsy samples.

BACKGROUND: The out-of-hospital cardiac arrest (OHCA) in the young may be associated with a genetic predisposition which is relevant even for genetic counseling of relatives. The identification of genetic variants depends on the availability of intact genomic DNA. DNA from autopsy may be not available due to low autopsy frequencies or not suitable for high-throughput DNA sequencing (NGS). The emergency medical service (EMS) plays an important role to save biomaterial for subsequent molecular autopsy. It is not known whether the DNA integrity of samples collected by the EMS is better suited for NGS than autopsy specimens. MATERIAL AND METHODS: DNA integrity was analyzed by standardized protocols. Fourteen blood samples collected by the EMS and biomaterials from autopsy were compared. We collected 172 autopsy samples from different tissues and blood with postmortem intervals of 14-168 h. For comparison, DNA integrity derived from blood stored under experimental conditions was checked against autopsy blood after different time intervals. RESULTS: DNA integrity and extraction yield were higher in EMS blood compared to any autopsy tissue. DNA stability in autopsy specimens was highly variable and had unpredictable quality. In contrast, collecting blood samples by the EMS is feasible and delivered comparably the highest DNA integrity. CONCLUSIONS: Isolation yield and DNA integrity from blood samples collected by the EMS is superior in comparison to autopsy specimens. DNA from blood samples collected by the EMS on scene is stable at room temperature or even for days at 4 °C. We conclude that the EMS personnel should always save a blood sample of young fatal OHCA cases died on scene to enable subsequent genetic analysis.

Frequency-dependent signaling in cardiac myocytes.

Background: Recent experimental data support the view that signaling activity at the membrane depends on its geometric parameters such as surface area and curvature. However, a mathematical, biophysical concept linking shape to receptor signaling is missing. The membranes of cardiomyocytes are constantly reshaped due to cycles of contraction and relaxation. According to constant-volume behavior of cardiomyocyte contraction, the length shortening is compensated by Z-disc myofilament lattice expansion and dynamic deformation of membrane between two adjacent Z-discs. Both morphological changes are strongly dependent on the frequency of contraction. Here, we developed the hypothesis that dynamic geometry of cardiomyocytes could be important for their plasticity and signaling. This effect may depend on the frequency of the beating heart and may represent a novel concept to explain how changes in frequency affect cardiac signaling. Methods: This hypothesis is almost impossible to answer with experiments, as the in-vitro cardiomyocytes are almost two-dimensional and flattened rather than being in their real in-vivo shape. Therefore, we designed a COMSOL multiphysics program to mathematically model the dynamic geometry of a human cardiomyocyte and explore whether the beating frequency can modulate membrane signal transduction. Src kinase is an important component of cardiac mechanotransduction. We first presented that Src mainly localizes at costameres. Then, the frequency-dependent signaling effect was studied mathematically by numerical simulation of Src-mediated PDGFR signaling pathway. The reaction-convection-diffusion partial differential equation was formulated to simulate PDGFR pathway in a contracting sarcomeric disc for a range of frequencies from 1 to 4 Hz. Results: Simulations exhibits higher concentration of phospho-Src when a cardiomyocyte beats with higher rates. The calculated phospho-Src concentration at 4, 2, and 1 Hz beat rates, comparing to 0 Hz, was 21.5%, 9.4%, and 4.7% higher, respectively. Conclusion: Here we provide mathematical evidence for a novel concept in biology. Cell shape directly translates into signaling, an effect of importance particularly for the myocardium, where cells continuously reshape their membranes. The concept of locality of surface-to-volume ratios is demonstrated to lead to changes in membrane-mediated signaling and may help to explain the remarkable plasticity of the myocardium in response to biomechanical stress.

Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation.

Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.

Targeted therapies in genetic dilated and hypertrophic cardiomyopathies: from molecular mechanisms to therapeutic targets. A position paper from the Heart Failure Association (HFA) and the Working Group on Myocardial Function of the European Society of Cardiology (ESC).

Genetic cardiomyopathies are disorders of the cardiac muscle, most often explained by pathogenic mutations in genes encoding sarcomere, cytoskeleton, or ion channel proteins. Clinical phenotypes such as heart failure and arrhythmia are classically treated with generic drugs, but aetiology-specific and targeted treatments are lacking. As a result, cardiomyopathies still present a major burden to society, and affect many young and older patients. The Translational Committee of the Heart Failure Association (HFA) and the Working Group of Myocardial Function of the European Society of Cardiology (ESC) organized a workshop to discuss recent advances in molecular and physiological studies of various forms of cardiomyopathies. The study of cardiomyopathies has intensified after several new study setups became available, such as induced pluripotent stem cells, three-dimensional printing of cells, use of scaffolds and engineered heart tissue, with convincing human validation studies. Furthermore, our knowledge on the consequences of mutated proteins has deepened, with relevance for cellular homeostasis, protein quality control and toxicity, often specific to particular cardiomyopathies, with precise effects explaining the aberrations. This has opened up new avenues to treat cardiomyopathies, using contemporary techniques from the molecular toolbox, such as gene editing and repair using CRISPR-Cas9 techniques, antisense therapies, novel designer drugs, and RNA therapies. In this article, we discuss the connection between biology and diverse clinical presentation, as well as promising new medications and therapeutic avenues, which may be instrumental to come to precision medicine of genetic cardiomyopathies.

Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.

Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell­derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9­generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis.

Nitro-oleic acid (NO2-OA), a nitric oxide (NO)- and nitrite (NO2-)-derived electrophilic fatty acid metabolite, displays anti-inflammatory and anti-fibrotic signaling actions and therapeutic benefit in murine models of ischemia-reperfusion, atrial fibrillation, and pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp-/-) develop dilated cardiomyopathy (DCM), characterized by impaired left ventricular function and increased ventricular fibrosis at the age of 8 weeks. This study investigated the effects of NO2-OA on cardiac function in Mlp-/- mice both in vivo and in vitro. Mlp-/- mice were treated with NO2-OA or vehicle for 4 weeks via subcutaneous osmotic minipumps. Wildtype (WT) littermates treated with vehicle served as controls. Mlp-/- mice exhibited enhanced TGFß signalling, fibrosis and severely reduced left ventricular systolic function. NO2-OA treatment attenuated interstitial myocardial fibrosis and substantially improved left ventricular systolic function in Mlp-/- mice. In vitro studies of TGFß-stimulated primary cardiac fibroblasts further revealed that the anti-fibrotic effects of NO2-OA rely on its capability to attenuate fibroblast to myofibroblast transdifferentiation by inhibiting phosphorylation of TGFß downstream targets. In conclusion, we demonstrate a substantial therapeutic benefit of NO2-OA in a murine model of DCM, mediated by interfering with endogenously activated TGFß signaling.

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy.

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.

Titin M-line insertion sequence 7 is required for proper cardiac function in mice.

Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation-contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation-contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.

Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere.

Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168-M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross-talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1-mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down-regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.

The long noncoding RNA TUNAR modulates Wnt signaling and regulates human ß-cell proliferation.

Many long noncoding RNAs (lncRNAs) are enriched in pancreatic islets and several lncRNAs are linked to type 2 diabetes (T2D). Although they have emerged as potential players in ß-cell biology and T2D, little is known about their functions and mechanisms in human ß-cells. We identified an islet-enriched lncRNA, TUNAR (TCL1 upstream neural differentiation-associated RNA), which was upregulated in ß-cells of patients with T2D and promoted human ß-cell proliferation via fine-tuning of the Wnt pathway. TUNAR was upregulated following Wnt agonism by a glycogen synthase kinase-3 (GSK3) inhibitor in human ß-cells. Reciprocally, TUNAR repressed a Wnt antagonist Dickkopf-related protein 3 (DKK3) and stimulated Wnt pathway signaling. DKK3 was aberrantly expressed in ß-cells of patients with T2D and displayed a synchronized regulatory pattern with TUNAR at the single cell level. Mechanistically, DKK3 expression was suppressed by the repressive histone modifier enhancer of zeste homolog 2 (EZH2). TUNAR interacted with EZH2 in ß-cells and facilitated EZH2-mediated suppression of DKK3. These findings reveal a novel cell-specific epigenetic mechanism via islet-enriched lncRNA that fine-tunes the Wnt pathway and subsequently human ß-cell proliferation.NEW & NOTEWORTHY The discovery that long noncoding RNA TUNAR regulates ß-cell proliferation may be important in designing new treatments for diabetes.

The Degree of Cardiac Remodelling before Overload Relief Triggers Different Transcriptome and miRome Signatures during Reverse Remodelling (RR)-Molecular Signature Differ with the Extent of RR.

This study aims to provide new insights into transcriptome and miRome modifications occurring in cardiac reverse remodelling (RR) upon left ventricle pressure-overload relief in mice. Pressure-overload was established in seven-week-old C57BL/6J-mice by ascending aortic constriction. A debanding (DEB) surgery was performed seven weeks later in half of the banding group (BA). Two weeks later, cardiac function was evaluated through hemodynamics and echocardiography, and the hearts were collected for histology and small/bulk-RNA-sequencing. Pressure-overload relief was confirmed by the normalization of left-ventricle-end-systolic-pressure. DEB animals were separated into two subgroups according to the extent of cardiac remodelling at seven weeks and RR: DEB1 showed an incomplete RR phenotype confirmed by diastolic dysfunction persistence (E/e' ≥ 16 ms) and increased myocardial fibrosis. At the same time, DEB2 exhibited normal diastolic function and fibrosis, presenting a phenotype closer to myocardial recovery. Nevertheless, both subgroups showed the persistence of cardiomyocytes hypertrophy. Notably, the DEB1 subgroup presented a more severe diastolic dysfunction at the moment of debanding than the DEB2, suggesting a different degree of cardiac remodelling. Transcriptomic and miRomic data, as well as their integrated analysis, revealed significant downregulation in metabolic and hypertrophic related pathways in DEB1 when compared to DEB2 group, including fatty acid ß-oxidation, mitochondria L-carnitine shuttle, and nuclear factor of activated T-cells pathways. Moreover, extracellular matrix remodelling, glycan metabolism and inflammation-related pathways were up-regulated in DEB1. The presence of a more severe diastolic dysfunction at the moment of pressure overload-relief on top of cardiac hypertrophy was associated with an incomplete RR. Our transcriptomic approach suggests that a cardiac inflammation, fibrosis, and metabolic-related gene expression dysregulation underlies diastolic dysfunction persistence after pressure-overload relief, despite left ventricular mass regression, as echocardiographically confirmed.

Distinct Myocardial Transcriptomic Profiles of Cardiomyopathies Stratified by the Mutant Genes.

Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely LMNA (lamin A/C), RBM20 (RNA binding motif protein 20), TTN (titin) and PKP2 (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in RBM20 and TTN as the splice factor RBM20 targets amongst other genes TTN, leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in LMNA is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in PKP2 lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.

Epigenetics and Heart Failure.

Epigenetics refers to changes in phenotypes without changes in genotypes. These changes take place in a number of ways, including via genomic DNA methylation, DNA interacting proteins, and microRNAs. The epigenome is the second dimension of the genome and it contains key information that is specific to every type of cell. Epigenetics is essential for many fundamental processes in biology, but its importance in the development and progression of heart failure, which is one of the major causes of morbidity and mortality worldwide, remains unclear. Our understanding of the underlying molecular mechanisms is incomplete. While epigenetics is one of the most innovative research areas in modern biology and medicine, compounds that directly target the epigenome, such as epidrugs, have not been well translated into therapies. This paper focuses on epigenetics in terms of genomic DNA methylation, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) modifications. These appear to be more dynamic than previously anticipated and may underlie a wide variety of conditions, including heart failure. We also outline possible new strategies for the development of novel therapies.

An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level.

Different types of cardiac hypertrophy have been associated with an increased volume of cardiac myocytes (CMs), along with changes in CM morphology. While the effects of cell volume on gene expression are well known, the effects of cell shape are not well understood. This paper describes a method that has been designed to systematically analyze the effects of CM morphology on gene expression. It details the development of a novel single-cell trapping strategy that is then followed by single-cell mRNA sequencing. A micropatterned chip has also been designed, which contains 3000 rectangular-shaped fibronectin micropatterns. This makes it possible to grow CMs in distinct length:width aspect ratios (AR), corresponding to different types of heart failure (HF). The paper also describes a protocol that has been designed to pick up single cells from their pattern, using a semi-automated micro-pipetting cell picker, and individually inject them into a separate lysis buffer. This has made it possible to profile the transcriptomes of single CMs with defined geometrical morphotypes and characterize them according to a range of normal or pathological conditions: hypertrophic cardiomyopathy (HCM) or afterload/concentric versus dilated cardiomyopathy (DCM) or preload/eccentric. In summary, this paper presents methods for growing CMs with different shapes, which represent different pathologies, and sorting these adherent CMs based on their morphology at a single-cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of HF.

Cell shape: effects on gene expression and signaling.

The perception of biophysical forces (mechanosensation) and their conversion into chemical signals (mechanotransduction) are fundamental biological processes. They are connected to hypertrophic and atrophic cellular responses, and defects in these processes have been linked to various diseases, especially in the cardiovascular system. Although cardiomyocytes generate, and are exposed to, considerable hemodynamic forces that affect their shapes, until recently, we did not know whether cell shape affects gene expression. However, new single-cell trapping strategies, followed by single-cell RNA sequencing, to profile the transcriptomes of individual cardiomyocytes of defined geometrical morphotypes have been developed that are characteristic for either normal or pathological (afterload or preload) conditions. This paper reviews the recent literature with regard to cell shape and the transcriptome and provides an overview of this newly emerging field, which has far-reaching implications for both biology, disease, and possibly therapy.

Control of p21Cip by BRCA1-associated protein is critical for cardiomyocyte cell cycle progression and survival.

AIMS: Identifying the key components in cardiomyocyte cell cycle regulation is of relevance for the understanding of cardiac development and adaptive and maladaptive processes in the adult myocardium. BRCA1-associated protein (BRAP) has been suggested as a cytoplasmic retention factor for several proteins including Cyclin-dependent-kinase inhibitor p21Cip. We observed profound expressional changes of BRAP in early postnatal myocardium and investigated the impact of BRAP on cardiomyocyte cell cycle regulation. METHODS AND RESULTS: General knockout of Brap in mice evoked embryonic lethality associated with reduced myocardial wall thickness and lethal cardiac congestion suggesting a prominent role for BRAP in cardiomyocyte proliferation. αMHC-Cre driven cardiomyocyte-specific knockout of Brap also evoked lethal cardiac failure shortly after birth. Likewise, conditional cardiomyocyte-specific Brap deletion using tamoxifen-induced knockout in adult mice resulted in marked ventricular dilatation and heart failure 3 weeks after induction. Several lines of evidence suggest that Brap deletion evoked marked inhibition of DNA synthesis and cell cycle progression. In cardiomyocytes with proliferative capacity, this causes developmental arrest, whereas in adult hearts loss of BRAP-induced apoptosis. This is explained by altered signalling through p21Cip which we identify as the link between BRAP and cell cycle/apoptosis. BRAP deletion enhanced p21Cip expression, while BRAP overexpression in cardiomyocyte-specific transgenic mice impeded p21Cip expression. That was paralleled by enhanced nuclear Ki-67 expression and DNA synthesis. CONCLUSION: By controlling p21Cip activity BRAP expression controls cell cycle activity and prevents developmental arrest in developing cardiomyocytes and apoptosis in adult cardiomyocytes.

Myopalladin promotes muscle growth through modulation of the serum response factor pathway.

BACKGROUND: Myopalladin (MYPN) is a striated muscle-specific, immunoglobulin-containing protein located in the Z-line and I-band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss-of-function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. METHODS: Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. RESULTS: MKO mice were 13% smaller compared with wild-type controls and exhibited a 48% reduction in myofibre cross-sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild-type mice, MKO mice showed progressively decreased exercise capability, Z-line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z-line widening starting from 8 months of age. RNA-sequencing analysis revealed down-regulation of serum response factor (SRF)-target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF-cofactor myocardin-related transcription factor A (MRTF-A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF-A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF-A-mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. CONCLUSIONS: Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z-line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.

Cell shape determines gene expression: cardiomyocyte morphotypic transcriptomes.

Cardiomyocytes undergo considerable changes in cell shape. These can be due to hemodynamic constraints, including changes in preload and afterload conditions, or to mutations in genes important for cardiac function. These changes instigate significant changes in cellular architecture and lead to the addition of sarcomeres, at the same time or at a later stage. However, it is currently unknown whether changes in cell shape on their own affect gene expression and the aim of this study was to fill that gap in our knowledge. We developed a single-cell morphotyping strategy, followed by single-cell RNA sequencing, to determine the effects of altered cell shape in gene expression. This enabled us to profile the transcriptomes of individual cardiomyocytes of defined geometrical morphotypes and characterize them as either normal or pathological conditions. We observed that deviations from normal cell shapes were associated with significant downregulation of gene expression and deactivation of specific pathways, like oxidative phosphorylation, protein kinase A, and cardiac beta-adrenergic signaling pathways. In addition, we observed that genes involved in apoptosis of cardiomyocytes and necrosis were upregulated in square-like pathological shapes. Mechano-sensory pathways, including integrin and Src kinase mediated signaling, appear to be involved in the regulation of shape-dependent gene expression. Our study demonstrates that cell shape per se affects the regulation of the transcriptome in cardiac myocytes, an effect with possible implications for cardiovascular disease.

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.