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Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat–containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.

Background: Doxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence.Objectives: The aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target.Methods: Biopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell–derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol.Results: Senescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function.Conclusions: Senescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 ( PKP2 ). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell–derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.

Aims Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that is characterized by progressive loss of myocardium that is replaced by fibro-fatty cells, arrhythmias, and sudden cardiac death. While myocardial degeneration and fibro-fatty replacement occur in specific locations, the underlying molecular changes remain poorly characterized. Here, we aim to delineate local changes in gene expression to identify new genes and pathways that are relevant for specific remodelling processes occurring during ACM. Methods and results Using Tomo-Seq, genome-wide transcriptional profiling with high spatial resolution, we created transmural epicardial-to-endocardial gene expression atlases of explanted ACM hearts to gain molecular insights into disease-driving processes. This enabled us to link gene expression profiles to the different regional remodelling responses and allowed us to identify genes that are potentially relevant for disease progression. In doing so, we identified distinct gene expression profiles marking regions of cardiomyocyte degeneration and fibro-fatty remodelling and revealed Zinc finger and BTB domain-containing protein 11 (ZBTB11) to be specifically enriched at sites of active fibro-fatty replacement of myocardium. Immunohistochemistry indicated ZBTB11 to be induced in cardiomyocytes flanking fibro-fatty areas, which could be confirmed in multiple cardiomyopathy patients. Forced overexpression of ZBTB11 induced autophagy and cell death-related gene programmes in human cardiomyocytes, leading to increased apoptosis. Conclusion Our study shows the power of Tomo-Seq to unveil new molecular mechanisms in human cardiomyopathy and uncovers ZBTB11 as a novel driver of cardiomyocyte loss.

Cardiac allograft vasculopathy (CAV) and antibody-mediated rejection are immune-mediated, long-term complications that jeopardize graft survival after heart transplantation (HTx). Interestingly, increased plasma levels of immunoglobulins have been found in end-stage heart failure (HF) patients prior to HTx. In this study, we aimed to determine whether increased circulating immunoglobulin levels prior to transplantation are associated with poor post-HTx survival. Pre-and post-HTx plasma samples of 36 cardiac transplant recipient patients were used to determine circulating immunoglobulin levels. In addition, epicardial tissue was collected to determine immunoglobulin deposition in cardiac tissue and assess signs and severity of graft rejection. High levels of IgG1 and IgG2 prior to HTx were associated with a shorter survival post-HTx. Immunoglobulin deposition in cardiac tissue was significantly elevated in patients with a survival of less than 3 years. Patients with high plasma IgG levels pre-HTx also had significantly higher plasma levels after HTx. Furthermore, high pre-HTX levels of IgG1 and IgG2 levels were also significantly increased in patients with inflammatory infiltrate in CAV lesions. Altogether the results of this proof-of-concept study suggest that an activated immune response prior to transplantation negatively affects graft survival.

Carriers of the R14del pathogenic variant in the phospholamban (PLN) gene develop severe cardiomyopathy with extracellular adipocyte infiltration and intracellular cardiomyocyte mitochondrial disturbances. However, the basis of this metabolic dysregulation tailoring potential treatment targets is unknown. Here, we present a combined approach of transcriptional regulation analysis in human primary tissue and validation in a unique long-term (160 days) matured human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We demonstrate a dysregulated PPARA-mediated mitochondrial fatty acid oxidation (FAO) signalling in PLN-R14del hearts and hiPSC-CMs. PLN-R14del hiPSC-CMs also displayed a higher preference for glycolysis over FAO and presented limited flexibility in energy substrate switching leading to enhanced lipid droplet storage. By activating PPARA in PLN-R14del hiPSC-CMs using bezafibrate, we observed an improved mitochondrial structure and calcium handling function, further indicating the importance of FAO in the disease and the potential of PPARA agonists as a novel therapeutic strategy in cardiomyopathies.

Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

AIMS Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndromes and in rare cases sudden cardiac death (SCD). Connective tissue abnormalities, coronary inflammation, increased coronary vasa vasorum density and coronary fibromuscular dysplasia have all been implicated in the pathophysiology of SCAD but have not previously been systematically assessed. We designed a study to investigate the coronary histological and dermal collagen ultrastructural findings in SCAD. METHODS AND RESULTS 36 autopsy SCAD cases were compared with 359 SCAD survivors. Coronary and myocardial histology and immunohistochemistry were undertaken. Transmission electron microscopy (TEM) of dermal extracellular matrix components (ECM) of n = 31 SCAD survivors and n = 16 healthy volunteers were compared. Autopsy cases were more likely male (19% versus 5%; p = 0.0004) with greater proximal left coronary involvement (56% versus 18%; p

We present the first case in the literature of a patient with a histology-proven intimal sarcoma of the heart, recurrent after surgery, treated with stereotactic MR-guided online adaptive radiation therapy on an MR-Linac machine. The treatment was feasible and well tolerated. The CT scan 6 months after the last treatment showed stable disease.

Since cardiac hypertrophy may be considered a cause of death at autopsy, its assessment requires a uniform approach. Common terminology and methodology to measure the heart weight, size, and thickness as well as a systematic use of cut off values for normality by age, gender, and body weight and height are needed. For these reasons, recommendations have been written on behalf of the Association for European Cardiovascular Pathology. The diagnostic work up implies the search for pressure and volume overload conditions, compensatory hypertrophy, storage and infiltrative disorders, and cardiomyopathies. Although some gross morphologic features can point to a specific diagnosis, systematic histologic analysis, followed by possible immunostaining and transmission electron microscopy, is essential for a final diagnosis. If the autopsy is carried out in a general or forensic pathology service without expertise in cardiovascular pathology, the entire heart (or pictures) together with mapped histologic slides should be sent for a second opinion to a pathologist with such an expertise. Indication for postmortem genetic testing should be integrated into the multidisciplinary management of sudden cardiac death. Supplementary Information The online version contains supplementary material available at 10.1007/s00428-021-03038-0.

Genetic cardiomyopathy is caused by mutations in various genes. The accumulation of potentially proteotoxic mutant protein aggregates due to insufficient autophagy is a possible mechanism of disease development. The objective of this study was to investigate the distribution in the myocardium of such aggregates in relation to specific pathogenic genetic mutations in cardiomyopathy hearts. Hearts from 32 genetic cardiomyopathy patients, 4 non‐genetic cardiomyopathy patients and 5 controls were studied. Microscopic slices from an entire midventricular heart slice were stained for p62 (sequestosome‐1, marker for aggregated proteins destined for autophagy). The percentage of cardiomyocytes with p62 accumulation was higher in cardiomyopathy hearts (median 3.3%) than in healthy controls (0.3%; P

Background - Variants within the alpha-tropomyosin gene (TPM1) cause dominantly inherited cardiomyopathies, including dilated (DCM), hypertrophic (HCM) and restrictive (RCM) cardiomyopathy. Here we investigated whether TPM1 variants observed in DCM and HCM patients affect cardiomyocyte physiology differently.Methods - We identified a large family with DCM carrying a recently identified TPM1 gene variant (T201M) and a child with RCM with compound heterozygote TPM1 variants (E62Q and M281T) whose family members carrying single variants show diastolic dysfunction and HCM. The effects of TPM1 variants (T201M, E62Q or M281T) and of a plasmid containing both the E62Q and M281T variants on single-cell Ca2+ transients (CaT) in HL-1 cardiomyocytes were studied. To define toxic threshold levels, we performed dose-dependent transfection of TPM1 variants. In addition, cardiomyocyte structure was studied in human cardiac biopsies with TPM1 variants.Results - Overexpression of TPM1 variants led to time-dependent progressive deterioration of CaT, with the smallest effect seen for E62Q and larger and similar effects seen for the T201M and M281T variants. Overexpression of E62Q/M281T did not exacerbate the effects seen with overexpression of a single TPM1 variant. T201M (DCM) replaced endogenous tropomyosin dose-dependently, while M281T (HCM) did not. Human cardiac biopsies with TPM1 variants revealed loss of sarcomeric structures.Conclusion - All TPM1 variants result in reduced cardiomyocyte CaT amplitudes and loss of sarcomeric structures. These effects may underlie pathophysiology of different cardiomyopathy phenotypes. (C) 2020 The Authors. Published by Elsevier B.V.

Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.

Calcifications are common in the tunica intima and tunica media of leg arteries. There is growing interest in medial arterial calcifications, as they may be modifiable with treatment. We aimed to investigate radiography and computed tomography (CT) for the detection and characterization of both types of arterial calcification in leg arteries in relation to histology. In a postmortem study we therefore investigated 24 popliteal and 24 tibial arteries. The reference standard was presence of arterial calcification and the dominance of intimal or medial calcification on histology. Radiographs and CT scans were scored for presence of calcification and for dominant intimal or medial pattern based on prespecified criteria (annularity, thickness, continuity). Both radiography and CT detected 87% of histologically proven calcifications but missed mild calcifications in 13%. When only the arteries with detected calcifications were included, a moderate agreement was observed on intimal/medial location of calcifications between histology and radiography (correct in 19/24 arteries (79%); Kappa 0.58) or CT (correct in 33/46 arterial segments (72%); Kappa 0.48). With both modalities there was a slight tendency to classify intimal calcifications as being located in the media and to miss media calcification. Our study demonstrates the potential and limitations of both radiography and CT to detect and classify arterial calcifications in leg arteries.

The 2020 COVID-19 crisis has had and will have many implications for healthcare, including pathology. Rising number of infections create staffing shortages and other hospital departments might require pathology employees to fill more urgent positions. Furthermore, lockdown measures and social distancing cause many people to work from home. During this crisis, it became clearer than ever what an asset digital diagnostics is to keep pathologists, residents, molecular biologists and pathology assistants engaged in the diagnostic process, allowing social distancing and a ‘need to be there’ on-the-premises policy, while working effectively from home. This paper provides an overview of our way of working during the 2020 COVID-19 crisis with emphasis on the virtues of digital pathology.

Aims Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. Methods and results The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. Conclusion Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

This editorial refers to ‘An automated computational image analysis pipeline for histological grading of cardiac allograft rejection’†, by E.G. Peyster et al., on page 2356.

Electroporation ablation creates deep and wide myocardial lesions. No data are available on time course and characteristics of acute lesion formation.For the acute phase of myocardial lesion development, seven pigs were investigated. Single 200 J applications were delivered at four different epicardial right ventricular sites using a linear suction device, yielding a total of 28 lesions. Timing of applications was designed to yield lesions at seven time points: 0, 10, 20, 30, 40, 50, and 60 min, with four lesions per time point. After killing, lesion characteristics were histologically investigated. For the chronic phase of myocardial lesion development, tissue samples were used from previously conducted studies where tissue was obtained at 3 weeks and 3 months after electroporation ablation.Acute myocardial lesions induce a necrosis pattern with contraction band necrosis and interstitial edema, immediately present after electroporation ablation. No further histological changes such as hemorrhage or influx of inflammatory cells occurred in the first hour. After 3 weeks, the lesions consisted of sharply demarcated loose connective tissue that further developed to more fibrotic scar tissue after 3 months without additional changes. Within the scar tissue, arteries and nerves were unaffected.Electroporation ablation immediately induces contraction band necrosis and edema without additional tissue changes in the first hour. After 3 weeks, a sharply demarked scar has been developed that remains stable during follow-up of 3 months. This is highly relevant for clinical application of electroporation ablation in terms of the electrophysiological endpoint and waiting period after ablation.

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p &lt, 0.05), increased contractility (QRS and QTc, p &lt, 0.05), and less inflammation (e.g., interleukins 6 and 1β, p &lt, 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p &lt, 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency exacerbates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

Background The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. Methods Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. Results First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. Conclusions In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.

Background: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb , encoding B-type natriuretic peptide, after infarction. Methods: Transgenic reporter mice were used to identify the Nppb -positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. Results: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB , ANKRD1 , DES , UCHL1, JUN , and FOXP1 . Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. Conclusions: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1–driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

AIMS Programmed death-ligand 1 (PD-L1) immunostaining is used to predict which non-small-cell lung cancer (NSCLC) patients will respond best to treatment with programmed cell death protein 1/PD-L1 inhibitors. PD-L1 immunostaining is sometimes performed on alcohol-fixed cytological specimens instead of on formalin-fixed paraffin-embedded (FFPE) biopsies or resections. We studied whether ethanol prefixation of clots from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) results in diminished PD-L1 immunostaining as compared with formalin fixation. METHODS AND RESULTS FFPE cell blocks from EBUS-TBNA specimens of 54 NSCLC patients were identified. For each case, paired samples were available, consisting of clots directly immersed in formalin and clots prefixed in Fixcyt (50% ethanol). Serial sections were immunostained for PD-L1 by use of the standardised SP263 assay and the 22C3 antibody as a laboratory-developed test (LDT). PD-L1 positivity was determined with two cut-offs (1% and 50%). Concordance of PD-L1 positivity between the formalin-fixed (gold standard) and ethanol-prefixed material was assessed. When the 22C3 LDT was used, 30% and 36% of the ethanol-prefixed specimens showed false-negative results at the 1% and 50% cut-offs, respectively (kappa 0.64 and 0.68). When SP263 was used, 22% of the ethanol-prefixed specimens showed false-negative results at the 1% cut-off (kappa 0.67). At the 50% cut-off, concordance was higher (kappa 0.91), with 12% of the ethanol-prefixed specimens showing false-negative results. CONCLUSION Ethanol fixation of EBUS-TBNA specimens prior to formalin fixation can result in a considerable number of false-negative PD-L1 immunostaining results when a 1% cut-off is used and immunostaining is performed with SP263 or the 22C3 LDT. The same applies to use of the 50% cut-off when immunostaining is performed with the 22C3 LDT.

Aims Pathological cardiac remodeling is characterized by cardiomyocyte hypertrophy and fibroblast activation, which can ultimately lead to maladaptive hypertrophy and heart failure (HF). Genome-wide expression analysis on heart tissue has been instrumental for the identification of molecular mechanisms at play. However, these data were based on signals derived from all cardiac cell types. Here we aimed for a more detailed view on molecular changes driving maladaptive cardiomyocyte hypertrophy to aid in the development of therapies to reverse pathological remodeling. Methods and results Utilizing cardiomyocyte-specific reporter mice exposed to pressure overload by transverse aortic banding and cardiomyocyte isolation by flow cytometry, we obtained gene expression profiles of hypertrophic cardiomyocytes in the more immediate phase after stress, and cardiomyocytes showing pathological hypertrophy. We identified subsets of genes differentially regulated and specific for either stage. Among the genes specifically upregulated in the cardiomyocytes during the maladaptive phase we found known stress markers, such as Nppb and Myh7, but additionally identified a set of genes with unknown roles in pathological hypertrophy, including the platelet isoform of phosphofructokinase (PFKP). Norepinephrine-angiotensin II treatment of cultured human cardiomyocytes induced secretion of NT pro-BNP and recapitulated the upregulation of these genes, indicating conservation of the upregulation in failing cardiomyocytes. Moreover, several genes induced during pathological hypertrophy were also found to be increased in human heart failure, with their expression positively correlating to the known stress markers NPPB and MYH7. Mechanistically, suppression of Pfkp in primary cardiomyocytes attenuated stress-induced gene expression and hypertrophy, indicating that Pfkp is an important novel player in pathological remodeling of cardiomyocytes. Conclusions Using cardiomyocyte-specific transcriptomic analysis we identified novel genes induced during pathological hypertrophy that are relevant for human HF, and we show that PFKP is a conserved failure-induced gene that can modulate the cardiomyocyte stress response. Translational perspective Maladaptive cardiac remodeling is a consequence of pathological hypertrophy which includes cardiomyocytes changes and a decline in contractility. Our cardiomyocyte-specific gene expression studies revealed a gene program specific for pathological hypertrophy that is conserved in diseased mouse and human cardiomyocytes. We identified PFKP as a novel gene actively involved in cardiomyocyte remodeling, indicating PFKP as a potential therapeutic target to block the progression of heart failure.

Background Active infection is generally considered a contraindication for heart transplantation. The rare combination of a patient with an active Coxiella burnetii infection and a congenital corrected transposition of the great arteries requiring heart transplantation impose challenging treatment decisions. We would like to demonstrate that if Q fever is restricted to the heart only, heart transplantation is also beneficial from an infectious point of view, therefore treating two severe conditions simultaneously. Case summary A patient with end-stage heart failure due to congenital corrected transposition of the great arteries and requiring heart transplantation developed chronic Q fever and endocarditis. Different antibiotic regimes were tried due to severe adverse reactions. Antibiotic treatment was precisely monitored by measuring Q fever polymerase chain reaction (PCRs) and phase I IgG antibody titres. A positron emission tomography scan revealed that Q fever was confined to the heart only after which it was decided to perform heart transplantation. Based on the results of PCR and antibody testing, antibiotic treatment was stopped after 1 year. After 5 years of follow-up, patient is still in an optimal condition. Discussion In case of a patient with end-stage heart failure and chronic Q fever, a combined treatment with PCR-/antibody monitored antibiotics and heart transplantation can cure both conditions.

BackgroundThe R14del mutation in the phospholamban (PLN) gene is associated with various types of cardiomyopathies and increases the risk of developing life-threatening ventricular arrhythmias. In this study, we focused on a homogeneous Dutch founder cohort of genetic cardiomyopathy due to PLN R14del mutation and aimed to study the influence of epigenetic changes from a multi-dimensional perspective.ResultsUsing cardiac tissue of PLN R14del patients and donors, we identified differentially acetylated promoters and enhancers (H3K27ac ChIPseq), annotated enriched transcription factor (TF) binding motifs located in those regions, and identified differentially expressed genes (RNA-seq). In line with the fibrofatty replacement in PLN R14del hearts at the histological level, our integrative analysis detected the downregulation of key TF regulators in fatty acid oxidation (FAO) metabolisms and their downstream target in PLN R14del hearts as compared to controls. We further examined heart tissue using immunofluorescence staining (IF) and to confirm the mitochondrial lipid abnormalities in the PLN R14del hearts. Furthermore, we observed the accumulation and deformation of lipid droplets and a disrupted morphology of mitochondria, the key organelle where FAO takes place, in PLN R14del heart using transmission electron microscopy (TEM).ConclusionUsing multi-omics approaches, we successfully obtained a unique list of chromatin regions and genes, including TF-coding genes, which played important roles in the metabolism-related signalling in PLN R14del hearts.

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.

For in situ tissue engineering (TE) applications it is important that implant degradation proceeds in concord with neo-tissue formation to avoid graft failure. It will therefore be valuable to have an imaging contrast agent (CA) available that can report on the degrading implant. For this purpose, a biodegradable radiopaque biomaterial is presented, modularly composed of a bisurea chain-extended polycaprolactone (PCL2000-U4U) elastomer and a novel iodinated bisurea-modified CA additive (I-U4U). Supramolecular hydrogen bonding interactions between the components ensure their intimate mixing. Porous implant TE-grafts are prepared by simply electrospinning a solution containing PCL2000-U4U and I-U4U. Rats receive an aortic interposition graft, either composed of only PCL2000-U4U (control) or of PCL2000-U4U and I-U4U (test). The grafts are explanted for analysis at three time points over a 1-month period. Computed tomography imaging of the test group implants prior to explantation shows a decrease in iodide volume and density over time. Explant analysis also indicates scaffold degradation. (Immuno)histochemistry shows comparable cellular contents and a similar neo-tissue formation process for test and control group, demonstrating that the CA does not have apparent adverse effects. A supramolecular approach to create solid radiopaque biomaterials can therefore be used to noninvasively monitor the biodegradation of synthetic implants.

Background H3K27ac histone acetylome changes contribute to the phenotypic response in heart diseases, particularly in end-stage heart failure. However, such epigenetic alterations have not been systematically investigated in remodeled non-failing human hearts. Therefore, valuable insight into cardiac dysfunction in early remodeling is lacking. This study aimed to reveal the acetylation changes of chromatin regions in response to myocardial remodeling and their correlations to transcriptional changes of neighboring genes. Results We detected chromatin regions with differential acetylation activity (DARs; Padj. < 0.05) between remodeled non-failing patient hearts and healthy donor hearts. The acetylation level of the chromatin region correlated with its RNA polymerase II occupancy level and the mRNA expression level of its adjacent gene per sample. Annotated genes from DARs were enriched in disease-related pathways, including fibrosis and cell metabolism regulation. DARs that change in the same direction have a tendency to cluster together, suggesting the well-reorganized chromatin architecture that facilitates the interactions of regulatory domains in response to myocardial remodeling. We further show the differences between the acetylation level and the mRNA expression level of cell-type-specific markers for cardiomyocytes and 11 non-myocyte cell types. Notably, we identified transcriptome factor (TF) binding motifs that were enriched in DARs and defined TFs that were predicted to bind to these motifs. We further showed 64 genes coding for these TFs that were differentially expressed in remodeled myocardium when compared with controls. Conclusions Our study reveals extensive novel insight on myocardial remodeling at the DNA regulatory level. Differences between the acetylation level and the transcriptional level of cell-type-specific markers suggest additional mechanism(s) between acetylome and transcriptome. By integrating these two layers of epigenetic profiles, we further provide promising TF-encoding genes that could serve as master regulators of myocardial remodeling. Combined, our findings highlight the important role of chromatin regulatory signatures in understanding disease etiology.