The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Spatial multi-omic map of human myocardial infarction.

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.

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity.

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.

Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy.

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.

Characteristics and time course of acute and chronic myocardial lesion formation after electroporation ablation in the porcine model.

INTRODUCTION: Electroporation ablation creates deep and wide myocardial lesions. No data are available on time course and characteristics of acute lesion formation. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

P62-positive aggregates are homogenously distributed in the myocardium and associated with the type of mutation in genetic cardiomyopathy.

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 < .0001). p62 accumulation was highest in the desmin (15.6%) and phospholamban (7.2%) groups. P62 accumulation was homogeneously distributed in the myocardium. Fibrosis was not associated with p62 accumulation in subgroup analysis of phospholamban hearts. In conclusion, accumulation of p62-positive protein aggregates is homogeneously distributed in the myocardium independently of fibrosis distribution and associated with desmin and phospholamban cardiomyopathy. Proteotoxic protein accumulation is a diffuse process in the myocardium while a more localized second hit, such as local strain during exercise, might determine whether this leads to regional myocyte decay.

False-negative programmed death-ligand 1 immunostaining in ethanol-fixed endobronchial ultrasound-guided transbronchial needle aspiration specimens of non-small-cell lung cancer patients.

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.

Histopathological characterization of intimal lesions and arterial wall calcification in the arteries of the leg of elderly cadavers.

INTRODUCTION: Although arteries of the leg have been studied in extensively diseased amputation specimens, little is known about the composition of vascular lesions present in the general population. The aim of this study was to describe the natural development of adaptive intimal thickening, atherosclerotic lesion development and vascular calcification in the leg of a general elderly population. MATERIALS AND METHODS: Two hundred and seventy postmortem samples from the popliteal and posterior tibial arteries of 14 elderly cadavers were studied histologically. RESULTS: Atherosclerotic lesions were more frequently observed in the popliteal (60%) than in the posterior tibial artery (34%; p < .0005). These atherosclerotic plaques were most often nonatheromatous (80% and 83% for popliteal and posterior tibial plaques, respectively). The atheroma's that were present were small (most <25% of plaque area). Atherosclerotic plaque calcification was observed more often in the popliteal (39%) than in the posterior tibial samples (17%; p < .0005). Medial arterial calcification was observed more often in the posterior tibial (62%) than in the popliteal samples (46%; p = .008). Plaque calcification and medial arterial calcification were not associated with lumen stenosis. CONCLUSIONS: In the leg of elderly cadavers, the presence of atherosclerotic plaque and intimal calcification decreases from the proximal popliteal artery to the more distal posterior tibial artery and most atherosclerotic lesions are of the fibrous nonatheromatous type. In contrast, the presence and severity of medial calcification increases from proximal to distal.

Ventricular TLR4 Levels Abrogate TLR2-Mediated Adverse Cardiac Remodeling upon Pressure Overload in Mice.

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 < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1ß, p < 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 < 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 ameliorates 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.

Conserved NPPB+ Border Zone Switches From MEF2- to AP-1-Driven Gene Program.

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.

Flotillins in the intercalated disc are potential modulators of cardiac excitability.

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.

Increased circulating IgG levels, myocardial immune cells and IgG deposits support a role for an immune response in pre- and end-stage heart failure.

The chronic inflammatory response plays an important role in adverse cardiac remodelling and the development of heart failure (HF). There is also evidence that in the pathogenesis of several cardiovascular diseases, chronic inflammation is accompanied by antibody and complement deposits in the heart, suggestive of a true autoimmune response. However, the role of antibody-mediated immune responses in HF progression is less clear. We assessed whether immune cell infiltration and immunoglobulin levels are associated with HF type and disease stage, taking sex differences into account. We found IgG deposits and increased infiltration of immune cells in the affected myocardium of patients with end-stage HF with reduced ejection fraction (HFrEF, n = 20). Circulating levels of IgG1 and IgG3 were elevated in these patients. Furthermore, the percentage of transitional/regulatory B cells was decreased (from 6.9% to 2.4%) compared with healthy controls (n = 5). Similarly, increased levels of circulating IgG1 and IgG3 were observed in men with left ventricular diastolic dysfunction (LVDD, n = 5), possibly an early stage of HF with preserved EF (HFpEF). In conclusion, IgG deposits and infiltrates of immune cells are present in end-stage HFrEF. In addition, both LVDD patients and end-stage HFrEF patients show elevated levels of circulating IgG1 and IgG3, suggesting an antibody-mediated immune response upon cardiac remodelling, which in the early phase of remodelling appear to differ between men and women. These immunoglobulin subclasses might be used as marker for pre-stage HF and its progression. Future identification of auto-antigens might open possibilities for new therapeutic interventions.

Myofilament Remodeling and Function Is More Impaired in Peripartum Cardiomyopathy Compared with Dilated Cardiomyopathy and Ischemic Heart Disease.

Peripartum cardiomyopathy (PPCM) and dilated cardiomyopathy (DCM) show similarities in clinical presentation. However, although DCM patients do not recover and slowly deteriorate further, PPCM patients show either a fast cardiac deterioration or complete recovery. The aim of this study was to assess if underlying cellular changes can explain the clinical similarities and differences in the two diseases. We, therefore, assessed sarcomeric protein expression, modification, titin isoform shift, and contractile behavior of cardiomyocytes in heart tissue of PPCM and DCM patients and compared these with nonfailing controls. Heart samples from ischemic heart disease (ISHD) patients served as heart failure control samples. Passive force was only increased in PPCM samples compared with controls, whereas PPCM, DCM, and ISHD samples all showed increased myofilament Ca2+ sensitivity. Length-dependent activation was significantly impaired in PPCM compared with controls, no impairment was observed in ISHD samples, and DCM samples showed an intermediate response. Contractile impairments were caused by impaired protein kinase A (PKA)-mediated phosphorylation because exogenous PKA restored all parameters to control levels. Although DCM samples showed reexpression of EH-myomesin, an isoform usually only expressed in the heart before birth, PPCM and ISHD did not. The lack of EH-myomesin, combined with low PKA-mediated phosphorylation of myofilament proteins and increased compliant titin isoform, may explain the increase in passive force and blunted length-dependent activation of myofilaments in PPCM samples.

A Novel Cardiovascular Prosthesis Made from Woven Ultrahigh-Molecular-Weight Polyethylene Fibers, Proof of Concept in a Sheep Model.

BACKGROUND: A patch made of woven ultrahigh-molecular-weight polyethylene (UHMWPE) fibers is thin, strong, and flexible and may be attractive for use in cardiovascular prostheses. This study assessed the hemocompatibility of this patch in a sheep model. METHODS: The UHMWPE patches were sutured in the right carotid artery and jugular vein in 12 adult sheep, and in the same animal, expanded polytetrafluoroethylene (ePTFE) patches were sutured as control patches in the left carotid artery and jugular vein. Follow-up (FU) was 4 hrs, 1 week, and 6 weeks (n = 4 for all time points). Patency of the vessels was assessed with qualitative observational short-axis echography, and thrombus formation and tissue deposition were assessed with histology. RESULTS: All vessels were fully patent at the end of the study. Thrombus formation was comparable for the UHMWPE and the ePTFE patches. Tissue deposition was not significantly different on the UHMWPE patches, except for patches in the jugular vein at 1 week of FU where it was significantly thicker. CONCLUSIONS: These results suggest the noninferiority of the UHMWPE patch compared with the clinically used ePTFE patch; therefore, this novel cardiovascular prosthesis might be attractive for use as a cardiovascular patch.

Genotype-specific pathogenic effects in human dilated cardiomyopathy.

KEY POINTS: Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy. TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+ -sensitivity and reduced length-dependent activation. TNNT2p.K217del caused increased passive tension. A mutation in the gene encoding Lamin A/C (LMNAp.R331Q ) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy. Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. ABSTRACT: Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non-sarcomeric genes. In this study we defined the pathogenic effects of three DCM-causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation ) and cardiac troponin T (TNNT2p.K217deletion ; also known as the p.K210del) and the non-sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q ). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane-permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+ -sensitivity and impaired length-dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild-type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.

A potential role for glycated cross-links in abdominal aortic aneurysm disease.

BACKGROUND: Diabetes is a risk factor for atherosclerotic disease but negatively associated with the development and progression of abdominal aortic aneurysm (AAA). Advanced glycation end products (AGEs) are increased in diabetes and renders the vascular matrix more resistant to proteolysis. We assessed the concentration of AGEs in AAA biopsies obtained from diabetic and nondiabetic patients and hypothesized that (nonenzymatic) glycation of AAA tissue protects against proteolytic breakdown of collagen. METHODS: AAA biopsies were collected from 30 diabetic and 30 matched nondiabetic AAA patients at the time of open repair. Aortic control samples from 10 nondiabetic and 16 diabetic patients were collected, and concentrations of the AGE cross-link pentosidine was measured. Furthermore, noncross-linking AGEs (adducts), as well as proteolytic enzymes known to play a role in aneurysm development (matrix metalloproteinase [MMP]-2, MMP-9, cathepsin B and S) were quantified. Ex vivo, nondiabetic AAA biopsies were glycated and measured subsequently for collagen type I release. RESULTS: Pentosidine concentrations in AAA wall biopsies were increased in patients with diabetes compared with nondiabetics 9.4 (5.0-13.5) vs 6.0 (2.5-9.6) pmol/µmol lysine (P = .02). Increased pentosidine concentrations were also observed in nonaneurysmatic aortic wall biopsies from diabetic patients. In diabetic AAA vascular wall tissue, pentosidine concentration was negatively correlated with aortic diameter (r = -0.43; P = .02). Ex vivo glycated AAA biopsies were resistant against MMP-induced collagen type I degradation as compared with controls (7.0 vs 10.4 µg/L; P = .02). No differences were observed for AGEs that are not forming cross-links. CONCLUSIONS: These findings suggest that cross-linking AGEs like pentosidine play a protective role in AAA progression in diabetic patients.

Proof-of-Concept Evaluation of the SailValve Self-Expanding Deep Venous Valve System in a Porcine Model.

PURPOSE: To evaluate the SailValve, a new self-expanding deep venous valve concept based on a single polytetrafluoroethylene cusp floating up and down in the bloodstream like a sail, acting as a flow regulator and allowing minimal reflux to reduce thrombogenicity. METHODS: Both iliac veins of 5 pigs were implanted with SailValve devices; the first animal was an acute pilot experiment to show the feasibility of accurately positioning the SailValve via a femoral access. The other 4 animals were followed for 2 weeks (n=2) or 4 weeks (n=2) under a chronic implantation protocol. Patency and valve function were evaluated directly in all animals using ascending and descending phlebography after device placement and at termination in the chronic implant animals. For reasons of clinical relevance, a regimen of clopidogrel and calcium carbasalate was administered. Histological analysis was performed according to a predefined protocol by an independent pathologist. RESULTS: Deployment was technically feasible in all 10 iliac veins, and all were patent directly after placement. No perioperative or postoperative complications occurred. Ascending phlebograms in the follow-up animals confirmed the patency of all valves after 2 or 4 weeks. Descending phlebograms showed full function in 5 of 8 valves. Limited reflux was seen in 1 valve (4-week group), and the function in the remaining 2 valves (2-week group) was insufficient because of malpositioning. No macroscopic thrombosis was noted on histology. Histology in the follow-up groups revealed a progressive inflammatory reaction to the valves. CONCLUSION: This animal study shows the potential of the SailValve concept with sufficient valve function after adequate positioning and no (thrombogenic) occlusions after short-term follow-up. Future research is essential to optimize valve material and long-term patency.

Predominance of Nonatherosclerotic Internal Elastic Lamina Calcification in the Intracranial Internal Carotid Artery.

BACKGROUND AND PURPOSE: Calcification of the intracranial internal carotid artery (iICA) is an independent risk factor for stroke. These calcifications are generally seen as manifestation of atherosclerosis, but histological investigations are limited. The aim of this study is to determine whether calcifications in the iICA are present in atherosclerotic plaques, or in other parts of the arterial wall. METHODS: Thirty-nine iICAs were histologically assessed, using digital microscopy to quantify the amount of calcification in the different layers of the arterial wall. RESULTS: Calcifications were found in the intima, around the internal elastic lamina and in the medial layer of the arterial wall. In 71% of the arteries, internal elastic lamina calcification contributed most to the total calcified cross-sectional surface area. Internal elastic lamina calcification was unrelated to the occurrence of atherosclerotic intimal lesions. Intimal calcifications were most often associated with atherosclerotic lesions, but also many noncalcified atherosclerotic lesions were found. CONCLUSIONS: In the iICA, calcifications are predominantly present around the internal elastic lamina, suggesting that this nonatherosclerotic type of calcification contributes to the previously observed increased risk of stroke in patients with iICA calcifications.