Myocardial PD-L1 Expression in Patients With Ischemic and Non-ischemic Heart Failure

Objective: Immune checkpoints inhibitors are promising and wide-spread agents in anti-cancer therapy. However, despite their efficacy, these agents could cause cardiotoxicity, a rare but life-threatening event. In addition, there are still no well-described predictive factors for the development of immune-related adverse events and information on high risk groups. According to known experimental studies we hypothesized that cardiovascular diseases may increase myocardial PD-L1 expression, which could be an extra target for Checkpoint inhibitors and a potential basis for complications development.Methods: We studied patterns of myocardial PD-L1 expression in non-cancer-related cardiovascular diseases, particularly ischemic heart disease (n = 12) and dilated cardiomyopathy (n = 7), compared to patients without known cardiovascular diseases (n = 10) using mouse monoclonal anti-PD-L1 antibody (clone 22C3, 1:50, Dako). Correlation between immunohistochemical data and echocardiographic parameters was assessed. Statistical analyses were performed using R Statistical Software—R studio version 1.3.1093.Results: In the myocardium of cardiac patients, we found membranous, cytoplasmic, and endothelial expression of PD-L1 compared to control group. In samples from patients with a history of myocardial infarction, PD-L1 membrane and endothelial expression was more prominent and frequent, and cytoplasmic and intercalated discs staining was more localized. In contrast, samples from patients with dilated cardiomyopathy displayed very faint endothelial staining, negative membrane staining, and more diffuse PD-L1 expression in the cytoplasm and intercalated discs. In samples from the non-cardiac patients, no convincing PD-L1 expression was observed. Moreover, we discovered a significant negative correlation between PD-L1 expression level and left ventricular ejection fraction and a positive correlation between PD-L1 expression level and left ventricular end-diastolic volume.Conclusions: The present findings lay the groundwork for future experimental and clinical studies of the role of the PD-1/PD-L1 pathway in cardiovascular diseases. Further studies are required to find patients at potentially high risk of cardiovascular adverse events associated with immune checkpoint inhibitors therapy.

Accelerated Growth, Differentiation, and Ploidy with Reduced Proliferation of Right Ventricular Cardiomyocytes in Children with Congenital Heart Defect Tetralogy of Fallot

The myocardium of children with tetralogy of Fallot (TF) undergoes hemodynamic overload and hypoxemia immediately after birth. Comparative analysis of changes in the ploidy and morphology of the right ventricular cardiomyocytes in children with TF in the first years of life demonstrated their significant increase compared with the control group. In children with TF, there was a predominantly diffuse distribution of Connexin43-containing gap junctions over the cardiomyocytes sarcolemma, which redistributed into the intercalated discs as cardiomyocytes differentiation increased. The number of Ki67-positive cardiomyocytes varied greatly and amounted to 7.0–1025.5/106 cardiomyocytes and also were decreased with increased myocytes differentiation. Ultrastructural signs of immaturity and proliferative activity of cardiomyocytes in children with TF were demonstrated. The proportion of interstitial tissue did not differ significantly from the control group. The myocardium of children with TF under six months of age was most sensitive to hypoxemia, it was manifested by a delay in the intercalated discs and myofibril assembly and the appearance of ultrastructural signs of dystrophic changes in the cardiomyocytes. Thus, the acceleration of ontogenetic growth and differentiation of the cardiomyocytes, but not the reactivation of their proliferation, was an adaptation of the immature myocardium of children with TF to hemodynamic overload and hypoxemia.

Phosphoproteomic Analysis Reveals Downstream PKA Effectors of AKAP Cypher/ZASP in the Pathogenesis of Dilated Cardiomyopathy

Background: Dilated cardiomyopathy (DCM) is a major cause of heart failure worldwide. The Z-line protein Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is closely associated with DCM, both clinically and in animal models. Our earlier work revealed Cypher/ZASP as a PKA-anchoring protein (AKAP) that tethers PKA to phosphorylate target substrates. However, the downstream PKA effectors regulated by AKAP Cypher/ZASP and their relevance to DCM remain largely unknown.Methods and Results: For the identification of candidate PKA substrates, global quantitative phosphoproteomics was performed on cardiac tissue from wild-type and Cypher-knockout mice with PKA activation. A total of 216 phosphopeptides were differentially expressed in the Cypher-knockout mice; 31 phosphorylation sites were selected as candidates using the PKA consensus motifs. Bioinformatic analysis indicated that differentially expressed proteins were enriched mostly in cell adhesion and mRNA processing. Furthermore, the phosphorylation of β-catenin Ser675 was verified to be facilitated by Cypher. This phosphorylation promoted the transcriptional activity of β-catenin, and also the proliferative capacity of cardiomyocytes. Immunofluorescence staining demonstrated that Cypher colocalised with β-catenin in the intercalated discs (ICD) and altered the cytoplasmic distribution of β-catenin. Moreover, the phosphorylation of two other PKA substrates, vimentin Ser72 and troponin I Ser23/24, was suppressed by Cypher deletion.Conclusions: Cypher/ZASP plays an essential role in β-catenin activation via Ser675 phosphorylation, which modulates cardiomyocyte proliferation. Additionally, Cypher/ZASP regulates other PKA effectors, such as vimentin Ser72 and troponin I Ser23/24. These findings establish the AKAP Cypher/ZASP as a signalling hub in the progression of DCM.

Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Rejuvenating the Aging Heart by Enhancing the Expression of the Cisd2 Prolongevity Gene

Aging is the major risk factor for cardiovascular disease, which is the leading cause of mortality worldwide among aging populations. Cisd2 is a prolongevity gene that mediates lifespan in mammals. Previously, our investigations revealed that a persistently high level of Cisd2 expression in mice is able to prevent age-associated cardiac dysfunction. This study was designed to apply a genetic approach that induces cardiac-specific Cisd2 overexpression (Cisd2 icOE) at a late-life stage, namely a time point immediately preceding the onset of old age, and evaluate the translational potential of this approach. Several discoveries are pinpointed. Firstly, Cisd2 is downregulated in the aging heart. This decrease in Cisd2 leads to cardiac dysfunction and impairs electromechanical performance. Intriguingly, Cisd2 icOE prevents an exacerbation of age-associated electromechanical dysfunction. Secondly, Cisd2 icOE ameliorates cardiac fibrosis and improves the integrity of the intercalated discs, thereby reversing various structural abnormalities. Finally, Cisd2 icOE reverses the transcriptomic profile of the aging heart, changing it from an older-age pattern to a younger pattern. Intriguingly, Cisd2 icOE modulates a number of aging-related pathways, namely the sirtuin signaling, autophagy, and senescence pathways, to bring about rejuvenation of the heart as it enters old age. Our findings highlight Cisd2 as a novel molecular target for developing therapies targeting cardiac aging.

Abstract P447: Layer-by-layer Fabrication Of Thick Cardiac Muscle Patches With High Viability And Maturity

Conventionally manufactured human cardiac muscle patches (hCMPs) typically rely on diffusion for oxygen and nutrient delivery to the cells and, consequently, the thickness of the hCMP is limited to 100-200 μm. Here, we tested our hypothesis that our layer-by-layer (lbl) manufacturing protocol could increase hCMP thickness without compromising the viability of individual cell populations. Human induced-pluripotent stem cells were differentiated into cardiomyocytes (CMs) or endothelial cells (ECs); then, layers composed of CMs or ECs were fabricated by mixing a cell-containing fibrinogen solution with thrombin and pouring the mixture into a mold, and the layers were stacked into a CM-EC-CM “sandwich”. hCMP viability was assessed via TUNEL staining, vascularization via expression of the EC marker CD144, and maturation via immunofluorescence and qPCR assessments of the expression of contractile (cTnT, cTnI), connective (Cx43, N-cad), and calcium-handling (RyR2, SERCA2, BIN1, Kir2.1) proteins. Sarcomere length was evaluated by staining for α-sarcomeric actinin, and hCMP ultrastructure was examined via transmission electron microscopy. The lbl-manufactured hCMP was 10 mm x 10 mm and 1.8 mm thick after fabrication. Minimal cells in the lbl-hCMP were apoptotic or necrotic. Compared to assessments in a single-layered patch containing the same number and proportion of cells, the lbl-hCMP was vascularized with higher levels of contractile, connective, and calcium-handling protein expression. Contractile proteins were also better aligned in the lbl-hCMP (deviation from global alignment 12.3 ± 3° vs. 24.3° ± 1.9°, p<0.05). The lbl-hCMP contained longer sarcomeres (2.05 ± 0.015 vs. 1.74 ± 0.026 um, p<0.05) with well-ordered I-bands, A-bands, M-lines, Z-lines, desmosomes, and intercalated discs. Thus, our lbl-manufacturing technique produced thicker and more mature patches that can be potentially clinically applicable for hearts with postinfarction LV remodeling.

Targeting the Microtubule EB1-CLASP2 Complex Modulates Na V 1.5 at Intercalated Discs

Rationale: Loss-of-function of the cardiac sodium channel Na V 1.5 causes conduction slowing and arrhythmias. Na V 1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I Na ) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I Na reduction, but the mechanisms underlying microdomain-specific targeting of Na V 1.5 remain largely unknown. Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating Na V 1.5 trafficking and subcellular distribution in cardiomyocytes. Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell I Na , increased action potential (AP) upstroke velocity (V max ), and enhanced Na V 1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na V 1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell I Na , decreased V max and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V max as well as increased APD. STORM imaging and macropatch I Na measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na V 1.5 and I Na preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I Na or Na V 1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on Na V 1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I Na within the ID region of cardiomyocytes.

Disorganization of intercalated discs in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a primary myocardial disease, the pathology of which is left ventricular or biventricular dilation and impaired myocardial contractility. The clinical and pathological diagnosis of DCM is difficult, and other cardiac diseases must be ruled out. Several studies have reported pathological findings that are characteristic of DCM, including cardiomyocyte atrophy, nuclear pleomorphism, and interstitial fibrosis, but none of these findings are DCM-specific. In this study, we examined the morphological differences in the intercalated discs (ICDs) between three groups of patients, a DCM group, a chronic heart failure group, and a control group. A total of 22 autopsy cases, including five DCM cases, nine CHF cases and eight control cases, were retrieved from the archives of the Department of Pathology at Akita University, Japan. The morphological differences were examined using multiple methods: macroscopic examination, light microscopy, immunohistochemistry, electron microscopy, and gene expression analyses. We observed disorganized ICDs, clearly illustrated by N-cadherin immunostaining in the DCM group. “Reduction of N-cadherin immunostaining intensity” and “ICD scattering” was DCM-specific. The results suggest that disorganized ICDs contribute to the development of DCM, and that N-cadherin immunostaining is useful for determining the presence of disorganized ICDs and for the pathological diagnosis of DCM.

Disorganization of Intercalated Discs in Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a primary myocardial disease, the pathology of which is left ventricular or biventricular dilation and impaired myocardial contractility. The clinical and pathological diagnosis of DCM is difficult, and other cardiac diseases must be ruled out. Several studies have reported pathological findings that are characteristic of DCM, including cardiomyocyte atrophy, nuclear pleomorphism, and interstitial fibrosis, but none of these findings are DCM-specific. In this study, we examined the morphological differences in ICDs between three groups of patients, a DCM group, a chronic heart failure group, and a control group. A total of 21 autopsy cases, including five DCM cases, eight CHF cases and eight control cases, were retrieved from the archives of the Department of Pathology at Akita University, Japan. The morphological differences were examined using multiple methods: macroscopic examination, light microscopy, immunohistochemistry, electron microscopy, and gene expression analyses. We observed disorganized intercalated discs (ICDs), clearly illustrated by N-cadherin immunostaining in the DCM group. “Reduction of N-cadherin immunostaining intensity” and “ICD scattering” was DCM-specific. The results suggest that disorganized ICDs contribute to the development of DCM, and that N-cadherin immunostaining is useful for determining the presence of disorganized ICDs and for the pathological diagnosis of DCM.

GLYCOGEN AND ATRIAL FIBRILLATION ATRIAL MYOCYTES CONTRACTILITY AS MECHANICAL FACTOR SHIFTING INTRACELLULAR GLYCOGEN AGAINST INTERCALATED DISCS

The purpose of this communication is to introduce in the medical literature an additional factor until now hypothesized action of atrial cells depolarization as factor in intracellular flow of glycogen molecules coalescing against the gap junctions. The demonstrated effect of gap junction blockers on paired cells contractility combined with gap junction’s selectivity towards glycogen molecules; and the visualization of contrasting intracellular glycogen images during atrial fibrillation are shown. Published data supporting atrial myocytes contraction as a mechanism in intracellular glycogen molecules migration and its deleterious effects leading into atrial fibrillation (AF) is proposed.