bluuebunny/crossref_metadata_2025 huggingface dataset:
Glu298Asp variant of endothelial nitric oxide synthase gene and acute or early presentation of coronary artery disease: An updated systematic review and meta-analysis
Authors: Rai H , Fitzgerald S , Coughlan J J , Spence M , Colleran R , Joner M , Byrne R A
Published: November 2023

Background

Several published reports have attested the association of Glu298Asp polymorphism (rs1799983) residing in the endothelial nitric oxide synthase (NOS3) gene with lower levels of circulating nitric oxide as well as coronary artery disease (CAD). However, its association status with acute or early presentation of CAD such as acute/sub-acute myocardial infarction, acute coronary syndrome, premature CAD and unstable angina is still unclear.

Purpose

Against this background, we conducted an updated systematic review and a study level meta-analysis to assess the association of NOS3 Glu298Asp polymorphism with acute or early CAD.

Methods

A comprehensive online search to identify relevant studies and sub-studies was performed on several databases including PubMed, EMBASE, MEDLINE, Scopus, Cochrane library and Web of Science and later stratified into two ancestral subgroups: ‘European ancestry’ and ‘All other ancestries combined’. Study level odds ratios (ORs) and their 95% confidence intervals (CI) were pooled using random effects employing a Z test.

Results

Out of a total of 193 distinct records identified through online search, 36 articles with 38 different studies, with a total sample of 27,244 (11,444 cases / 15,800 controls) were included for quantitative synthesis. (Figure 1.) Pooled results suggested significant associations of NOS3 Glu298Asp polymorphism with acute or early CAD through dominant as well as allelic genetic models (p≤0.004), driven by the ‘All other ancestries combined’ subgroup. Our ‘All other ancestries combined’ subgroup demonstrated an additional risk of 36% and 37% for acute or early CAD, through dominant and allelic genetic models respectively (OR= 1.36, 95%CI= 1.12, 1.65, p= 0.002 and OR= 1.37, 95%CI= 1.14, 1.64, p= 0.0007 respectively). On the other hand, the European ancestry subgroup failed to show any meaningful associations. Sensitivity analysis (Figure 2.) and a substudy for myocardial infarction endpoint further attested our observed associations.

Conclusions

Our present meta-analysis indicates the association of NOS3 Glu298Asp polymorphism with acute or early presentation of CAD, predominantly driven by ‘All other ancestries combined’ subgroup. In contrast, ‘European ancestry’ subgroup does not demonstrate any significant association. Further large-scale investigations are required to confirm our derived results.Figure 1.Figure 2.
Upregulation of mitochondrial fusion as potential cardioprotective strategies against trastuzumab-induced cardiotoxicity in rats
Authors: Maneechote C , Khuanjing T , Ongnok B , Arinno A , Prathumsap N , Chunchai T , Arunsak B , Kerdphoo S , Chattipakorn S C , Chattipakorn N
Published: November 2023

Background

Trastuzumab (TRZ) is a commonly used chemotherapeutic drug for treating advanced malignancies that express human epidermal growth factor receptor type 2. However, TRZ has been linked with cardiotoxicity, which is an unfavourable clinical side effect of cancer treatment. Alterations in mitochondrial dynamic mechanisms, particularly reduced fusion, have been shown to be a key mechanistic insight into the progression of chemotherapy-induced cardiotoxicity. Despite the cardioprotective benefits of a mitochondrial fusion promoter (M1) that have been established in various cardiovascular settings, its ability to counteract the cardiotoxicity caused by TRZ remains unknown.

Purpose

To examine the effect of M1 treatment on mitochondrial fusion, myocardial oxidative stress, apoptosis, and cardiac injury biomarker in TRZ-induced cardiotoxicity rats.

Methods

Male Wistar rats (n=18) were divided into the TRZ group (4 mg/kg/day for 7 days, I.P., n=12) and the control group (CON, n=6). In the TRZ group, at the time of first TRZ injection all rats also received the intervention with either the vehicle injection (VEH, n=6) or M1 (M1, 2 mg/kg/day, n=6) via I.P. injection for 7 days. The left ventricular ejection fraction (LVEF) was measured using echocardiography, and troponin-I levels were determined using blood serum. Then, the cardiac tissue was processed to assess the levels of malondialdehyde (MDA) for oxidative stress, mitochondrial fusion, and apoptotic proteins.

Results

TRZ induced cardiotoxicity via downregulation of mitochondrial fusion along with upregulation of myocardial oxidative stress and apoptosis, as indicated by decreasing Mfn1 protein expression and increasing the levels of MDA and Bax to Bcl-2 ratio, respectively (Fig. 1A, B, C, and E). TRZ also showed markedly higher levels of serum Troponin-I (Fig. 1D) and impaired LV function (77% LVEF for TRZ vs. 89% LVEF for CON). M1 co-treatment promoted Mfn1-mediated mitochondrial fusion, resulting in a reduction in MDA-associated oxidative stress, apoptosis, Troponin-I, and cardiac dysfunction (16% improvement over the TRZ group) (Fig. 1A-E).

Conclusion

M1 effectively protected the heart against TRZ-induced cardiotoxicity by promoting mitochondrial fusion and preventing oxidative stress and apoptosis-mediated myocardial injury. These findings suggest that the pharmacological promotion of mitochondrial fusion could be a novel anti-cardiotoxicity strategy in TRZ-induced cardiotoxicity.
Overexpression of cardiac calsequestrin as a novel gene-therapy approach to treat CPVT1: in silico and in vivo proves of principle
Authors: Santiago Castillo D , Bongianino R , Trancuccio A , Kukavica D , Tarifa C , Denegri M , Mazzanti A , Priori S G
Published: November 2023

Background

More than 200 gain-of-function mutations in the cardiac ryanodine receptor (RyR2) cause catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1). From a molecular point of view, 3 types of mutations exist [1]: Class 1 mutants enhance RyR2 opening in response to sarcoplasmic reticulum (SR) free Ca2+; Class 2 and Class 3 mutants enhance RyR2 opening in response to both cytosolic and SR free Ca2+.

Purpose

The study's objective was to evaluate in silico and in vivo, in CPVT1 knock-in mice carriers of the Class 1 mutation RyR2(R4496C/+), whether overexpression of cardiac calsequestrin (CASQ2) rescues the arrhythmic phenotype in CPVT1 mice.

Rationale: Several studies performed in the 2000’s have collectively demonstrated that an increase in CASQ2 promotes the closure of RyR2 channels [2]: inspired by this evidence, here we test the hypothesis that CASQ2 gene therapy might be a novel treatment for CPVT1.

Methods

(1) In silico study: Properties of Class 1 and Class 2 RyR2s mutants (Figure, panels A-C) were introduced into an in silico model of the murine ventricular myocyte [3]. Virtual myocytes were paced either at baseline or during simulated delivery of 100 nM Isoproterenol at 1 Hz for 180 seconds. To simulate CASQ2 overexpression, protein levels were progressively increased, and the changes in the arrhythmic burden were documented.

(2) In vivo study (performed in compliance with ethical regulations): an adeno-associated viral vector containing the cDNA sequence of CASQ2 was injected into RyR2(R4496C/+) mice (tail iv, 3×10^13 vg/kg) at 4 weeks of age. Eight weeks post-infection, animals were treated with an ip bolus of caffeine (120mg/kg) and epinephrine (2 mg/kg) to elicit arrhythmias (Epi/Caff Test).

Results

(1) In silico: dose-response curves were simulated to correlate CASQ2 levels with arrhythmia inducibility during virtual Isoproterenol delivery. For the simulated prototypical Class 1 mutation, 1.4-fold CASQ2 expression suppressed arrhythmias. For the prototypical Class 2 mutation, 1.8-fold CASQ2 expression was needed (Figure, panel D).

(2) In vivo: At 12 weeks of age, both RyR2(R4496C/+) treated and non-treated littermates received the ip Epi / Caff bolus. Arrhythmias were observed in 17/32 (53%) untreated mice and in 0/12 treated animals (p=0.0012).

Conclusion(s)

We provide proof-of-principle demonstrating that in mice, the overexpression of CASQ2 may be a powerful and safe therapeutic strategy for CPVT1.
Smart-device ECGs and inconclusive results: impact of ECG anomalies
Authors: Weidlich S , Mannhart D , Serban T , Krisai P , Knecht S , Du Fay De Lavallaz J , Schaer B , Osswald S , Kuehne M , Sticherling C , Badertscher P
Published: November 2023

Background

Evermore smart-devices are capable of recording single-lead (SL) electrocardiograms (ECGs). Each recording comes with an automated rhythm interpretation usually consisting of sinus rhythm (SR), atrial fibrillation (AF) or inconclusive. The impact of common ECG anomalies possibly leading to an inconclusive interpretation by the devices algorithm is largely unknown.

Purpose

to assess the impact of baseline ECG anomalies on automated smart-device rhythm classification.

Methods

In this prospective study, consecutive hospitalized patients undergoing electrophysiological procedures in a tertiary referral center were included. Each participant obtained a 12-lead ECG followed by SL-ECGs with 5 different smart-devices (AliveCor KardiaMobile, Apple Watch 6, Fitbit Sense, Samsung Galaxy Watch3 and Withings ScanWatch). The smart-device generated rhythm determination was compared to manual interpretation. ECG anomalies were assessed by 2 independent cardiologists and included atrial Flutter (AFlut), pacing and conduction delay consisting of AV-block or left/right bundle branch block, artifacts and ectopy. Inconclusive tracings were labelled false-positive if the goldstandard was SR in the 12-lead ECG and false-negative if the goldstandard was AF in the 12-lead ECG. Relative Risk (RR) for anomalies to result as inconclusive diagnosis by the smart-device was calculated.

Results

245 participants were included (34% female, mean age 64±13 years) resulting in a total of 1’165 recorded SL-ECGs. Among these, 247 SL-ECGs (21%) were labeled as inconclusive by at least one smart-device (Figure 1). Overall, 834 ECG anomalies were present. 169 inconclusive SL-ECGs were labelled false-positive with 192 anomalies present. Among these, 34 (18%) had ventricular pacing, RR 2.9 (CI 2.2-3.8), 52 (27%) showed conduction delay, RR 1.5 (CI 1.1-2), 44 (23 %) had ectopy, RR 2.3 (CI 1.7-3), and 62 (32%) some sort of artifacts, RR 1.9 (CI 1.5-2.5). Among 78 inconclusive SL-ECGs with false-negative results, 84 anomalies were present. 18 (21%) were in AFlut, RR 1.5 (CI 0.94-2.3), 12 (14%) had ventricular pacing RR, 4.2 (CI 3.1-5.6), 14 (17%) showed conduction delay, RR 1.3 (CI 0.8-2.1), 9 (11%) had ectopy, RR 0.99 (CI 0.54-1.8), and 31 (37%) some sort of artifacts, RR 1.7 (CI 1.2-2.5). There were no differences between assessed smart-devices.

Conclusion

Underlying ECG anomalies are common in inconclusive SL-ECGs and impact automated rhythm classification.Figure 1
Betaine therapy of cardiac hypertrophy through inhibition of Hydroxy-carboxylic Acid Receptor 1 signaling
Authors: Fan M , Yao L
Published: November 2023

Background

Hydroxy-carboxylic Acid Receptor 1 (HCAR1), formerly known as G protein-coupled Receptor 81 (GPR81), is a cell membrane receptor for lactate, and essential for activation of a variety of intracellular signaling pathways. We found a correlation between the activation of lactate-HCAR1 signaling and occurrence of cardiac hypertrophy. However, the regulatory mechanism remains unclear.

Purpose

Our purpose is to investigate the regulatory mechanism between the activation of lactate-HCAR1 signaling and occurrence of cardiac hypertrophy.

Methods

Neonatal rat cardiomyocytes (NRCMs) were used for preparing cardiac hypertrophy model in vitro by phenylephrine (PE) stimulation, and C57 BL/6 mice were used for preparing pressure overloaded-induced cardiac hypertrophy model in vivo by TAC (transverse aortic constriction) surgery. Adenovirus containing shRNA-HCAR1 (ADV-shHCAR1) were constructed to knockdown HCAR1 in NRCMs. Binding interaction between HCAR1 and Betaine was predicted by AUTODOCK 4 and further validated with RNA-seq analysis.

Results

Lactate concentration and HCAR1 expression were both increased in the NRCMs upon PE treatment. Activation of HCAR1 by its agonist 3-Chloro-5-hydroxybenzoic acid (3Cl-HBA) specially induced hypertrophy in NRCMs. Furthermore, the hypertrophy phenotypes in NRCMs showed alleviation by transfection of ADV-shHCAR1. An AI-based molecule-docking screening analysis identified Betaine as a potential molecule to bind HCAR1. Treatment with Betaine significantly attenuated PE- or 3Cl-HBA- induced hypertrophy in NRCMs in vitro. In addition, Betaine administration of TAC mice (i.g. 50 mg/kg/day) for 4 weeks in vivo led to increase of ejection fraction (EF) and decreases of left ventricular (LV) posterior wall thickness, heart weight/tibia length ((HW/TL), cross-sectional area and cardiac fibrosis area as well. RNA-seq analysis and functional assays demonstrated that Betaine could bind to HCAR1, activated Hippo signaling pathway, and thereby blocked the HCAR1-mediated hypertrophy in cardiomyocytes.

Conclusions

In summary, the current study not only identified HCAR1 as a therapeutic target of cardiac hypertrophy, but also demonstrated Betaine as a therapeutic candidate molecule holding potential to benefit cardiac hypertrophy patients.
Rimeporide, a first in class Na/H exchanger-1 inhibitor, ameliorates right ventricular dysfunction and pulmonary arterial hypertension in a SuHx model
Authors: Milano G , Reinero M , Sameere A , Porte-Thome F , Beghetti M
Published: November 2023

Background

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and vascular remodeling leading to right ventricular (RV) failure. Although RV function is the major prognostic factor in PAH, no RV-specific therapies exist. The Na+/H+ exchanger type 1 (NHE1) regulates the intra- and extracellular pH balance mainly through the 1:1 exchange of intracellular H+ with extracellular Na+. Increased NHE-1 activity is involved in several diseases, including hypoxic PAH. Inhibition of NHE-1 activity attenuates pulmonary vascular remodeling in rodents exposed to hypoxia but its potential direct contribution in RV remodeling remains unclear.

Aim

In the current study, we aimed to investigate the effect of NHE-1 inhibition by Rimeporide a preclinical hypoxic models of PH: Sugen5416/hypoxia.

Methods

Sugen/hypoxia model: PAH was induced in adult Sprague-Dawley rats with a single injection of SU5416 (20 mg/kg) followed by 3 weeks of hypoxia (10% O2; Sugen/Hypoxia model: SuHx) and then return to normoxia. Control rats were injected with equal volume of vehicle. At week 5 post-injection, control and SuHx rats were treated with rimeporide (100 mg/Kg daily in drinking water, n=10/group) or placebo (n=11/group) for 3 weeks. Echocardiography and blood sampling were performed. RV phenotype was monitored by echocardiography and hemodynamic analysis. Lung and heart were collected for histological and protein expression analysis, such as NHE-1 (target of Rimeporide).

Results

SuHx caused severe PAH and RV dysfunction. RV dysfunction was accompanied by an increase in RV hypertrophy and RV fibrosis and PA remodeling (increase in the arteriolar wall thickness and fibrosis). A marked increase in macrophages was found in lung and RV tissues but not in LV. Moreover, Western blot analysis showed that RV protein levels of NHE-1 was higher in SuHx rats than Normoxia rats. Treatment with Rimeporide reversed SuHx-induced PAH as well as RV dysfunction and blunted pulmonary and RV inflammation, RV NHE-1 protein expression.

Conclusion

Our results indicate that NHE-1 levels are increased in RV tissues from SuHx-induced PAH rats. Rimeporide, through NHE-1 inhibition, might be a potential new target for treatment of RV dysfunction and PAH.
CaMKIIdelta-mediated SR Ca leak contributes to long-term Doxorubicin-induced cardiotoxicity
Authors: Feder A L , Tarnowski D , Maier L S , Sag C M
Published: November 2023

Background

Cardiotoxicity represents a major complication of cancer therapy with anthracyclines, whose main known compound is doxorubicine (DOX). Although mechanisms of DOX-induced toxic cardiomyopathy (DICM) are not entirely understood, CaMKIIδ-activity is known to be increased upon acute DOX exposure, contributing to impaired Calcium (Ca) handling. However, currently no studies have addressed the question, whether CaMKIIδ is involved in DOX-induced long-term cardiotoxicity.

Purpose

We aimed to investigate cardiac function and intracellular Ca handling using an in-vivo DOX treated mouse model, examining whether CaMKIIδ is involved in DOX-induced long-term cardiotoxicity.

Methods

Cardiac function and excitation-contraction coupling of CaMKIIdWT and CaMKIId-/- (global CaMKIId knockout) mice were tested in an in-vivo DOX treated mouse model (cumulative dose 12 mg/kg, intraperitoneal injection). Cardiac function was determined by echocardiography after 12 weeks. Ca handling of freshly isolated cardiomyocytes was assessed by epifluorescence microscopy (Fura2-AM) and confocal microscopy (Fluo4-AM). Histological analyses regarding cardiac fibrosis and cell dimensions were examined.

Results

Long-term DOX exposure reduced left ventricular ejection fraction (EF) by ∼13% and markedly increased cardiac fibrosis in CaMKIIδWT mice. Moreover, CaMKIIdWT hearts displayed decreased cell volume indicating atrophy following DOX treatment. Coinciding with the reduction in EF, CaMKIIdWT mice showed depressed Ca transient amplitudes as well as slowed Ca transient decay indicating depressed Ca uptake into the sarcoplasmic reticulum (SR). In addition, spontaneous diastolic Ca leakage from the SR was dramatically increased. In contrast, CaMKIId-/- mice showed preserved EF following 12 weeks of DOX treatment as compared to CaMKIIdWT in line with maintained Ca transient amplitudes. As an underlying cause, SR Ca leakage was not increased in CaMKIId-/- myocytes despite DOX-treatment. Pharmacological CaMKII inhibition using AIP (1µM) prevented SR Ca leak and restored Ca transient amplitudes in DOX-treated CaMKIIdWT myocytes.

Conclusion

Our study shows that CaMKIId contributes to long-term DOX-induced cardiotoxicity. We found that chronic DOX treatment induced depressed cardiac function and myocardial fibrosis, associated with impaired calcium handling as a consequence of CaMKIId-mediated SR Ca leak, which was absent in our CaMKIId-/- mouse model. In line, pharmacological inhibition of CaMKII rescued impaired Ca handling in DOX-treated CaMKIIdWT myocytes.
The anti-fibrotic effect of atrial natriuretic peptide depends on myocardial accumulation of the peptide in rat heart
Authors: Hatem S N , Bakhos J J , Saliba Y , Hajal J , Achkouty G , Semaan A , Fares N
Published: November 2023

Background

The atrial natriuretic peptide (ANP) has an anti-fibrotic function for the heart. However, during heart failure (HF), which is characterized by marked myocardial fibrosis, ANP has a time-limited effect due to the rapid peptide degradation. Angiotensin-receptor-neprilysin inhibitors are a new class of drug that inhibits ANP degradation and improves cardiac performance, raising the question of the impact of increased ANP bioavailability on this pharmacological effect.

Purpose

We aim to assess the impact of intracardiac accumulation of ANP on cardiac function and myocardial fibrosis. An original strategy consisted of using a mutated ANP, MANP, also known as fsANP (frameshift), which is highly resistant to enzymatic degradation.

Methods

Type 2 diabetes was induced in adult male rats by a high fat diet coupled to low dose streptozotocin injections (25 mg/kg). MANP and ANP were administered each at a dose of 2 pmol/kg/min by subcutaneous osmotic minipumps. Sacubitril was given orally at a dose of 18 mg/kg. The protocol duration was 42 days. Cardiac fibrosis and cultured cardiac fibroblasts (CFs) were evaluated in the presence or absence of ANP, MANP, and angiotensin-receptor-neprilysin inhibitors; sacubitril. In addition to classical immunohistological and biochemical approaches, cGMP production was measured by real-time imaging in live cultured CFs (n=477). Results: MANP and sacubitril preserved cardiac performance more efficiently than ANP (p<0.05). Moreover, MANP, as well as sacubitril attenuated myocardial, epicardial, and perivascular fibrosis were more efficient than ANP (p<0.05). There was an increase in the bioavailability of MANP compared to ANP in cardiac tissue (p<0.05). Furthermore, a high-level expression of protein kinase G, and inhibition of the SMADs signaling pathway were identified in CFs. Moreover, cGMP bio-sensing in CFs, showed that similarly to ANP, MANP enhances cGMP production and activates PKG signaling, resulting in similar SMADs pathway inhibition (p<0.05). These results indicate similar biological efficiency as ANP and MANP.

Conclusion

MANP and sacubitril are more efficient than ANP to improve cardiac function and to reduce myocardial fibrosis in HF. Both molecules increase ANP bioavailability. This study proves that tissue accumulation of ANP is an important therapeutic target during HF.
Effects of pericyte and smooth muscle specific knock-out of CXCL12 on cardiac function after myocardial ischemia in mice
Authors: Staggl S , Messner M , Seiringer H , Ghadge S K , Maurer T , Poelzl G , Bauer A , Zaruba M M
Published: November 2023

Introduction

Ischemic cardiomyopathy as a result of myocardial infarction represents one of the most common causes of heart failure (1). The C-X-C motif chemokine ligand 12 (CXCL12) and its receptor CXCR4 facilitate cardiac repair after myocardial ischemia (MI) and play fundamental roles in cardiovascular development (2). However, the cell- and tissue-specific actions of CXCL12 are poorly understood, limiting the introduction of targeted therapies. Here, we aimed to examine the role of pericyte and smooth muscle cell-specific CXCL12 expression on myocardial function after MI.

Methods

We generated a smooth muscle protein 22-alpha-Cre and a pericyte protein NG2-Cre driven mouse model to ablate CXCL12 specifically in smooth muscle cells (SM22-Cre x CXCL12flox/flox) and in pericytes (NG2-Cre x CXCL12flox/flox). Genotyping was performed using ear hole biopsy by PCR. Hearts were analysed morphologically by histology and immunofluorescence. Cardiac function and dimensions of the wall thicknesses and cardiac cavities were determined by echocardiography. Myocardial infarction was induced in the animals and cardiac function was further investigated 28 days after MI.

Results

In immunofluorescence, we found that CXCL12 is highly expressed in SM22+ smooth muscle cells and in cells surrounding arterial blood vessels (3). NG2-CXCL12-/- mice did not show increased mortality, they behaved inconspicuously and no obvious developmental defects could be detected. Echocardiographic analysis of cardiac function in NG2-Cre x CXCL12flox/flox mice without infarction showed no significant difference in left ventricular (LV) performance and no evidence of abnormal geometry of the ventricles compared with controls. Assessment of SM22-Cre x CXCL12flox/flox mice after MI revealed reduced ejection fraction (EF) (29.0%±9.7 vs. 40.0%±8.1; n=10; p=0.027) and stroke volume (SV) (19.0µl±3.3 vs. 32.7µl±10.3; n=10; p=0.002) compared to non-transgenic controls. In contrast NG2-Cre x CXCL12flox/flox mice showed no significant changes in EF (34.6%±11.4 vs. 40.0%±8.1; n=10; p=0.368) and SV (27.7µl±7.3 vs. 32.7µl±10.3; n=10; p=0.303).

Conclusion

Our findings suggest that pericyte specific CXCL12 plays no major role in cardiac development and function after MI. In contrast, KO of CXCL12 in smooth muscle seems to deteriorate cardiac function after ischemia.
Protein tyrosine phosphatase sigma and zeta1 regulate cardiac fibroblast contractile function through TGF-beta dependent and independent pathways
Authors: Weidner J , Faist D , Irengbam R , Theilmeier G , Hasselbach L
Published: November 2023

Background

Heart failure is a frequently fatal cardiovascular disease with limited therapeutic options. To improve our understanding of signaling pathways in affected cells is therefore important. Protein tyrosine phosphatase sigma (PTPRσ) and zeta 1 (PTPRz1) have been shown to be involved in cardiac development and disease [1,2].

Both phosphatases are upregulated in patients with end stage heart failure. We demonstrated that PTPRz1 inhibits while PTPRσ is required for migration of neonatal cardiac fibroblasts (nCF) [3].

Purpose

To further evaluate the influence of PTPRσ and PTPRz1 on fibroblast phenotype with regard to stress fiber formation and collagen contraction and their interaction with the TGFβ pathway.

Methods

nCF were transfected with either scrambled-, PTPRσ-, PTPRz1-, or both siRNAs. mRNA lysates were harvested 24h after transfection with subsequent rt-PCR analysis for the phosphatases. Transfected cells were seeded and stained with labeled phalloidin to measure the formation of stress fibers or in collagen gel matrices to evaluate their contractile capacities. To evaluate the nuclear translocation of SMAD3 immunohistochemical colocalization of SMAD3 and DAPI was quantified. Data were analyzed by Kruskal-Wallis and Mann-Whitney-U or ANOVA followed by t-test with Welsh correction using GraphPad prism.

Results

PTPRz1 and -σ knockdown (kd) was highly efficient (>85%). PTPRz1 mRNA was upregulated by 48% after PTPRσ kd (p < 0.05, n=4) but not vice versa and not after combined kd. Compared to control, nCF showed a 26% increase in the number of stress fibers in response to the PTPRz1 kd (p = 0.057, n=3-4) and an increase of 29% after PTPRσ/-z1 kd (p < 0.05, n=4). Collagen gels were contracted to about 70% of their surface by TGFβ in control (p<0.05, n=4) or any kd condition. They were also contracted by 24,5% after PTPRz1 knockdown (p < 0.01, n=4), whereas the additional kd of PTPRσ obliterated the effect. Nuclear translocation of SMAD3 was roughly doubled upon TGFβ stimulation in controls and all kd conditions. In unstimulated nCF, PTPRσ kd induced a 28%, PTPRz1 kd a 45%, and their combination a 114% increase of nuclei positive for SMAD3 (p < 0.05).

Conclusion

SMAD3 nuclear translocation is additively induced by PTPRz1 and PTPRσ kd independent of TGFβ. Stress fiber formation, however, seems to be inhibited by PTPRz1 independent of PTPRσ, while PTPRz1-kd-induced collagen contraction does depend on the presence of PTPRσ. PTPRσ and PTPRz1 thus have separate, overlapping and antagonistic effects that are TGFβ dependent and independent. The elucidation of the involvement of the phosphatases in fibroblast signaling and function could improve our understanding of adverse remodeling during heart failure and deliver novel options for therapeutic interventions.
Interleukin-22 deficiency exacerbates right ventricular remodeling during pressure overload
Authors: Shibao K , Yasukawa H , Akagaki D , Okabe K , Yanai T , Shima K , Ymamoto M , Fukumoto Y
Published: November 2023

Background

Fibrosis and remodeling of right ventricle (RV) are associated with prognosis in patients with RV failure. Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, play roles in tissue protection and wound repair. We have previously shown that IL-22 plays an important role in cardiac remodeling after acute myocardial infarction; however, the role of IL-22 in RV fibrosis and remodeling remains elusive.

Purpose

We investigated the role of IL-22 in the pathogenesis of RV remodeling during pressure overload.

Methods

Pulmonary artery banding (PAB) is performed by placing a 6-0 suture around the pulmonary artery over a 24 G needle in wild type mice (C57BL/6J) and IL-22 knockout mice (IL-22 KO). Only mice with moderate pulmonary artery stenosis (peak pressure gradient across the pulmonary band: 25–40 mmHg at 1 week after surgery by echo Doppler) were included in the study protocol. Two weeks after PAB, RV function was measured by echocardiography and real-time PCR analysis was performed to measure IL-22, IL-22 receptor subunit alpha 1 (IL-22RA1), and IL-22 signaling inhibitor IL-22 binding protein (IL-22BP).

Results

2 weeks after PAB, IL-22 KO mice showed markedly increased RV weight (IL-22 KO mice vs. wild-type mice; RV/LV+IVS: 0.48±0.05 vs 0.40±0.02, P=0.002). IL-22 KO mice exhibited significant RV enlargement and RV dysfunction 2 weeks after PAB. (IL-22 KO mice vs wild type mice; RVEDV: 10±2.3mm2 vs 12.9±3.1mm2, P=0.005, TAPSE: 10.8±1.8mm vs 9.5±1.7mm, P=0.04, FAC: 35.2±7.8% vs 23±6.0%, P<0.001). The expression of IL-22, IL-22RA1 and IL-22BP in the RV tended to increase 2 weeks after PAB (wild-type mice with PAB (n=3) vs. wild-type mice with sham; IL-22: 0.0023 vs 0.0028, P=0.18 IL-22RA1: 1.489 vs 1.773, P=0.175, IL-22BP: 0.025 vs 0.067, p=0.005).

Conclusion

These results suggest that IL-22 is involved in RV remodeling during RV pressure overload.
Population-specific and cross-ancestry genome-wide association study identifies shared genetic architecture and 6 new risk loci including CAMK2D associated for Brugada syndrome
Authors: Makita N , Ishikawa T , Masuda T , Hachiya T , Dina C , Simonet F , Tank M W T , Wilde A A , Redon R , Bezzina C R , Tanaka T , Barc J , Okada Y , Schott J J
Published: November 2023

Introduction

The Brugada syndrome (BrS) is an inherited arrhythmia characterized by coved-type ST-segment elevation in the right precordial leads and an increased risk for sudden cardiac death. Genome-wide association studies (GWAS) of BrS, mainly performed in European ancestry, have illustrated its complex genetic architecture as an polygenic disease besides a monogenic trait due to mutations in the major responsible cardiac sodium channel gene SCN5A. Clinical manifestations of BrS including the disease prevalence and the incidence of sudden death are known to be variable between Asian and European populations, however, its underlying mechanisms are largely unknown.

Objectives

We aimed to identify novel BrS-associated loci and to assess ancestry-dependent genetic heterogeneity that may underlie the variable clinical manifestations of BrS patients across different populations.

Methods

We performed a genome-wide meta-analysis of BrS in the Japanese ancestry (940 cases and 1,634 controls), followed by a cross-ancestry meta-analysis of Japanese and European GWAS (total of 3,760 cases and 11,635 controls) to identify novel risk loci and compared the allelic effects between two ancestries. We characterized the novel loci based on fine-mapping of potential causal variants, and expression and splicing quantitative trait associations using the Genotype-Tissue Expression (GTEx) database.

Results

The Japanese-specific genome-wide meta-analysis identified a novel association signal near the ZSCAN20 (lead variant, rs2336244; P=1.0E-08) besides previously reported 4 association signals at the SCN5A-SCN10A and HEY2 loci. Cross-ancestry genome-wide meta-analysis identified 17 association signals including 6 novel loci. The effect directions were consistent for 94.1% of the variants (16 of 17; P for sign test = 0.00027) and their allelic effects were highly correlated across ancestries (Pearson’s R=0.91 [P=2.9E-07]). The genetic risk score (GRS) derived from European-ancestry GWAS of BrS was significantly associated with the risk of BrS in the Japanese populations (odds ratio, 2.12 [95% confidence interval, 1.94–2.31]; P=1.2E-61), suggesting the shared genetic architecture across ancestries. The functional characterization of the 6 novel loci showed that one lead SNP on CAMK2D promotes an alternative splicing resulting in an isoform switch of calmodulin kinase II that favors up-regulation of the pro-inflammatory isoform δ9 and down-regulation of the pro-survival isoform δ3.

Conclusions

Our results identified 6 novel susceptible loci associated with BrS and revealed shared genetic architecture across ancestries.GWAS Manhattan plotShared genetic architecture
In vitro model of atrial fibrillation: investigating the initiation and maintenance mechanisms of atrial remodeling using hiPSC-derived atrial cardiomyocytes
Authors: Casini M , Fambuena Santos C , Emig R , Peyronnet R , Ravens U , Ontoria Oviedo I , Climent A , Sepulveda P
Published: November 2023

Despite atrial fibrillation (AF) being a common, clinically important, and economically significant public health problem, the initiation mechanisms of atrial remodeling during AF is still unclear. Furthermore, really few experimental models have the power to investigate these mechanisms in a human invitro two-dimensional (2D) monolayer of atrial cardiomyocytes.

The purpose of this work is to use human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes to develop a new human in vitro atrial model of AF able to reproduce and evaluate the arrhythmia initiation and maintenance mechanisms.

Two different hiPSC lines were differentiated into atrial cardiomyocytes using 1 µM retinoic acid from day 3 to day 8. Protocol to differentiate atrial cells was compared with the classical method to generate ventricular hiPSC to demonstrate atrial specificity. On day 20 of the differentiation cells were seeded into plates with different sizes: 0.32 cm2 and 9.6 cm2, a larger size that allows rotor formation. After 10 days qPCR and optical mapping experiments were performed at 37˚C. These experiments aimed to evaluate the electrophysiologic and genetic remodeling the

Cells undergo during AF initiation and maintenance.

The atrial phenotype was assessed by patch clamp recordings that showed cells treated with RA had significantly shorter action potential duration at 90% repolarization compared to the no RA treated, 182.3.5 ± 68 ms and 371.6 ± 82 ms, faster depolarization rate and higher spontaneous beating frequency (n=14, ± SD). Optical mapping experiments allowed the evaluation of activation rate depending on the size of the dish: larger wells activated spontaneously at 1.6 ± 1.9 Hz, whereas smaller wells presented 0.9 ± 0.5 Hz of activation rate. The fast activation rate on larger wells was associated higher number of reentrant wavefronts: 11.0 ± 8.2 reentries/cm2, vs 0.9 ± 0.5 reentries/cm2 in the smaller wells, demonstrating an AF phenotype. In fact, depending on the culture conditions cells showed different reentries occurrence: 100% of large dishes exhibited fibrillation compared to only 10% of small dishes. Furthermore, qPCR studies demonstrated that the spontaneous initiation and maintenance of rotors in the larger well sizes lead to an expression remodeling of the channels SCN5A, KCNJ2, GJA5, ATP2A2, and RYR2 as previously demonstrated on isolated tissue from AF patients.

We conclude that atrial cardiomyocytes obtained from hiPSC can recapitulate the remodeling that atrial tissue undergoes during AF initiation in patients. Providing a unique opportunity to study the initiation of fibrillatory activities and their maintenance in a human-relevant in-vitro model.hiPSC-aCM characterizationElectrophysiologic & genetic remodeling
Ethanol-induced atrial fibrillation results from late INa and can be prevented by ranolazine
Authors: Baier M , Mustroph J , Unsin D , Lebek S , Tarnowski D , Provaznik Z , Voigt N , Wagner S , Maier L , Neef S
Published: November 2023

Background

Ethanol consumption is the most important self-triggered cause of atrial fibrillation (AF). As abstinence often fails, alternative therapies are needed. CaMKII-activation upon ethanol with consecutive Ca2+-leak from the sarcoplasmic reticulum (SR) appears to be an important pathomechanism for ethanol-induced AF, but is not a druggable target. CaMKII has previously been shown to induce late sodium current (late INa) which could be proarrhythmogenic.

Purpose

We hypothesized that late INa might be involved in the development of AF upon acute ethanol exposition and that thus, the antianginal drug ranolazine (an inhibitor of late INa) could be repurposed to prevent ethanol-induced AF.

Methods

Acute effects of ethanol were investigated in vivo and in vitro as compared to vehicle. The occurrence of AF in vivo was assessed by programmed electrical stimulation in mice. To test the role of late INa, we used ranolazine in vivo and in vitro, as well as tetrodotoxin in vitro. We measured late INa and cytosolic sodium concentration, recorded stimulated action potentials and spontaneous delayed afterdepolarizations (DAD), and assessed SR Ca2+-leak by recording Ca2+-sparks. Mechanistically, we inhibited CaMKII using AIP and used NaV1.5 S571A mice to specifically investigate the role of CaMKII-mediated late INa. Human biopsies were acquired during surgery and were used for cell isolation or chunk incubation.

Results

Ethanol acutely induced late INa, sodium overload, SR Ca2+-leak and delayed afterdepolarizations in isolated atrial cardiomyocytes, as well as atrial fibrillation in vivo (10 of 10 mice). All of these cellular ethanol effects were prevented by pharmacologic inhibition of late INa. Importantly, ranolazine prevented the development of AF upon ethanol exposure in vivo. Mechanistically, CaMKII-activation and, accordingly, CaMKII-dependent NaV1.5 phosphorylation (S571) were increased by ethanol exposure. Indeed, the phosphoresistant NaV1.5 S571A mutation prevented late INa, SR Ca2+-leak and in vivo AF upon ethanol, demonstrating a critical role of CaMKII-mediated NaV1.5 phosphorylation for acute ethanol effects.

Conclusion

We show that ethanol acutely induces late INa upstream of arrhythmogenic SR Ca2+-leak and delayed afterdepolarizations, dependent on NaV1.5 S571 phosphorylation, leading to atrial fibrillation in vivo. Importantly, these effects as well as AF in vivo can be prevented using the late INa inhibitor ranolazine. We suggest that this antianginal drug could be repurposed to prevent ethanol-induced AF.Late INa (Patch-Clamp)In vivo AF (programmed stimulation)
Cardiosphere derived cell secretome therapy after experimental myocardial infarction: effects of intracoronary administration on ventricular tachycardia inducibility and cardiac function
Authors: Baez Diaz C , Blanco Blazquez V , Sanchez Margallo F M , Torrescusa A , Pulido M , Lopez E , Arenal A , Crisostomo V
Published: November 2023

Background

Myocardial infarction (MI) carries high risk of sudden cardiac death due to the development of ventricular tachycardia (VT) which is caused by viable myocytes located within the infarct scar tissue [1]. Cardiosphere derived cell secretome (S-CDCs) has been demonstrated to possess antiarrhythmic properties when delivered transendocardially [2]. Further, S-CDCs has been proven to reduce scar size and increase viable myocardial mass in chronic MI [3].

Purpose

The objective of this study was to evaluate the effects of an intracoronary (IC) administration of S-CDCs on VT inducibility and cardiac function in a porcine MI model.

Methods

After obtaining the approval of the Institutional Animal Care and Use Committee, 14 Large White swine were subjected to closed chest MI induction carrying out balloon occlusion of the left anterior descending coronary artery (below the second diagonal branch) for 150 minutes. One month after MI creation, pigs received an IC administration of saline (CON; 5mL; n=7) or S-CDCs (S-CDCs; 9.16 mg of protein in 5mL of saline; n=7). VT inducibility and magnetic resonance (MR) studies were performed pre- and four months post-injection determining ejection fraction (EF), infarct size (IS) and indexed end diastolic and systolic volumes (EDVi, ESVi).

Results

Before injection VT was inducible in all cases and no differences were observed between groups in any MR-derived parameter (figure). IC infusion was successful in all swine. At four months VT inducibility study revealed lower inducibility rates in S-CDCs compared to CON (57% versus 100%; p=0.05). At this timepoint EF was higher in S-CDCs (35±10% versus 29±10%) although differences between groups were not statistically significant. Conversely, IS was significantly lower in S-CDCs (12±3% versus 16±2% (p=0.03)). Moreover, a trend towards lower ventricular volumes (EDVi: 83±18mL/m² versus 88±29mL/m² and ESVi: 56±20mL/m² versus 64±27mL/m²) was detected in this group.

Conclusions

The IC administration of S-CDCs suggests a potential reduction on post-MI VT development as well as a beneficial effect on cardiac function. This therapy seems to reduce IS in this porcine MI model.MR derived parameters
Chd4 and Zbtb7b cooperate to inhibit Cardiac Arrhythmia by repressing Cornifin-A expression
Authors: El Abdellaoui Soussi F A S , Novillo A N , Redondo J M R , Gomez-Del Arco P
Published: November 2023

Cardiac arrhythmias are life-threatening pathologies worldwide, but their etiology and development are still under debate. We have recently described that the Chd4/NuRD chromatin-remodeling complex is one of the epigenetic regulators involved in the maintenance of cardiac muscle identity. Indeed, conditional cardiomyocyte deletion of Chd4 (Chd4cKO) in mice induces arrhythmias, in part due to aberrant expression of the skeletal muscle sarcomeric gene program in differentiating and terminally differentiated cardiomyocytes (Gomez-del Arco et al. 2016 and El Abdellaoui-Soussi, 2022). In addition, we also discovered that numerous cardiac conduction system (CCS)-specific genes such as Hcn4, Cntn2, Cx43, Cx40, among others, were deregulated in these mutant mice. To better characterize the triggers of arrhythmogenesis in these Chd4cKO mice, we performed an in silico search for transcription factors (TFs) interacting with Chd4 in the heart and found that Zbtb7b could be one of these TFs. We therefore generated cKO mice for both genes and found that the arrhythmias appearing in the Chd4/Zbtb7b double KO (dKO) were much more severe than in the Chd4cKO and, therefore, dKO animals succumbed to sudden death faster than their Chd4cKO counterparts. In RNA-Seq experiments, we found that cornifin-A was synergistically overexpressed in dKO vs Chd4cKO and that the aberrant expression of this protein increased stiffness and apoptosis of cardiomyocytes, which could explain the observed exacerbated phenotype. Moreover, this overexpression was, at least in part, due to the concomitant downregulation of the micro-RNA miR-150-5p. Thus, our data provide paramount mechanistic evidence for a direct control of heart homeostasis in adult hearts by the Chd4/NuRD chromatin-remodeling complex in cooperation with the transcription factor Zbtb7b.
Vitronectin promotes TTR deposition and mitochondrial damage in ATTR-CA mice
Authors: Fan Y Z , Qiu Z P , Wang Z Y , Yang W B , Huang F Y , Chen Y J , Yang K , Jin W
Published: November 2023

Background

Transthyretin cardiac amyloidosis (ATTR-CA) is an increasingly recognized disorder that leads to heart failure and cardiovascular death. However, the pathogenesis of ATTR-CA is still poorly understood. Vitronectin (VTN), a common component of the extracellular matrix, has been reported to be associate with amyloidosis. However, the role of VTN in TTR deposition has not been clarified.

Purpose

Here we aimed to elucidate the TTR deposition mechanism in ATTR-CA and test therapeutic interventions using a gene-edited ATTR-CA mice model.

Methods

We used CRISPR/Cas9-mediated genome engineering to create humanized wild-type TTR knock-in mice (hTTRWT). Cardiac function was examined by echocardiography and pressure-volume analysis. VTN expression was assessed by immunofluorescence and western blotting. Interactions among VTN and TTR are analyzed with in vitro pull-down and co-immunoprecipitation (Co-IP) assays. Recombinant TTR protein was added in the culture of VTN overexpressed mice primary cardiomyocytes (CMs) to simulate the TTR deposition process in vitro. Mitochondrial function was determined by the Seahorse assay and mitochondrial ultrastructure was evaluated with the transmission electron microscope.Additionally, we assessed the mitochondrial damage with mitoSOX staining.

Results

In the hTTRWT group, pressure-volume analysis and echocardiography revealed significant diastolic dysfunction and increased left ventricle chamber stiffness at 52 weeks. Compared to WT group, the hTTRWT group showed a dramatic elevation of VTN in hearts. Co-IP and pull-down assays confirmed a direct interaction between VTN and TTR. In cultured CMs exposed to recombinant TTR protein, VTN overexpression promotes the deposition of TTR. Damaged mitochondrial ultrastructure, elevated oxidative stress and decreased mitochondrial numbers were found. In accordance with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity.

Conclusion

These results suggest that the VTN contributes to ATTR-related pathological and cardiac function changes and mitochondrial dysfunction as a contributor of disease progression from ATTR-CA to heart failure.
Mechanomics study of cardiomyocytes with filamin c truncations
Authors: Lazzarino M , Chen S N , Gao S , Zanetti M , Taylor M R G , Mestroni L , Sbaizero O
Published: November 2023

Background

Filamin C (FLNC) is expressed in cardiomyocytes, where it binds to actin and localizes to Z-discs, sarcolemma, and intercalated discs. Although FLNC truncation variants (FLNCtv) are an established cause of arrhythmias and heart failure (arrhythmogenic cardiomyopathy), the underlying mechanisms, in particular changes in mechanical properties of cardiomyocytes, are mostly unknown.

Purpose

In this study, we applied a biophysical approach to investigate the interaction between altered mechanical properties and biological response in human induced pluripotent stem cells–derived cardiomyocytes (hiPSC-CMs) carrying FLNCtv.

Methods

CRISPR/Cas9 genome-edited FLNC-/- hiPSC-CMs, a homozygous condition that is lethal in animal models, and FLNC+/- hiPSC-CMs, recapitulating the heterozygous human disease, were analyzed. Wild-type FLNC hiPSC-CMs were used as controls. Atomic Force microscopy (AFM) was used to perform single-cell force spectroscopy (SCFS) microindentation, to evaluate passive and dynamic mechanical properties. From the loading curves, two cell Young’s Moduli were calculated: E1, related to the compression of the plasma membrane and actin cortex, and E2, including the rest of the inner cell layers (cytoskeleton and nucleus). Cell adhesion was assessed using the retraction curve of the SCFS. Data were processed in terms of adhesion force (the highest value of the force exerted by the cell-surface contact to the cantilever during retraction) and work of adhesion (integrating the area under the retraction curve). Finally, FLNC-/- hiPSC-CMs were incubated with 1 µM Crenolanib (a PDGFRA signaling inhibitor) for 3 days, and the microindentation was performed again using DMSO-only-treated FLNC-/- cells as controls.

Results

The AFM indentation curve for the cells analyzed showed significant differences. Both elastic contributions (E1 and E2) show that wild-type FLNC iPSC-CMs are stiffer than both mutant FLNC+/- and FLNC-/- iPSC-CMs (see Figure upper panel). The adhesion showed the same trend as the elasticity: mutant FLNC iPSC-CMs showed a progressive decrease of work of adhesion as well as max adhesion force from the FLNC+/- to the FLNC-/- models. Rescue of the latter with Crenolanib showed to moderately increase their stiffness. Moreover, a qualitative analysis of the beating traces showed that FLNC+/- but mostly FLNC-/- display "irregular" small peaks, resembling those of delayed after depolarizations (see Figure lower panel).

Conclusions

We found that FLNCtv causes a decrease in cell stiffness which declines progressively from the heterozygous status, which is associated with FLNC haploinsufficiency, to the homozygous status, which lacks FLNC. This indicates a progressive softening of the cardiomyocytes, suggesting a damaged cytoskeleton. Crenolanib rescue suggests that the inhibition of PDGFRA signaling could partially restore such damage (see Figure upper panel - right corner).