bluuebunny/crossref_metadata_2025 huggingface dataset:
PCSK9 modulates the Wnt/beta-catenin signaling pathway in myocardial ischemia/reperfusion injury
Authors: Cha Y , Kim H E , Jeon S B , Park S W , Lee S H , Lee C J
Published: October 2022

Background

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein that affects cholesterol homeostasis. Recent research has found that PCSK9 has various effects on the heart that are unrelated to LDL cholesterol regulation. The Wnt/β-catenin signaling pathway plays a crucial role during heart development, and it is re-activated in response to cardiac injury. Low-density lipoprotein receptor-related proteins 5 (LRP5) act as co-receptors of Wnt ligands and are indispensable for Wnt/β-catenin signal transduction. However, it is not fully elucidated whether other members of the LDLR-superfamily may be targets of PCSK9.

Purpose

This study aimed to determine if LRP5 is a PCSK9 target, study the association between PCSK9 and Wnt/β-catenin signaling, and elucidate its effect on myocardial infarction in patients with ischemic cardiomyopathy.

Methods

The expression of Lrp5, phospho-β-catenin, total β-catenin was evaluated by western blot analysis, and the effects of overexpressed PCSK9 were tested under normoxia, hypoxia, or hypoxia/re-oxygenation (H/R) in mouse cardiomyocytes (HL-1). The transcriptional activity of β-catenin was assessed using the TOP-Flash/FOP-Flash luciferase reporter assay. In addition, the impact on various downstream targets of the Wnt/β-catenin signaling pathway was assessed using a quantitative real-time polymerase chain reaction. To examine whether PCSK9 regulates injury of cardiomyocytes in vivo, we subjected transgenic mice with cardiac-specific overexpression of PCSK9 (PCSK9 TG) and wild-type (WT) mice to either sham surgery or ischemia/reperfusion (I/R) surgery.

Results

Under hypoxic conditions, the Wnt/β-catenin signaling pathway-related genes were downregulated in HL-1 cells, as evidenced by lower Lrp5 and active phospho-β-catenin expression levels (0.5-fold, n=3, p<0.01). After H/R, the Wnt/β-catenin-related genes were recovered (1.5-fold, p<0.01) in the control group but not in the PCSK9 overexpressed group. In the luciferase reporter assay results, PCSK9 overexpression inhibited the recovery of β-catenin transcriptional activity after H/R, in contrast to the control group. Furthermore, mRNA levels of Axin2, Cyclin D1, which are the Wnt/β-catenin signaling downstream pathway targets, were down-regulated under hypoxia and recovered after H/R but did not recover in PCSK9 overexpressed cells. In the mouse I/R model, the overall protein levels of the Wnt/β-catenin signaling-related genes were down-regulated in PCSK9 TG mice compared to WT mice after I/R injury.

Conclusions

These results indicated that the regulation of PCSK9 is closely associated with the Wnt/β-catenin signaling pathway which may play a crucial role in damaged cardiomyocytes. It suggests that the regulation of PCSK9 could be a therapeutic target in patients with ischemic cardiomyopathy.

Funding Acknowledgement

Type of funding sources: None.
Red blood cells induce endothelial dysfunction in patients with ST-elevation myocardial infarction and elevated C-reactive protein
Authors: Tengbom J , Collado A , Jiao T , Yang J , Zhou Z , Mahdi A , Pernow J
Published: October 2022

Background

The important role of inflammation in atherosclerotic plaque progression and instability leading to myocardial infarction has been widely demonstrated. C-reactive protein (CRP) has been shown to be of predictive value in atherosclerotic cardiovascular disease. The red blood cell (RBC) is an important regulator of cardiovascular function through nitric oxide bioactivity and oxidative stress in ischemic heart disease. Also, arginase-1 has been shown to greatly influence nitric oxide bioactivity in RBCs and to cause endothelial dysfunction. However, the mechanisms by which RBCs regulate vascular function in patients with myocardial infarction and its relation to inflammation and arginase-1 remain unknown.

Objective

The study aimed to investigate the effect of RBCs on endothelial function in patients with ST-elevation myocardial infarction (STEMI) and its possible association with systemic inflammation and arginase-1.

Material and methods

Blood samples were collected from patients with STEMI within 36 h after admission and from age-matched healthy controls. RBCs were incubated with isolated rat aortic segments for 18 h after which the aortas were assessed for endothelium-dependent and endothelium-independent relaxations using wire myographs and application of acetylcholine and nitroprusside, respectively. The vascular response was evaluated in relation to the level of inflammation defined as CRP <2 and ≥2 mg/L at admission. The levels of 4-hydroxynonenal (4-HNE; a marker of oxidative stress formed by lipid peroxidation) and the expression of arginase-1 were visualized in rat aortas following incubation with RBCs by immunohistochemistry. All handling and procedures regarding human subjects and sampling were performed according to the Declaration of Helsinki. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996).

Results

RBCs from patients with STEMI and elevated CRP (≥2 mg/L, mean of 9.6 mg/L) induced significant impairment of endothelium-dependent relaxation compared to RBCs from patients with STEMI and low CRP (<2 mg/L, mean of 1.0 mg/L) and to the healthy controls (Fig. 1). Endothelium-independent relaxations did not differ between the groups. Immunohistochemical staining of the aorta revealed that incubation with RBCs from patients with STEMI and high CRP increased the expression of 4-HNE and arginase-1 compared to incubation of RBCs from healthy controls and patients with STEMI and low CRP (Fig. 2).

Conclusion

RBCs from patients with STEMI and elevated CRP induce endothelial dysfunction and increase the expression of 4-HNE and arginase-1, indicating that inflammation is involved in the mechanism by which RBCs induce endothelial dysfunction in STEMI.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung foundation
Inhibition of coagulation factor XI attenuates inflammation in myocardial ischemia/reperfusion injury
Authors: Luo Q , Molitor M , Aluia M , Finger S , Wenzel P
Published: October 2022

Background

Increasing evidence suggests that FXI is an attractive target for antithrombotic therapy because it reduces thrombosis without increasing bleeding risk. Patients with decreased levels of FXI are at reduced risk of cardiovascular diseases and thromboembolic events. We have already revealed that depletion of FXI inhibits the vascular coagulation-inflammatory circuit in angiotensin II-induced arterial hypertension. However, the effect of FXI depletion on cardiac inflammation and vascular function in ischemia/reperfusion (I/R) injury is unknown.

Purpose

Using FXI-depleted mice to investigate the role of FXI in inflammation response post cardiac I/R injury.

Methods

8–12 weeks old C57BL/6J male mice were injected intraperitoneally with FXI antisense oligonucleotide (FXI ASO) or scrambled controls for a period of 2 weeks. Then we temporarily ligated the left anterior descending (LAD) for 45 minutes to induce myocardial ischemia, followed by the reperfusion for 72 hours (ischemia/reperfusion injury, IRI). Myocardial inflammation and vascular function were analyzed by flow cytometry, real-time PCR, vascular relaxation studies from isolated aortic segment in organ chamber and chemiluminescence photon counting of oxidative burst in whole blood.

Results

FXI ASO treatment reduced hepatic FXI mRNA levels and prolonged activated partial thromboplastin time. Compared to scrambled ASO injected mice, oxidative burst from whole blood and endothelial dysfunction were decreased in FXI ASO injected mice with IRI. Compared to control groups, depletion of FXI attenuated cardiac infiltration of CD45+CD11b+ cells, especially LyG-LyChigh monocytes and LyG+ neutrophils at day 3 in I/R injury. Furthermore, the expression levels of adhesion molecules and expression of pro-inflammatory cytokines, such as Vcam-1, CCL2, IL-6 and IL-1b, in ischemic myocardium were significantly decreased in the FXI depleted mice of I/R injury.

Conclusion

Our results suggest that Inhibition of FXI leads to reduction of vascular dysfunction and ROS production in I/R injury, as well as attenuation of the inflammatory response and the influx of inflammatory cells in ischemic myocardium. This indicates that FXI could be a potential target for further treatment in cardiac IRI.

Funding Acknowledgement

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): TICARDIO project has received funding from the European Union's Horizon 2020 research and Innovation programme under the Marie Skłodowska-Curie grant agreement No 813409.
Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes: potential implication in Doxorubicin-induced cardiotoxicity
Authors: Shimizu M , Ohwada W , Kouzu H , Sato T , Osanami A , Ogawa T , Ino S , Toda Y , Kuno A , Tanno M , Yano T
Published: October 2022

Background

The treatment with doxorubicin, a powerful chemotherapeutic agent, has been shown to be associated with an increased risk of lethal heart failure. Although various types of cell death pathway such as apoptosis and ferroptosis have been shown to be involved in the development of doxorubicin-induced cardiotoxicity, DIC, the involvement of necroptosis, a novel programmed necrosis induced by translocation of activated mixed lineage kinase domain-like protein, MLKL, to plasma membrane, remains unclear.

Purpose

The aim of this study was to determine whether necroptosis is involved in the development of DIC.

Methods and results

DIC was induced in C57BL/6J mice by intraperitoneal injection of doxorubicin at a dose of 10 mg/kg 3 times for a week. Eight days after the commencement of injection, echocardiographic analyses showed that left ventricular ejection fraction assessed by echocardiography was significantly lower in the doxorubicin-treated mice than in the vehicle-treated mice (44.0±13.7 vs. 70.5±3.7%), indicating the development of DIC. Immunoblot analysis showed that MLKL protein level was higher by 1.6 fold in the doxorubicin-treated mice than in the vehicle-treated mice. Interestingly, immunohistochemical analysis showed that signals of phospho-Ser345-MLKL, an activated form of MLKL, was found in the nuclei in addition to cytosol and intercalated discs of cardiomyocytes in the doxorubicin-treated mice. To get novel insight into significance of nuclear MLKL accumulation, a leucine-rich nuclear export signal (NES) spanning amino acids 280–284 of rat MLKL was identified by site-directed mutation analyses, and H9c2 cells, cultured rat cardiomyoblasts, were transfected with expression constructs for nucleus-directed MLKL (FLAG-mtNES-MLKL) or its wild type (FLAG-WT-MLKL). Percentage of FLAG-positive cells stained with Zombie Red, a fluorescent dye that is non-permeant to live cells, was higher in FLAG-mtNES-MLKL-transfected cells than in FLAG-WT-MLKL-transfected cells (80.0±3.5% vs. 6.3±1.3%, p<0.05), whereas percentage of cells immunostained with cleaved caspase-3 to FLAG-positive cells was similar in the two groups. The effect of the MLKL mutant on necroptosis was attenuated by treatment with GppNHp, an inhibitor of Ran-mediated nuclear protein import.

Conclusion

Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes, which may contribute to progression of DIC.

Funding Acknowledgement

Type of funding sources: None.
Doxorubicin-induced upregulation of follistatin-like 3 (FSTL3): a new therapeutic target
Authors: Chen D , Croft A , Kelly C , Haw T J , Leong A , Sverdlov A , Ngo D
Published: October 2022

Introduction

Doxorubicin (DOX) is among the most used anticancer drugs with associated cardiotoxicity. Follistatin-like 3 (FSTL3), a secreted member of the follistatins family that can selectively bind to members of the TGF-β superfamily, is involved in regulation of cardiac hypertrophy and heart failure. FSTL3 is also upregulated in breast and colorectal cancer tumours, is also an unfavourable prognostic indicator for various cancers.

Purpose

We aim to determine the dual role of FSTL3 in prevention of DOX-induced cardiotoxicity and synergistic anti-cancer effects.

Methods

Human cardiomyocytes (HCMs) were treated with DOX at 1uM (EC50) for 72 hours. Cell viability was assessed via CellTiter-Glo®. Secreted FSTL3 levels, as measured by ELISA (R&D systems). FSTL3 and TGF-β mRNA levels were measured by qPCR. Co-treatment of DOX with human anti-FSTL3 antibodies (Aviva Systems Biology) at 10ug/mL were introduced for 72hrs treatment.

Results

Secreted FSTL3 levels were significantly increased in DOX-treated HCMs at 72hrs compared to control (n=5, p<0.001). Consistently, FSTL3 and TGF-β mRNA levels, in collected HCMs were significantly increased in DOX-treated cells. Co-treatment of DOX with human anti-FSTL3 antibodies at 10ug/mL significantly improved HCM viability compared to IgG control group. Conversely, anti-FSTL3 antibodies provided synergistic anti-cancer effects with DOX: MCF-7 breast cancer cells were significantly reduced when co-treated with DOX and anti-FSTL3 antibody vs. IgG controls.

Conclusion

We show, for the first time, that: 1) FSTL3 is secreted directly from HCMs; 2) FSTL3 levels (both circulating and mRNA) is markedly elevated with DOX treatment; 3) neutralisation of FSTL3 in DOX-treated HCMs, restored HCM viability; and 4) exhibit synergistic anti-cancer effects with DOX. Taken together, FSTL3 is a potential target for dual anti-cancer and cardioprotective effects.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Heart Foundation of Australia Future Leader FellowshipsNSW Ministry of Health EMC FellowshipNSW Ministry of Health Translational Research Grant
Nucleic acid-based therapies using living myocardial slices
Authors: Nunez-Toldra R , Del Canizo A , Secco I , Nicastro L , Giacca M , Terracciano C M
Published: October 2022

Background

Gene therapy based on viral vectors is a promising new approach and offers great potential for the study and the treatment of cardiac diseases. Nucleic acid-based transfection of human heart tissue could be considered a valid and translational alternative to the use of transgenic animal models in cardiovascular research.

Purpose

Here we explore the use of Living Myocardial Slices (LMS) as a platform for nucleic acid-based therapies. Rat LMS and Adeno-Associated viruses (AAV) were used to optimise and analyse gene transfer efficiency, viability, tissue functionality, and cell tropism in cardiac tissue. Human cardiac samples from failing (dilated cardiomyopathy) hearts were also used to validate the human model. In addition, we transfected and tested for the first time the regenerative effect of AAV-miR-199a in both healthy and pathologically overloaded LMS.

Methods

LMS (300 μm thick) were prepared and glued to PTFE-coated holders, mounted on custom stretchers, and cultured at physiological sarcomere length for 72-hours under electrical stimulation. Two recombinant AAV serotypes (AAV-6 and AAV-9) at different multiplicity of infection (MOI) expressing green fluorescent protein (GFP) or the miR-199 transgene were added to the surface of the LMS.

Results

AAV6 proved to be the most suitable serotype and 20,000 MOI the most efficient dose, as these conditions were proven not to affect LMS contractility and to have the highest transduction and penetrability efficiency. This serotype exhibited preferential cell tropism to cardiomyocytes and stromal cells (40% of transduction in both) in rat LMS, with lower efficiency in endothelial cells, (20% of transduction). In contrast, AAV6 in human failing LMS showed higher transduction efficiency in endothelial cells and myofibroblasts. Treatment with miR-199a significantly increased the active force of pathologically overstretched LMS, downregulated the expression of its target genes, and affected the proliferative capacity of LMS.

Conclusion

LMS can be used as a model for nucleic acid-based therapies and their transfection protocol can be adapted to samples obtained from failing human hearts. With great translational relevance, this model would accelerate preclinical studies of novel nucleic acid-based therapies for heart failure.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation
Autoantibodies against the cardiovascular protective BPIFB4 in hospitalized patients with COVID-19
Authors: Ciaglia E , Montella F , Lopardo V , Esposito R M , Guarracino F , Spinetti G , Maciag A A , Ciccarelli M , Vitale C , Pellegrino S G , Polverino B , Izzo C , Vecchione C , Puca A A
Published: October 2022

Background/Introduction

The bactericidal/permeability-increasing fold-containing family-B-member-4 (BPIFB4) serves as a biomarker of healthy aging [1,2] and displays prognostic relevance in vascular pathology [3–5]. We recently described a drop in plasma BPIFB4 level in patients with severe COVID-19 compared to low-grade disease patients [6].

Purpose

As COVID-19 is associated with autoimmune features, we developed the methods for determination of Anti-BPIFB4 IgG (autoAbs) and then characterized their neutralizing activity in COVID-19 patients.

Methods

A sandwich ELISA-based colorimetric assay followed by immunoblot analysis detected the presence of autoAbs against BPIFB4 in 60 hospitalized COVID-19 patients and in 30 healthy volunteers. Compared to the healthy controls, the optical density (OD) level of autoAbs in COVID-19 showed considerable variability distributing over a range between 0.13 and 0.85. We thus divided the patients into two groups, one with OD >0,29 and the other one with a OD >0,29, where 0,29 represents the OD mean value of autoAbs against BPIFB4 in physiological conditions.

Results

Since patients with higher OD are mainly those who spend in average a higher number of days in hospital, we stratified the patients according to the Length of Stay (LoS) in hospital (Figure 1), and found a trend towards a positive correlation between AutoAbs OD level and length of hospitalization within COVID-19 patients.

When present, autoAbs exclusively target the WT-BPIFB4 autoantigens and neglect the recognition of the Longevity-associated-variant-(LAV) of the BPIFB4 gene known for its therapeutic efficacy in cardiomyopathy, atherosclerosis (4), diabetes (6) and platelets' reactivity.

As expected, the pre-treatment of human PrP with the recombinant rhLAV-BPIFB4 reduces platelets' aggregation in response to ADP and collagen in COVID-19 patients in vitro.

On the other hand, at functional level, the well established LAV-BPIFB4-regulated M2 macrophage polarization (4,7), is neutralized in presence of anti-BPIFB4 autoAbs-enriched plasma.

Conclusion

We conclude that a significant proportion of hospitalized COVID-19 patients displays BPIFB4-AutoAbs which are positively correlated with the Length of Stay (LoS) in hospital. In future, it will be of utmost importance to clarify if the 4 missense SNPs which distinguish LAV-BPIFB4 gene from its WT-counterpart, are instrumental to prevent the self-tolerance brake-down and the potential development of specific antibodies against endogenous cardiovascular protectors.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Cariplo Foundation (n.2016-0874) to AAP and CV; Ministry of Health (RF-2016-02364864) to AAP and CV
Hepatic T1-time predicts cardiovascular risk in all-comers referred for cardiovascular magnetic resonance
Authors: Mascherbauer K , Dona C , Koschutnik M , Dannenberg V , Nitsche C , Duca F , Beitzke D , Loewe C , Waldmann E , Trauner M , Bartko P , Goliasch G , Mascherbauer J , Hengstenberg C , Kammerlander A
Published: October 2022

Background

Liver damage is frequently observed in patients with cardiovascular disease (CVD) but infrequently quantified. We hypothesized that in patients with CVD undergoing cardiac magnetic resonance (CMR), liver T1-times indicate liver damage and are associated with cardiovascular outcome.

Methods

We measured hepatic T1-times, displayed on standard cardiac T1-maps, in an all-comer CMR-cohort. At the time of CMR, we assessed validated general liver fibrosis scores. Kaplan-Meier estimates and Cox-regression models were used to investigate the association between hepatic T1-times and a composite endpoint of non-fatal myocardial infarction, heart failure hospitalization, and death.

Results

1022 participants (58±18 y/o, 47% female) were included (972 patients, 50 controls). Hepatic T1-times were 590±89ms in patients and 574±45ms in controls (p=0.052). They were significantly correlated with cardiac size and function, presence of atrial fibrillation, NT-pro-BNP levels, and gamma-glutamyl-transferase levels (p<0.001 for all). During follow-up (58±31 months), a total of 280 (29%) events occurred. On Cox-regression, high hepatic T1-times yielded a significantly higher risk for events (adj.HR 1.66 [95% CI: 1.45–1.89] per 100ms increase, p<0.001), even when adjusted for age, sex, left and right ventricular ejection fraction, NT-proBNP, and myocardial T1-time. On restricted cubic splines, we found that a hepatic T1-time exceeding 610ms was associated with excessive risk.

Conclusion

Hepatic T1-times on standard CMR scans were significantly associated with cardiac size and function, comorbidities, natriuretic peptides, and independently predicted cardiovascular mortality and morbidity. A hepatic T1-time >610ms seems to indicate excessive risk.

Funding Acknowledgement

Type of funding sources: None.
LncRNA BACE1-AS: a link between heart failure and Alzheimer's disease
Authors: Greco S , Piella S N , Made' A , Tascini A S , Garcia-Manteiga J M , Castelvecchio S , Menicanti L , Martelli F
Published: October 2022

Background

BACE1-antisense RNA (BACE1-AS) is a lncRNA antisense to the Beta-Secretase-1 (BACE1) gene encoding a key enzyme in the production of the β-amyloid peptide, associated to Alzheimer's disease (AD). In a previous study1, we showed that the BACE1-AS/BACE1 axis is activated in heart failure (HF) patients, leading to β-amyloid accumulation in failing hearts. Accordingly, BACE1-AS expression in different cultured cardiac cell types induced the expression of BACE1 and β-amyloid that, in turn, triggered apoptosis. The mechanisms underlying BACE1-AS action are not yet fully elucidated. Indeed, current models based on miRNA “sponging” or “masking” inducing BACE1 post-transcriptional stabilization may not recapitulate all BACE1-AS functions.

Purpose

Our aim is to investigate the molecular mechanisms regulated by BACE1-AS in heart failure disease.

Methods

BACE1-AS-pull-down, followed by RNA-Seq, was used to identify RNAs interacting with BACE1-AS in AC16 cardiomyocytes. Moreover, accessible chromatin sites induced by BACE1-AS overexpression were assayed by ATAC-Seq.

Results

BACE1-AS pull-down identified 698 BACE1-AS interacting RNAs. Among these enriched transcripts, 69 were mapping to genomic enhancer regions that have been found to be hypo-methylated in AD brains2. After qPCR validation of the interactions identified by RNA-seq, the modulation of SEMA4D, ABCG1, GFRA2 and RIMBP2, which were under the control of a subset of these enhancers, was assayed upon BACE1-AS overexpression in cardiomyocytes. It was found that their expression was induced, supporting the functional interaction between BACE1-AS and the identified enhancer loci.

To gain further insight into BACE1-AS function in cardiomyocytes, accessible chromatin sites induced by BACE1-AS overexpression were assayed by ATAC-Seq. Interestingly, 17 regions identified by this approach overlapped with enhancers that are hypo-methylated in AD brains. One of the accessible chromatin sites was the locus encompassing RNF214/BACE1/BACE1-AS that maps to active enhancer marks, indicating that BACE1-AS may regulate the expression of BACE1 transcriptionally.

Conclusion

Collectively, these data suggest BACE1-AS as a node of shared disease-mechanisms between heart failure and AD.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health
Enhancing NO pathway improves diastolic function in a hamster model of heart failure with preserved ejection fraction associated with nonalcoholic steatohepatitis
Authors: Dubroca C , Maupoint J , Costard C , Brousseau E , Briand F , Sulpice T
Published: October 2022

Background and aims

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical phenotype frequently associated to the presence of multiple non-cardiac comorbidities including nonalcoholic steatohepatitis (NASH) commonly seen in metabolic syndrome and obesity. In this context, the enhanced production of ROS due to systemic inflammatory state participates to the myocardial structural and functional alterations by limiting nitric oxide bioavailability. We thus evaluated the effect of the phosphodiesterase type 5 (PDE5) inhibitor, vardenafil, on cardiac function and remodeling in a NASH hamster model.

Method

Male Golden Syrian hamsters were fed for 20 weeks with a free choice diet consisting in free access to control chow diet with normal drinking water or a high fat/high cholesterol diet with 10% fructose enriched drinking water. At 15 weeks hamsters were randomized on E/A ratio and ejection fraction to receive either vardenafil (10mg/kg/day orally for 5 weeks) or vehicle before cardiac and metabolic parameters evaluation.

Results

After 20 weeks of free choice diet, hamsters presented a large increase in hepatic lipid contents and developed NASH. Echocardiography showed that systolic function was preserved with ejection fraction in normal range whatever the diet. In contrast, E/A ratio was significantly increased in free choice fed hamsters (2,1±0,1 vs 1,4±0,04) and E'/A' ratio inverted when compared to control (0,8±0,02 vs 1,3±0,04). This was associated with a decreased isovolumic relaxation time (IVRT) and an increased E/E' ratio signing relaxation impairment and higher filling pressure, respectively. Moreover, pro-BNP was largely increased in free choice fed hamsters when compared to controls. While vardenafil had no impact on NASH, diastolic function was largely improved as evidenced by the significant decrease in E/A and E/E' ratios along with the increase in E'/A' ratio and IVRT. This effect was corroborated by the normalization of the pro-BNP plasma levels induced by the 5 week-treatment with vardenafil.

Conclusion

The present data demonstrate that free choice fed hamsters developed NASH and showed the ultimate stage of diastolic dysfunction characterized by a restrictive profile combining compliance and relaxation impairments as well as higher filling pressure. While no effect on NASH was observed, enhancing NO pathway with vardenafil treatment significantly improves diastolic function in this hamster model of HFpEF.

Funding Acknowledgement

Type of funding sources: None.
Comparative effectiveness of the new calpain inhibitor NPO-2270 versus enalapril in pressure overload-induced heart failure
Authors: Delgado S , Aluja D , Llenas J , Vazquez E , Barrabes J A , Ferreira I , Inserte J
Published: October 2022

Background

Studies performed in multiple preclinical models support the contribution of the Ca2+-dependent cysteine proteases calpains to ventricular remodelling and heart failure (HF). However, pharmacological calpain inhibition has not yet been tested in patients with HF mainly due to the limitations of available inhibitors.

Purpose

To determine the effect of NPO-2270 (NPO), a new ketoamide derivative calpain inhibitor, in a mouse model of pressure-overload and compare its effectiveness with that of enalapril.

Methods

C57BL6 mice were subjected to transverse aortic constriction (TAC) for 4 weeks. Mice were randomised to receive orally administered NPO or enalapril at the dose of 10 mg/kg/day once a day, or vehicle, starting at day 7 after TAC surgery. The combination of both drugs and the effect of NPO starting 1 day after surgery were tested in additional TAC mice. Echocardiographic data, markers of hypertrophy, fibrosis, calpain activity and cleavage of calpain substrates were measured at different time points.

Results

TAC increased calpain-1 and -2 expression and activity. Administration of NPO and enalapril prevented the progression of hypertrophy and interstitial fibrosis induced by TAC with no statistically significant differences between the two treatments. However, ongoing ventricular dysfunction was less severe in the NPO group than in the enalapril group (27% of LVEF reduction in control group, 6% in NPO group and 16% in the enalapril group after TAC, P=0.024 between NPO and enalapril groups). The combined treatment or the administration of NPO from the first day after TAC surgery was not superior to NPO alone starting 7 days after TAC. These differences in LVEF correlated with better preservation of cadherin-based cell adhesion complex in mice treated with NPO-2270. No adverse effects associated with long-term NPO administration were observed in a sham group.

Conclusions

The new calpain inhibitor NPO-2270 prevents the development of hypertrophy and fibrosis with similar efficacy than enalapril but prevents cardiac dysfunction more effectively in a preclinical model of pressure overload when given orally at equivalent doses.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III of the Spanish Ministry of Health
Transcriptomic and proteomic profiling of human diabetic heart disease
Authors: Gollmer J , Potter L , Vosko I , Tomin T , Birner-Gruenberger R , Von Lewinski D , Sedej S , Scherr D , Wende A R , Rainer P , Zirlik A , Bugger H
Published: October 2022

Studies in animal models demonstrated the capability of type 2 diabetes (T2D) to induce cardiac dysfunction in the absence of vascular disease. However, whether and how T2D also impairs structure and function in human hearts remains poorly understood. Here, we performed transcriptional and proteomic profiling of left ventricular samples of 8 subjects with T2D, preserved EF (63,5%) and no history of ischemic heart disease (= diabetic cardiomyopathy; DbCM), 7 subjects with T2D, reduced EF (26,9%) and ischemic heart disease (= diabetic heart failure; DbHF), and 15 non-diabetic individuals with normal EF (64,7%) serving as controls. Among 1168 proteins identified by LC-MS/MS, 146 proteins were differentially regulated in DbHF, but only 66 in DbCM. Pathway analysis revealed downregulation of energy metabolic proteins, but upregulation of proteins involved in oxidative stress and inflammatory response. In DbCM, pathways of structural remodeling, cardiomyocyte proliferation, and mechanotransduction were upregulated. Bulk RNA sequencing revealed 1795 differentially regulated genes in DbHF, and 527 in DbCM, with only 128 genes being commonly regulated. DbHF, but not DbCM, could be clearly discriminated from controls by hierarchical clustering. While inflammation/immunity were major regulated pathways in DbHF, extracellular matrix remodeling and cellular growth were the most regulated pathways in DbCM. Thus, the differential regulation of biological pathways in DbCM versus DbHF suggests the existence of two distinct disease entities rather than DbHF being an advanced disease stage of DbCM.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Diabetes Society
A transgenic mouse model mimicking human cardiac amyloidosis
Authors: Fauvel C , Mulder P , Heron C , Nicol L , Brakenhielm E , Bellien J , Kalopissis A , Bauer F
Published: October 2022

Background

Heart failure (HF) pathophysiology complicating cardiac amyloidosis (CA) is poorly explored due to the lack of relevant animal model. A recently transgenic mouse expressing an amyloidogenic variant of human apolipoprotein AII was developed that showed ubiquitous amyloid deposit but limited data on cardiac involvement.

Purpose

To investigate heart structure and function in transgenic mice expressing an amyloidogenic variant of human apolipoprotein AII

Methods

Seventy-nine mice ageing 2 to 3 months were included in this study as follow: amyloidosis group (n=44) and sham (i.e. genetic mutation without phenotypic expression, n=35). Both were serially imaged by echocardiography (Vevo3100 Fujifilm) and cardiac magnetic resonance imaging (CMR, Biospec 4.7 tesla), and invasively explored by left–sided catheterization (pressure-volume loop [PV loop], Millar Catheterization), before sacrifice and histological investigations.

Results

As soon as 2–3 months of age, the amyloidosis group demonstrated significant left ventricular (LV) hypertrophy, diastolic dysfunction and left atrial dilatation compared with sham group (p<0.01). Left ventricular ejection fraction was initially normal in both groups but deteriorated in amyloidosis mice (p<0.001) before right ventricular function collapses (p<0.001). In amyloidosis group, PV loops showed significant LV end-diastolic pressure increase (p<0.001), stiffer LV (p<0.01) and reduced systolic function LV elastance (p<0.05). Invasive and non-invasive abnormalities paralleled severe CA deposits and subendocardial fibrosis matrix remodeling, both labeling by Congo red and Red Sirius coloration (p<0.001). CMR analysis showed significant T1 (p<0.05) and T2 (p<0.01) signal increase and significant decrease in myocardial perfusion (p<0.01) in the amyloidosis group. Clinically, mice with amyloidosis covered less distance during exercise test (p<0.001) and died earlier (log-rank test, p<0.01).

Conclusion

By mimicking human cardiac amyloidosis, the model of transgenic mouse expressing an amyloidogenic variant of human apolipoprotein AII is promising to investigate the underlying pathophysiology of heart failure due to amyloidosis.

Funding Acknowledgement

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Pfizer grant
Induction of periodontitis and oral microbiome dysbiosis in a mouse model with heart failure with preserved ejection fraction results in worsening hypertension and diastolic dysfunction
Authors: Da'na S , Abedat S , Nachman D , Rokach Y , Qadan A , Karram S , Tzach-Nahman R , Beeri R , Amir O , Houri-Haddad Y , Asleh R
Published: October 2022

Introduction

Heart Failure with Preserved Ejection Fraction (HFpEF) is characterized by increased inflammation and disruption of the nitric oxide (NO) pathway. Oral microbiota has an essential role in the generation of a large portion of the NO bioavailability pool in the human body. Periodontitis (PD) is a common inflammatory condition that contributes to the development of chronic low-grade inflammation, oral microbiota dysbiosis, and dysregulation of the NO hemostasis.

Purpose

In this study, we sought to investigate the direct effect of PD induction on HFpEF manifestation in a mouse model.

Methods

HFpEF was induced in 11-week-old C57/black male mice by high-fat diet and inhibition of NO synthase using L-NAME (Nω-nitro-l-arginine-methyl-ester) (a “2-hit-model”) for 10 weeks. PD was induced by oral infection with P. gingivalis. Diastolic function of the left ventricle (LV) was assessed by high-frequency echocardiography. Blood pressure (BP) was measured using CODA non-invasive tail-cuff system.

Results

Induction of PD resulted in a significantly impaired diastolic function demonstrated by more pronounced decrease in e' and increase in E/e' ratio compared to HFpEF without PD or control mice (E/e': 39.7±2.6 vs. 30.1±1.9 vs. 24.3±2.1 for PD-HFpEF [n=15], HFpEF [n=15], and controls [n=10], respectively, p<0.001). While LV ejection fraction (EF) was similar, global longitudinal strain (GLS) was decreased in the HFpEF group and further decreased in the PD-HFpEF group compared to controls (p<0.001). BP was elevated in the HFpEF mice and PD induction resulted in a more remarkable increase in BP (systolic BP: 124.7±3.3 vs. 112.7±3.8 vs. 94.8±2.2 mmHg, respectively, p<0.001).

Conclusions

Induction of PD in a mouse model with HFpEF results in a more pronounced BP elevation and diastolic dysfunction compared to HFpEF without PD. Extensive molecular experiments are ongoing to explore the mechanisms responsible for the increased HFpEF severity in the setting of PD.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): Israel Science Foundation
Human, mouse scRNA-seq and flow cytometry data in ischemic heart failure models predict immune cell activation signatures with altered endothelial cell cross talk
Authors: Merten M , Rasper T , Cremer S , Schuhmacher B , Tombor L , Zeiher A M , Hoffmann J , Abplanalp W T , Dimmeler S
Published: October 2022

Chronic ischemic Heart Failure (HF) with reduced ejection fraction is marked by adverse remodelling and sustained inflammation. The effects of monocytes and T cells in early state immune responses after myocardial infarction are known, to date their role in progression and maintenance of chronic HF remains elusive.

Single cell RNA sequencing of peripheral immune cells from healthy and HF donors revealed a significant increase of the antigen presentation (i.e. HLA-DRB5) and co-stimulatory molecules (i.e. ICAM-1) on monocytes, while the elevation of the T effector memory population in HF patients shows enhanced T cell activation. Subsequent flow cytometry validation in 55 healthy and 110 HF patients confirmed that HF derived monocytes have elevated levels of HLA-DRB5 predicts increased ICAM-1 and TREM-1 (p<0.0001 for both), indicating enhanced T cell activation capacity. In addition, the activated fraction of both CD4+ and CD8+ T cells, including central memory, effector memory and TEMRA cells, is significantly enriched in the HF patients (p<0.0001 for both) with reduction of the naïve T cells. T cell receptor (TCR) sequencing of age matched healthy and HF donors showed an increase in the relative TCR clonality in HF patients, indicating an expansion of the circulating T cells.

ScRNA-seq murine myocardial infarction (MI) time course data (day (d) 0/1/3/7/14/28/48 post-MI), found a progressive increase in Th17 cells and activated CD8+ T cells in the cardiac tissue at d48 (2.8x-, and 2.7x-fold, respectively) relative to d0. The T cell imbalance was marked by loss of immunosuppressive markers such as Lef1 (p=2e-30) in T cell clusters primarily populated by d48 relative to d0.

Mechanistically the downstream effects of the immune cell activation were analysed in the serum of HF patients, where increased levels of sICAM-1, IL-6 and TNFRI levels (1.75x-, 5.30x-, 2.63x-fold, respectively) could be detected. Furthermore, treatment of endothelial cells with the conditioning media from HF-immune cells induced capability to adhere with monocytes in a co-culture system but also decreased the checkpoint inhibitor molecule PD-L1, a known blocker of autoreactive T cells.

These data link HF with enhanced monocyte and T cell activation as well as T cell clonal expansion, along with increased T cell numbers in mouse hearts post-MI. These data suggest HF is may drive impaired resolution of inflammation in cardiac tissue and facilitate adverse remodelling and cardiac damage. Further studies will assess the specific role of chronic monocyte and T cell activation in the progression of heart failure.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG -Deutsche ForschungsgemeinschaftDZHK -Deutsches Zentrum für Herzkreislauferkrankungen
Desmoglein 2 mutant mice reproduce arrhythmogenic right ventricular cardiomyopathy patients' phenotype
Authors: Zankov D , Ohno S
Published: October 2022

Background

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is inherited cardiac disease with unresolved treatment. ARVC is progressive and leads to lethal arrhythmias and terminal heart failure. Most often encountered ARVC mutations are in desmosomal genes [1].

Purpose

We aimed to generate transgenic mice with exact copy of genetic defects found in Japanese cohort of ARVC patients, describe the phenotype, and identify target for curative therapy.

Methods

CRISPR/Cas9 genome editing was used to generate knock-in mice with the two most common point mutations in Japanese ARVC patients: DSG2 292R>C and 494D>A [2] (mouse equivalent positions are 297R and 499D, respectively). To analyze the phenotype of mice we used imaging techniques - cardiac echography and MRI as well as telemetry, treadmill, immunohistochemistry, confocal microscopy, histology, Western blot.

Results

In vivo observations of transgenic mice demonstrated inequalities in phenotypical presentation between the two mutations. Some of 297C mice (both hetero- and homozygous) died suddenly, starting from the age of 9 weeks, in contrast to surviving all 499A carriers. Dissection of suddenly died mice reveals enlarged cardiac cavities, mainly in the right heart. In addition, 297C homozygous hearts present with pale zones scattered all over the heart. Paraffin sections from this hearts stained with hematoxilin/eosin and Masson's trichrome show myocardial areas with absent myocytes, collagen accumulation and calcifications. With exception of homozygous 297C mice, spontaneous development of cardiac phenotype in Dsg2 knock-in mice starts after 25th weeks of age. Both mutant mice gradually developed cardiac dysfunction (echography and MRI, Fig. 1) and echographically visible left ventricular wall infiltrations. Heterozygous 297C mutation produces more severe phenotype that develops earlier. Similar to human ARVC, the degree of cardiac damage vary significantly. It is known from the human ARVC that physical activity aggravates the ARVC phenotype and may cause sudden cardiac death. To evaluate the effect of physical stress on the phenotype of Dsg2 knock-in mice we subjected 11 weeks old animals on treadmill exercise. Training for 8 weeks provoked development of heart failure in both 297C and 499A mutant mice, significantly earlier than natural progress of the phenotype. No mouse died suddenly. Telemetry experiment demonstrated electrical instability in 297C homozygous hearts showing conduction and rhythm abnormalities (Fig. 2). Apoptosis was detected in both mutant mice by TUNEL staining and confocal microscopy. Dsg2 protein expression was not affected by the mutations.

Conclusion

We generated mouse model of ARVC that reproduces exact genetic defect of the human disease. There are significant similarities between our model and ARVC patients' phenotype.

Funding Acknowledgement

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japanese KAKENHI funding
Divergent electrophysiologic safety profile of dapagliflozin and empagliflozin in a whole-heart model
Authors: Frommeyer G , Uphoff J , Wolfes J , Eckardt L , Ellermann C
Published: October 2022

Background

The SGLT2-inhibitors dapagliflozin and empagliflozin have emerged as standard therapy for the treatment of heart failure with reduced ejection fraction. However, experimental data on the electrophysiologic safety is sparse.

Purpose

Aim of this study was to characterize the safety profile of the SGLT2 inhibitors dapagliflozin and empagliflozin in a sensitive whole-heart model.

Methods

26 rabbit hearts of New Zealand white rabbits were excised and mounted to a Langendorff-apparatus. Hearts were divided up into two groups. The first group (n=12) was perfused with dapagliflozin in ascending concentrations (3, 5 and 10 μM). Further 14 hearts were treated with empagliflozin (1, 3 and 5 μM). Ventricular vulnerability was assessed by a predefined pacing protocol consisting of premature extra stimuli and burst stimulation.

Results

Dapagliflozin led to a significant reduction of the QT interval (3 μM: −27 ms, p<0.01; 5 μM: −47 ms, p<0.01; 10 μM: - 42 ms, p<0.01), action potential duration (APD90; 3 μM: −21 ms, p<0.01; 5 μM: −37 ms, p<0.01; 10 μM: −24 ms, p<0.01) and effective refractory periods (ERP; 3 μM: −34 ms, p<0.01.; 5 μM: −49 ms, p<0.01; 10 μM: −52 ms, p<0.01). Spatial dispersion of repolarization was not increased after dapagliflozin treatment (3 μM: −4 ms, p=ns; 5 μM: −4 ms, p<0.05; 10 μM: −8 ms, p=ns). Empagliflozin also led to a pronounced abbreviation of cardiac repolarization duration (QT: 1 μM: −28 ms, p<0.01.; 3 μM: −47 ms, p<0.01; 5 μM: −69 ms, p<0.01; APD90: 1 μM: −27 ms, p<0.01; 3 μM: −46 ms, p<0.01; 5 μM: −51 ms, p<0.01) and ERP (1 μM: −28 ms, p<0.05; 3 μM: −40 ms, p<0.01; 5 μM: −56 ms, p<0.01). In contrast to dapagliflozin, empagliflozin amplified spatial dispersion of repolarization (1 μM: −3 ms, p=ns.; 3 μM: +4 ms, p<0.05; 5 μM: +9 ms, p=ns). Dapagliflozin infusion did not provoke more ventricular arrhythmias (VA) compared to baseline conditions (baseline: 2 episodes; 3 μM: 6 episodes, p=ns.; 5 μM: 11 episodes, p=ns; 10 μM: 18 episodes, p=ns). In contrast, empagliflozin treatment significantly increased the occurrence of VA (baseline: 4 episodes; 1 μM: 12 episodes, p<0.05.; 3 μM: 8 episodes, p=ns; 5 μM: 29 episodes, p<0.05). Of note, none of the drugs provoked arrhythmias of the torsade de pointes type after lowering the potassium concentration.

Conclusion

The SGLT2 inhibitors dapagliflozin and empagliflozin shorten cardiac repolarization and effective refractory periods. While spatial dispersion remains stable after administration of dapagliflozin, empagliflozin amplifies spatial dispersion of repolarization. As a result, significantly more ventricular arrhythmias were inducible after empagliflozin treatment.

Funding Acknowledgement

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Hans and Gertie Fischer-Foundation (to C.E.)
Disease-associated calmodulin mutations disrupt L-type Ca2+ channel (Cav1.2) activity and CaMKIIdelta phosphorylation in long QT syndrome
Authors: Helassa N , Prakash O , Gupta N , McCormick L F , Antonyuk S , Dart C
Published: October 2022

Introduction

Long QT Syndrome (LQTS) is a major inherited arrhythmia syndrome that can cause sudden cardiac death. Using genome sequencing in human patients, mutations in the ubiquitous calcium (Ca2+) sensor protein calmodulin (CaM) have been associated to LQTS. CaM is an ion channel regulator and can modulate the activity of the voltage-gated calcium channel (Cav1.2) and Ca2+/CaM-dependent protein kinase II (CaMKIIδ), involved in cardiac muscle contraction. However the molecular mechanism by which CaM mutations contribute to irregular heartbeats remains unclear.

Methods

Interaction of CaM proteins with Cav1.2 and CaMKIId synthetic peptides (Cav1.2-NSCaTE51–68, Cav1.2-IQ1665–1685, Cav1.2-C1627–1652, CaMKIIδ294–315,) was investigated using Isothermal Titration Calorimetry (ITC) and X-ray crystallography. Whole-cell patch clamp electrophysiology was used to determine the effect of CaM mutations on L-type Ca2+ currents and Ca2+-dependent inactivation (CDI). CaMKIIδ phosphorylation activity was determined by western blot and fluorescence kinase assay.

Results

Binding affinity of CaMKIId and Cav1.2 peptides to the LQTS-associated CaM variants was significantly reduced, up to 7-fold. Interestingly, the Cav1.2-IQ1665–1685 peptide showed a stronger binding, up to 2-fold, towards LQTS-CaM mutants. Crystal structures of Ca2+-CaM:CaMKIId294–315 showed structural alterations induced by LQTS associated mutations. In addition, we demonstrated that CaMKIIδ autophosphorylation and kinase activity can be significantly reduced by LQTS-associated CaM mutants. Electrophysiological examination of Cav1.2 function revealed that CaM mutations significantly impaired channel CDI, without affecting the voltage dependence of activation and inactivation.

Conclusions

These data demonstrate a strong correlation between LQTS-associated CaM mutations and Cav1.2 activity. We provide molecular insights into the diverse factors contributing to CaM-mediated arrhythmias.

Funding Acknowledgement

Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship