bluuebunny/crossref_metadata_2025 huggingface dataset:
Simulation of Photovoltaic Water Pumping System in Sam Ratulangi University
Authors: Rumbayan Meita , Nelwan Adinda F. , Pang David , Kindangen Jefrey I. , Putro Muhamad Dwisnanto
Published: May 2024

The purpose of this study is to design and simulate a PV water pumping system at Sam Ratulangi University in Indonesia. The methods employed in this study consist of a literature review, design, and simulation using HOMER (Hybrid Optimization of Multiple Energy Resources) software. The comparison between PV water pumps and dieselpowered water pumps is analysed in terms of net present cost, cost of energy (COE), and emissions to meet the water requirements in the laboratory. The simulation results are based on two design scenarios for a solar photovoltaic water pumping system at a laboratory scale. In Scenario 1, the system has a daily load of 1.5 kWh/day, 0.52 kW PV modules, 4 batteries of 80 Ah each, and a 1 kW inverter. In Scenario 2, the system has a daily pump load of 2.5 kWh/day, 0.87 kW PV modules, 4 batteries of 80 Ah each, and a 1 kW inverter. The results indicate that the utilization of solar energy to power a PV water pumping system performs well and serves as a case study showcasing the use of solar energy. Further studies on the application of PV water pump should be implemented and tested to evaluate the system’s performance.
Enhancing Building Energy and Thermal Performance in Metro Manila, Philippines through Computational Fluid Dynamics-Assisted Analysis and Optimization
Authors: Mina Sherwin U. , Tayactac Reylina Garcia
Published: May 2024

This study presents a detailed investigation of the energy and thermal performance of buildings in the Philippines’ metropolitan cities, targeting sustainability and indoor comfort against the backdrop of the country’s tropical maritime climate. Utilizing a blend of occupant surveys, energy audits, climate data analysis, and building envelope assessments, the research employs Computational Fluid Dynamics (CFD) simulations to accurately evaluate energy use and thermal behaviors in residential and commercial structures. Key findings include distinct nighttime energy consumption patterns in residential buildings and the identification of climatic challenges such as high temperatures and humidity. The study proposes effective design interventions like enhanced ventilation, shading solutions, and the integration of Phase Change Materials (PCMs) for improved thermal efficiency. Significantly, the research contributes to policy development by suggesting updates in building energy codes and advocating for energy-efficient practices. Overall, the study provides actionable insights for enhancing building sustainability and occupant comfort in urban Philippine settings.
Ethnic diversity and divergent perceptions of climate change: a case study in Southwest China
Authors: Yang Hua , He Jun , Li Zhinong , Su Yufang , Xu Jianchu
Published: May 2024

Understanding divergent perceptions of ethnic groups to climate change in mountainous regions home to multi-ethnic cultures and the factors influencing these perceptions is crucial for policymakers to predict the trending impacts of climate change and make long-term decisions. Based on the case of Southwest China, 1216 households were interviewed by questionnaire surveys to gain insight into the perceptions of local people on the dynamic evolution characteristics of climate events in the uplands of Yunnan, China, which is an area home to rich ethnic diversity, and also to determine the factors that influence these perceptions. Results indicated that climate events have now become important events for farmers’ livelihoods, ranking only after family diseases and livestock diseases. Drought, long-term drought, and erratic rainfall are the three kinds of climatic events with the most significant increase in frequency and severity in mountainous areas. Farmers’ perceptions on whether drought, long-term drought, and erratic rainfall occurred 10 years ago as well as changes in frequency and severity are significantly influenced by characteristics of respondents, ethnic culture, geographical environment of farmer residences, farmland characteristics, and sources of livelihood. Ultimately, taking ethnic differences into consideration for long-term planning will be an important part of the local response to climate change in the future.
An encoding generative modeling approach to dimension reduction and covariate adjustment in causal inference with observational studies
Authors: Liu Qiao , Chen Zhongren , Wong Wing Hung
Published: May 2024

In this article, we develop CausalEGM, a deep learning framework for nonlinear dimension reduction and generative modeling of the dependency among covariate features affecting treatment and response. CausalEGM can be used for estimating causal effects in both binary and continuous treatment settings. By learning a bidirectional transformation between the high-dimensional covariate space and a low-dimensional latent space and then modeling the dependencies of different subsets of the latent variables on the treatment and response, CausalEGM can extract the latent covariate features that affect both treatment and response. By conditioning on these features, one can mitigate the confounding effect of the high dimensional covariate on the estimation of the causal relation between treatment and response. In a series of experiments, the proposed method is shown to achieve superior performance over existing methods in both binary and continuous treatment settings. The improvement is substantial when the sample size is large and the covariate is of high dimension. Finally, we established excess risk bounds and consistency results for our method, and discuss how our approach is related to and improves upon other dimension reduction approaches in causal inference.
Tourism as a “Tecnologia Social”: The Case of Ururay Group
Authors: Freitas João A. , Duarte Maurício Dias
Published: May 2024

The notion of social technology introduces a novel paradigm for technological advancement that emphasizes inclusivity, participation, and democracy. It is rooted in the idea of local citizens as empowered agents. Within this framework, we spotlight the initiatives of Grupo Ururay, a collective based in the East Zone of São Paulo. Through cultural, touristic, and educational endeavors, they have illuminated the pivotal role played by Black and Indigenous communities in shaping the city’s history. The crux of our argument lies in portraying the collective’s utilization of tourism as a social technology, effectively employing it as a catalyst for societal transformation. To substantiate this claim, we will delineate some of the group’s initiatives and delve into the significance of revitalizing and celebrating counter-hegemonic histories within São Paulo’s urban landscape.

Information

© The Authors 2024
Buenos Aires Youth Tourism Access Policy
Authors: Tapia Silvia A. , Catalano Bárbara , Xaus Maria
Published: May 2024

In Argentina, in the south of Latin America, student tourism has been associated with trips for young people who finish secondary school level. Such trips have a mountain and snow destination during the winter season and are organised privately between travel agencies and student groups, without the intervention of schools or state aid. Although sustained for decades, given their high costs, access to this type of tourism has become increasingly associated with upper-middle socio-economic sectors. During the COVID-19 pandemic, in 2021, the government of Buenos Aires, one of the largest and most populous provinces of Argentina, established the programme “End of Term Trip”, a local public policy of student tourism aimed at secondary school students of that province. Through a free trip package, it links the aims of universality and equality with a tourism policy that promotes new destinations, favours the work of travel agencies, and boosts off-season tourism. Based on documented data and an interview with a programme official this case study presents a tourism policy that seeks to boost the tourism sector and, at the same time, encourages the participation of young people from different social sectors in recreational tourism practices and their access to leisure and tourism rights.

Information

© The Authors 2024
Stage specific effects of insulin and Wnt on cell fate decisions in human induced pluripotent stem cell-derived cardiomyocytes
Authors: Yuan Q , Dinani R , Kuster D W D , Boon R A , Van Der Velden J , Buikema J W
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation

Background

Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes.

Purpose

This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs.

Methods

We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit.

Results

Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs.

Conclusions

Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.Graphical Summary
Phosphoinositide 3-Kinase C2alpha controls cardiac contractility through regulation of beta2-adrenergic receptor recycling
Authors: Cnudde S , Brand T , Fender J , Prever L , Murabito A , Russo M , Gulluni F , Lorenz K , Hirsch E , Ghigo A
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral fellowship

Introduction

Phosphoinositide 3-kinase C2α (PI3KC2α) is a ubiquitously expressed class II PI3K isoform, which has been previously shown to be involved in the control of vesicular trafficking. However, its role in the myocardium has not been investigated. The primary aim of this study is to elucidate the mechanism by which mechanism PI3KC2α controls cardiac pathophysiology.

Methods

Cardiomyocyte-specific PI3KC2α knockout (PI3KC2α KO) animals were generated by crossing mice expressing a tamoxifen-inducible Cre recombinase under the control of the αMHC promoter with PI3KC2αflox/flox mice. Zebrafish embryos were injected with a PI3KC2α (PI3KC2α morphants) or a control morpholino (control morphants) at 1-cell stage. HEK293 with stable overexpression of GFP-tagged β2-AR were generated and transfected with either scramble or PI3KC2α siRNAs.

Results

Our findings indicate that PI3KC2α KO mice display a reduced cardiac contractility compared to wildtype animals. Similarly, PI3KC2α morphants zebrafish exhibit lower heart rate and fractional shortening compared to controls, both at baseline and after isoproterenol (ISO) stimulation. A similar unresponsiveness to ISO was found in vivo in PI3KC2α KO mice where chronic treatment with the β-adrenergic receptor (β-AR) agonist failed to induce the classical β-AR-mediated remodeling, characterized by an increase of the left ventricular mass and of cardiomyocyte area. These findings suggest that PI3KC2α plays a critical role in the regulation of β-AR signaling. In agreement, cAMP levels failed to increase in response to ISO treatment in PI3KC2α morphants. Furthermore, silencing of PI3KC2α in HEK293-GFP-β2-AR cells resulted in an increased GFP-β2-AR internalization compared to control cells, both at baseline and after ISO stimulation. Interestingly, preliminary findings indicate that overexpression of constitutively active Rab11Q70L induced a redistribution of GFP-β2-AR at the plasma membrane in PI3KC2α-silenced cells.

Conclusion

Overall, we identify a key role for PI3KC2α in the control of cardiac contractility through the regulation of β2-AR trafficking to the plasma membrane through a Rab11-dependent mechanism.
Mitochondrial players as novel therapeutic options in arrhythmogenic cardiomyopathy
Authors: Volani C , Medici A , Philippe R , Lavdas A , Della Corte I , Lang M , Frommelt L S , Blumer M , Sommariva E , Pompilio G , Troppmair J , Rossini A
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Joint Project Südtirol- FWF (grant number 23623; Italy-Austria) and the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy)

Background

Mitochondria are dynamic organelles that sense and respond to environmental, nutritional, and pathological changes. These organelles can smartly adapt to different stressful conditions, by changing the way they network or the energy sources they use. Over the past years, numerous disorders have been associated with mitochondrial dysfunction, including cardiomyopathies. Arrhythmogenic Cardiomyopathy (ACM) is a cardiac disease characterized by the progressive replacement of the myocardium with fibro-fatty tissue. The current treatment primarily focuses on managing the symptoms rather than addressing the underlying issue of tissue substitution. Purpose: Therefore, in this study, we explored the mitochondrial and metabolic phenotype in ACM and tested the efficacy of a mitochondria-targeting library to rescue the fibro-fatty phenotype in ACM Methods: Human primary cardiac stromal cells (CStCs) were cultured in adipogenic medium (AM), a condition used to model the ACM fibro-fatty phenotype. Mitochondria-related analyses of ultrastructure, membrane potential, network connection, respiratory capacity, ROS production and redox system were performed at day (d) d0, d3, d7, d15 of AM exposure. A mitochondria-targeting library (MedChem Express) was tested on ACM-CStCs exposed to AM for 7 days. The extent of lipid accumulation was determined using the neutral lipid staining dye BODIPY 493/503, cell numbers were measured using Hoechst counterstaining, and images acquired using a high-content imaging system Results: At d0, the mitochondrial network was more fragmented in ACM compared to healthy CStCs (CTR). This result was in line with the observation of a decrease in mitochondrial respiratory capacity and an increase in ROS production. In addition, AM exposure led to faster and higher lipid accumulation in ACM CStCs, which was associated with higher mitochondrial oxygen consumption rates observed in ACM CStCs after 7 days of AM exposure. When ACM CStCs were simultaneously exposed to AM and 407 mitochondrial-directed compounds, about 25% of the compounds demonstrated the ability to reduce lipid accumulation Conclusion: ACM CStCs demonstrate an increased risk for mitochondrial network impairment. Moreover, prolonged exposure of ACM CStCs cells to AM negatively affects lipid deposition as well as mitochondrial and metabolic parameters. This highlights the potential for using specific mitochondria-targeting compounds to reduce lipid accumulation.
Caveolin-dependent endocytosis regulates arterial calcification
Authors: Gorgels A , Simon J , Hense N , Marx N , Goettsch C
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB219

Introduction

Patients with chronic kidney disease (CKD) have a significantly elevated cardiovascular risk, which is primarily due to premature vascular aging with arterial calcification - a substantial public health burden. Calcifying extracellular vesicles (EVs) are required for the onset and progression of calcification. Endocytosis and exocytosis are important for EV genesis and cargo loading, thus controlling EV release and propensities.

Purpose

We aim to investigate endocytosis, endosomal ripening, and exocytosis as critical mechanisms of EV-dependent mineralization.

Methods and Results

First, we characterized EVs from calcifying vascular smooth muscle cells (SMCs) compared to control SMCs. Western blot analysis revealed an increase in tissue nonspecific alkaline phosphatase (TNAP) among calcifying EVs but not in flotillin abundance. When compared to control EVs, calcifying EVs showed higher TNAP activity. Flow cytometry and a turbidity assay were then used to evaluate EV mineral cargo and aggregation potential. Compared to the control, EVs produced from SMCs cultivated under calcifying conditions had 5-fold more mineral-positive EVs (p=0.004) and a 3-fold higher nucleation potential (p<0.001). Next, endocytosis was blocked by targeting Dynamin-1 and –2, which inhibited caveolin- and clathrin-dependent endocytosis (Dynasore hydrate), or targeting clathrin-terminal domain, which inhibited solely clathrin-dependent endocytosis (Pitstop 2). Dynasore reduced matrix mineralization dose-dependently and lowered mineral-positive EVs by 18%, whereas Pitstop 2 had no effect, suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, Cav-1 silencing inhibited matrix mineralization and Cav-1 protein was more abundant in calcifying EVs than in control EVs. Dynasore and Pitstop-2 did not affect the EV number measured by nanoparticle tracking analysis. Inhibition of endosomal ripening by Rab7 inhibition did not affect SMC calcification. Exocytosis was inhibited by Rab27a inhibition, which reduced mineral deposition (-32%, p=0.017) without affecting EV mineral or EV quantity.

Conclusion

EVs released from calcifying SMCs showed a higher abundance of mineral and TNAP proteins, suggesting a role in mineral transport and deposition. Our data suggest that caveolin-dependent endocytosis is essential in EV-mediated SMC calcification. Mineral cargo in EVs is independent of Rab7 and Rab27a pathways.
HiPSC-derived cardiomyocyte cultivation and measurement of electrophysiological properties on gelatine/glucose/polypyrrole scaffold
Authors: Vaiciuleviciute R , Alaburda A , Shelest A , Elkhoury K , Maziz A , Bergaud C , Schubert M , Guan K , Uzieliene I , Bernotiene E
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EU Horizon 2020 Framework Programme (H2020) under Grant agreements No. 953138 (EMAPS-Cardio)

Purpose

Cardiotoxicity is one of the main side effects limiting development of new medicines and resulting in withdrawal of drugs from the market. There are different mechanisms of drug-induced cardiotoxicity, including arrhythmia, disruption of mitochondria function, apoptosis, altered growth factor signalling [1]. Heart on a chip model for drug testing seems attractive approach to evaluate the possible cardiotoxic effects on cell biological, but mostly electrophysiological properties. Our aim was to create a 3D system with a biocompatible scaffold, coated with a conductive layer, suitable for culturing and maturation of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) and compatible with electrophysiology parameter measurement techniques.

Methods

Elastic scaffold was fabricated by electrospinning of gelatin-glucose microfibers followed by the vapor-phase and electrochemical deposition of conductive polymer polypyrrole (PPy) to create electroconductive layer. hiPSC-CMs at different maturation levels (at 6 vs 14 weeks) were used to test biocompatibility of the scaffold. To increase cell attachment, scaffold was treated with 0.1 M HCl and coated with Geltrex. Cells were stained with fluorescent dyes (Calcein AM, DAPI). Electrophysiological properties were analysed using intracellular calcium imaging (Cal520) and action potential recordings with sharp electrodes. HiPSC-CMs on coverslips were used as a 2D control.

Results

Calcein AM staining showed that high number of hiPSC-CMs attached to gelatine/glucose/PPy scaffold proving that the scaffold is biocompatible and cells remain viable even after long term incubation (> 2 weeks). HiPSC-CMs at lower maturity (6 weeks) had better attachment properties to it, indicating that more mature cardiomyocytes (14 weeks) gradually lose their capacity to attach to PPy surface. Intracellular calcium imaging showed that both types of cells generated synchronous spontaneous calcium oscillations on gelatine/glucose/PPy scaffolds. Action potential recordings in 3D were similar to those in 2D, and confirmed differences in hiPSC-CMs maturation levels – at lower maturation level hiPSC-CMs had lower upstroke velocity as compared to more matured hiPSC-CMs.

Conclusions

Gelatine/glucose/PPy scaffold is biocompatible for hiPSC-CMs cultivation, and suitable for evaluation of cardiomyocyte electrophysiological properties. Such electromechanoactive scaffold can be further employed for development of heart-on-a-chip model.
FgF23 and soluble klotho effects on the myocardium: an ex vivo study
Authors: Sen P , D Alessio C , Hamers J , Sun Z , Dendorfer A , Merkus D
Published: May 2024

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): German Center for Cardiovascular research

Background

Fibroblast Growth Factor 23 (FGF23) and soluble Klotho (sKL) play a critical role in mineral homeostasis in the body by acting on the kidney to regulate phosphate levels. Circulating FGF23 levels rise in patients with kidney disease and dysregulation of the FGF23 –sKL- axis is a common phenomenon in uremic cardiomyopathy. Studies have shown that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4), while sKL mitigates these effects and may also exert an independent protective effect on the heart(1,2).

Purpose

We aimed to study the effects of FGF23 and sKL on myocardial contractility, calcium handling and molecular signaling pathways in the human heart using an ex vivo model of cultured contracting myocardial slices.

Methods

We obtained human heart slices from explanted hearts from patients undergoing cardiac transplantation at the LMU Klinikum Munich, Germany. The 300 µm thick slices were cultured under continuous stimulation (1 Hz, 3 ms pulse width) with 1 mN preload, and contractile function were recorded continuously. Slices with vehicle, recombinant FGF23 protein (100ug/ml), recombinant sKL protein (200ug/ml) and both FGF23((100ug/ml)+sKL (200ug/ml) were continuously monitored over 10 days. We checked for mRNA expression using RT PCR.

Results

Treatment of human left ventricular tissue with recombinant FGF23 protein resulted in enhanced mRNA expression of the FGFR4. sKL treatment alone induced downregulation of the FGFR4 gene and mitigated upregulation of FGFR4 in FGF23+ sKL treated slices. Post pause potentiation (PPP), assessed by pausing the stimulation of the slices for 120s and then measuring the relative height of the first beat after resumption of pacing, is an indirect measure of sarcoplasmic reticulum Ca2+ handling capacity. PPP was reduced in FGF23 treated slices and heightened in the slices with sKL compared to vehicle treated ones, indicating that FGF23 and sKL play a role in intracellular Ca2+ handling.

Conclusions

sKL and FGF23 have opposite effects on human cardiac muscle with sKL mitigating FGF23-induced upregulation of FGFR4. Furthermore, sKL improves, while FGF23 impairs myocardial calcium handling . As altered calcium handling may also be involved in hypertrophic signaling altered calcium handling may provide a means by which FGF23 promotes and sKL inhibits myocardial hypertrophy. sKL is advocated as a promising candidate in exploring it therapeutic role in FGF23 mediated cardiomyopathy. We intend to advance our understanding of sKL-FGF23signaling mechanisms by combining transcriptomic analysis with functional data in this model of myocardial slice culture.
Novel method for induction of myocardial infarction in rats by ultrasound guided electrocoagulation of the left anterior descending artery
Authors: Nordfalk M , Groenager S S , Flethoej M , Hunter I , Ripa R S , Kjaer A , Pedersen P J
Published: May 2024

Funding Acknowledgements

Type of funding sources: Private company. Main funding source(s): Minerva Imaging

Background

Murine models of acute myocardial infarction (MI) are commonly established through ligation of the left anterior descending artery (LAD). Despite inherent procedural challenges, including high mortality rates, limited improvements of the induction procedure have been made. Ultrasound-guided electrocoagulation of the LAD could be advantageous by improving animal welfare and decreasing procedural mortality.

Purpose

To investigate the feasibility of MI induction by ultrasound guided electrocoagulation in rats in comparison to traditional LAD ligation.

Methods

Thirty-eight female Sprague Dawley rats were randomized to MI by either of the two induction methods leading to 19 rats in each group. Infarct size and left ventricular ejection fraction (LVEF) were evaluated using positron emission tomography (PET) with [18F]fluorodeoxyglucose ([18F]FDG) 9 days post-MI. Myocardial infarct size was categorized as non-infarcted, mild, moderate, or severe based on [18F]FDG uptake. Animal welfare was assessed twice daily for the initial 48h post-surgery using a behavioural scoring system.

Results

There was no significant difference in the mortality rate of rats with MI induced by electrocoagulation compared to LAD ligation (16% vs 37%, P=0.27). Among the surviving animals both methods produced an equal number of myocardial infarcts (n=5). Electrocoagulation resulted in 1 mild, 2 moderate, and 2 severe MIs, while LAD ligation produced 1 mild, 0 moderate, and 4 severe MIs. Infarct size was similar in the electrocoagulation group compared to the LAD ligation group (mean±SD electrocoagulation: 15±5% vs ligation: 28±13%, P=0.13). The number of non-infarcted surviving rats in the electrocoagulation group was 11 compared to 7 in the LAD ligation group (P=0.33). The LVEF of infarcted rats showed no significant difference between groups (electrocoagulation: 47±8% vs ligation: 38±10%, P=0.14). The LVEF for non-infarcted rats was 53±5%. Post-surgical animal welfare appeared to be improved in the electrocoagulation group, however, the accumulated behavioural score did not reveal a significant difference (median [interquartile range] electrocoagulation: 0 [0-1] vs ligation 2 [0-2], P=0.19).

Conclusion

Induction of myocardial infarction by ultrasound-guided electrocoagulation is feasible in rats and should be considered as an alternative to LAD ligation. Further development is needed to improve the rate of success and consistency in infarct size.
Role of Orai1 and Ca2+-stimulated adenylyl cyclase 8 in the adverse cardiac remodeling after myocardial infarction
Authors: Falcon Boyano D , Calderon Sanchez E , Martin-Bornez M , Rosado J A , Ordonez A , Smani T
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación.

Background

It is well established that abnormalities in the regulation of the intracellular concentration of Ca2+ ([Ca2+]i) occurs in diseased heart such as under acute myocardial infarction (AMI). Recent studies have demonstrated that proteins related to the Store Operated Calcium Entry (SOCE), such as Orai1/2/3 and TRPC channels, play a role in the adverse cardiac remodelling. Previously, we demonstrated that the expression of Orai1 increased in heart subjected to ischemia and reperfusion (I/R) syndrome. However, the signalling pathway implicated in Orai1 regulation has not been addressed.

Objective

The aim of this study was to examine the molecular mechanism and signalling pathways involved in the of Orai1 upregulation after I/R in cardiomyocytes.

Materials and Methods

The experiments were performed in adult (ARVM) and neonatal rat ventricular cardiomyocytes (NRVM), in a rat model of myocardial I/R and in ventricular biopsies of patients with ischemic heart failure. Ca2+ studies were realized using microfluorimetric technique in FURA-2AM loaded cardiomyocytes. Immunofluorescence was used to investigate CREB activation using a specific phospho-CREB antibody and Orai1 and AC8 protein expression were analysed by Western Blot. This study was performed in accordance with the recommendations of the Royal Decree 53/2013 in agreement to the Directive 2010/63/EU of the European Parliament and approved by the local Ethics Committee on human Research and the Animal Research Committee.

Results

We found that under I/R diastolic level of [Ca2+]i was significantly increased in cardiac myocytes isolated from I/R hearts, which was inhibited by SOCE blockers. Moreover, we found that Orai1 and Ca2+-sensitive adenylyl cyclase 8 (AC8) are overexpressed in the heart isolated from I/R rat model, as compared to sham. Similarly, we found significant increase in the expression of Orai1 and AC8 in ventricle biopsies of post-infarction patients with HF. We also observed that I/R stimulated cAMP production, involving Orai1 and AC8 activation in rats, indicating that Ca2+ entry through Orai1 stimulated AC8. Moreover, I/R phosphorylated cAMP response element-binding protein (CREB) through PKA activation, but not via EPAC2 or ERK1/2. Interestingly, we found that CREB facilitated I/R-induced Orai1 upregulation and related exacerbated SOCE by its activation of Orai1 promoter under I/R. Finally, the intramyocardial administration of AAV9 holding AC8 shRNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i.

Conclusions

Our results show that the activation of Orai1/AC8/CREB axis plays a key role in the I/R-induced Orai1 upregulation, which participated in diastolic [Ca2+]i mishandling related to the adverse cardiac remodelling.
Long-range gene editing of LMNA
Authors: Rooijakkers A A M B , Pinto Y M , Tijsen A J M
Published: May 2024

Funding Acknowledgements

Type of funding sources: Foundation. Main funding source(s): Hartstichting (Dutch Heart Foundation)

Background

Mutations in the LMNA gene commonly affect the cardiac muscle tissue, which leads to the development of a severe dilated cardiomyopathy with life-threatening arrhythmias. The lack of response to conventional heart failure therapy in these patients indicates the urgent need for new therapies. Gene editing has the potential to become a curative treatment for these patients by correcting the genetic cause of the disease. Prime editing is a promising new gene editing technology, which uses a prime editing guide RNA (pegRNA) to target a specific genetic locus and creates edits using a reverse-transcriptase enzyme linked to a Cas9-nickase.

Purpose

Conventional prime-editing technology targets one mutation per pegRNA. However, with more than 600 disease-causing mutations described in LMNA, the feasibility of applying this technology to each mutation is limited. Therefore, we aim to target not a single, but a set of mutations with one single pegRNA.

Methods

The pegRNAs are optimised by selecting the optimal target regions, improving their stability and 3D structure. The in vitro editing efficiency is assessed using the fluoPEER reporter plasmid, transfected into HEK293T cells. Using flow cytometry, expression of mCherry fluorescent protein is detected following successful editing of the target mutation.

Results

Flow-cytometry analysis of treated cells revealed an editing efficiency of 12% with a long-range pegRNA targeting a full exon, compared to 15% with a conventional short pegRNA targeting the same mutation <10bp downstream of the Cas9 nick site. This editing efficiency using the same long-range pegRNA was retained at 9% when editing at a distant position >100bp downstream of the nick site. To increase the editing efficiency, we improved the stability of the pegRNAs by the addition of different 3’ structural motifs. This resulted in an increased editing efficiencies of 15% and 17% for nearby and distant mutations, respectively.

Conclusion

We show that it is possible to edit different mutations within a range of up to 120 bp with a single pegRNA. By optimisations of the pegRNA 3D structure and optimal selection of target locations we aim to further improve the editing efficiency of this system. This work could eventually provide a prime editing therapy able to cure disease in a subset of patients carrying different mutations in the same genetic region. For LMNA-associated diseases this would substantially reduce the number of pegRNAs that have to be developed to cover all mutations, greatly improving the feasibility of prime editing therapy.
Preoperative examination of inflammatory biomarkers in relation to the prognosis of postoperative complications after heart transplantation
Authors: Tarjanyi Z , Bernath D , Szappanos A , Teszak T , Kiraly A , Kugler S Z , Pallinger E , Nagy G Y , Sax B , Merkely B
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Public hospital(s). Main funding source(s): Semmelweis University

Heart transplantation (HTX) is the definitive therapy for end-stage heart failure. Although complications have drastically decreased with modern immunosuppressive therapy, surgical and anesthetic techniques, significant complications such as graft failure, acute rejection or infection must still be expected. The purpose of the study is to examine cytokine pathways linked to inflammation from direct preoperative serum samples, which can serve as prognostic factors in relation to septic events and mortality in the early postoperative period. The study was conducted using the blood samples of 102 patients who underwent HTX. Blood samples were taken from the patients immediately before HTX. The amounts of a total of 32 cytokines and related proteins were determined using LEGENDplex assays. After that, we correlated the biomarker amounts determined in the serum samples with the end points we indicated. In patients undergoing HTX who developed septic events during postoperative intensive care, the preoperative MMP2 serum concentration was significantly higher compared to other patients undergoing HTX (n=23, p=0.04). Independent predictors of mortality in the intensive care unit (n=5) were preoperative MRP8/14 (AUC=0.8), NGAL (AUC=0.75), IGFBP-4 (AUC=0.73), cystatin C (AUC=0.7) and the MMP2 (AUC=0.74) serum concentrations. Patients transplanted from left ventricular mechanical circulatory support (LVAD) (n=11) were examined as a separate group, where early postoperative mortality was significantly higher (n=4, p=0.02), the independent predictor of which was the concentration of IGFBP-4 (p=0.04 , AUC=0.73). Based on our results, it can be said that the differences in the preoperative concentration of certain inflammatory biomarkers that can be measured in the serum show correlations with the postoperative septic events and mortality of patients undergoing HTX.
Cardioprotective effects of Empagliflozin in cardiac volume overload-induced by aortocaval fistula in rats
Authors: Sykora M , Andelova K , Farkasova V , Honetschlagerova Z , Goncalvesova E , Tribulova N , Szeiffova Bacova B
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Slovak Research and Development Agency under the contract no. 21-0410

VEGA grants no. 2/0006/23

Heart failure is characterized by reduction in cardiac output and the occurrence of malignant arrhythmias, thereby contributing significantly to high morbidity and mortality worldwide. Myocardial structural remodelling is a key factor contributing to the development of these life-threatening events and belongs to unsolved clinical issues challenging to investigate in animal models. The aim of our study was to explore the impact of cardiac volume overload (VO) on structural remodelling and electrical coupling protein, connexin43 (Cx43) in the left and right heart ventricles as well as the efficacy of treatment with SGLT2i, Empagliflozin. Aortocaval fistula (ACF) was surgically induced in 6-month-old Wistar rats, to develop VO during 4 weeks, followed by oral administration of Empagliflozin 10 mg/kg/b.w. daily for 5 weeks. At the end of the experiment, biometric parameters were registered and left and right heart tissue samples were snap-frozen and kept at -80°C for western blot analysis and microscopic examination. Findings revealed an increase of heart, left and right ventricular mass due to ACF-VO versus sham controls, which was associated, with eccentric myocardial hypertrophy. Noteworthy, Empagliflozin reduced heart, left and right ventricular mass. Levels of myocardial protein Cx43, that determine electrical coupling among cardiomyocytes, were reduced by ACF-VO, while normalized by treatment with Empagliflozin. Moreover, treatment attenuated ACF-VO-induced increase of interstitial collagen visualized by van Gieson staining. It was in line with the protein expression of MMP-2, which is involved in structural remodeling. The protein levels of PKCε, concerned/associated with modulation of Cx43 and extracellular matrix, were decreased due to ACV-VO but normalized after treatment with Empagliflozin. On the other hand, protein levels of PKCδ, involved in proapoptotic and prohypertrophic pathways, were increased and normalized by Empagliflozin. The changes were more pronounced in the right ventricle compared to the left heart ventricle. In summary, our pilot results point out the benefit of Empagliflozin due to the suppression of myocardial structural remodelling and preservation of Cx43 in the rat ACF-VO model. Furthermore, this Empagliflozin-induced cardioprotection was associated with the normalization of Cx43, MMP-2, and PKC levels independent of glucose control. However, further analyses are needed to reveal the molecular mechanisms of the cardioprotective effects of Empagliflozin.
Unbiased clustering and molecular characterisation of novel metabolic phenotypes in a heart failure cohort
Authors: Esenkova E , Koeck T , Valentini G , Wild P , Casiraghi E , Araldi E
Published: May 2024

Funding Acknowledgements 

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DIASyM cluster

Heart Failure is a heterogeneous clinical syndrome characterized by the inability to pump enough blood and thus provide enough oxygen to the human body. The current classification of heart failure is based on the left ventricular ejection faction (LVEF). Although the classification clearly defines heart failure subtypes and stages, and clinical guidelines are available for their management, it is preferable to prevent the disorder or take control over it from its onset. Evidence suggests strong molecular connections between human metabolism and heart failure. Exploiting the unique and diverse molecular characteristics that drive heart failure could be an effective strategy for patient stratification, to identify new therapeutic targets and to personalize treatments.

The current research attempts to identify clinically relevant metabolic subgroups of individuals with the help of a similarity network-based approach, based on targeted proteomics data of a heart failure cohort. With Olink targeted proteomics technology, six protein abundance panels (547 distinct proteins) from 3063 subjects (1103 females) were obtained. Using nonlinear combination methods, the created networks were integrated into a single similarity network and then used for clustering with unbiased network-based approaches.

The results have distinguished the subgroups with worse outcome prediction, supported by significantly different clinical profiles. Surprisingly, the significant clinical parameters have shown the dominance of not only classical metabolic signatures (diabetes) but also parameters related to kidney and liver function. The differential protein abundance profiles were then compared among clusters to derive novel insights into clinical subtypes, and have highlighted a significant contribution of inflammatory proteins to the unbiased subtyping rationale.

The study attempted to improve previous patient stratification efforts, by having a large sample size with a broader variety of molecular variables. These results will pave the way to precision medicine, and will allow indirectly to integrate and/or adjust the analysis by adding clinically relevant social parameters such as lifestyle, parents’ anamnesis, demographics that are often left out due to their lower contribution at the pre-processing steps in comparison to more objective laboratory parameters. Previous studies have already clustered the heart failure population, based on various clinical and biochemical parameters. However, the clustering was guided by well-established metabolic parameters and thus, the results can be highly biased. The current project allows an unbiased, data-driven approach that holds more potential to identify novel and clinically relevant metabolic subgroups and to define new molecular targets.
Pirfenidone treatment reduces cardiac allograft vasculopathy in a mouse model
Authors: Graebner C , Scheunchen M , Theil F , Kuckhahn A , Vogg N , Gessner A , Fritz N , Ramsperger-Gleixner M , Dewald O , Heim C
Published: May 2024

Funding Acknowledgements

Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ISHLT research grant

Objectives

Chronic rejection in the form of cardiac allograft vasculopathy after heart transplantation remains without effective treatment. Main characteristics of cardiac allograft vasculopathy are immigration of leukocytes and proliferation of smooth muscle cells - collectively resulting in development of a neointima narrowing the vessel lumen and leading to graft ischemia. Pirfenidone is an anti-fibrotic drug with anti-inflammatory properties that is already approved for treatment of idiopathic pulmonary fibrosis.

The aim of this study was to evaluate whether application of pirfenidone leads to reduced luminal occlusion corresponding with decreased chronic rejection in a mouse transplant model.

Methods

Murine thoracic aortas were transplanted into the abdominal aorta of CBA mice (H2k). For allogeneic transplantation, C57BL/6 mice (H2b) were used as donors (n = 8 / group). After transplantation, mice were fed a 0.5% pirfenidone diet versus conventional diet. Preliminary tests were performed determining the blood pirfenidone level by mass spectrometry throughout the study period on days 1, 3, 7, 14, 21 and 28, respectively, as well as after obtaining the aortic grafts on day 30 for histological analysis.

Results

Preliminary tests confirmed the successful uptake of pirfenidone by achieving constant plasma levels of pirfenidone and its metabolite 5-carboxy-pirfenidone over the whole examination period.

Pirfenidone treated murine aortic grafts showed significantly reduced neointima formation compared to control allografts (luminal occlusion: 32.92 % ± 13.02 % vs. 42.44 % ± 19.81 %, p=0.046). As expected, syngeneic aortic grafts with and without pirfenidone treatment showed no luminal occlusion.

Conclusion

The results indicate that pirfenidone application after aortic transplantation reduces neointima formation and thus chronic rejection in a mouse aortic transplant model. Further analyses of underlying mechanisms are currently underway.
Hypertension-induced cardiac pro-arrhythmic disorders of connexin-43 and extracellular matrix are attenuated in hairless versus wild-type of spontaneously hypertensive rats
Authors: Andelova K , Sykora M , Szeiffova Bacova B , Farkasova V , Stankovicova T , Knezl V , Pravenec M , Tribulova N
Published: May 2024

Funding Acknowledgements

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Development Agency under the contract no. 21-0410

VEGA grants no. 2/0002/20, 2/0006/23

Background

Mechanical dysfunction and occurrence of malignant ventricular fibrillation (VF) are hallmarks of hypertensive heart disease and major health issue. Spontaneously hypertensive rats (SHR) that imitates human primary hypertension are prone to develop VF, mainly due to myocardial remodelling associated with disorders of electrical coupling protein connexin-43 (Cx43) and extracellular matrix (ECM) resulting in electrical instability. However, it is not known whether postnatal acclimation of hairless strain of SHR to surrounding temperature may induce cardio-protection. Therefore, we aimed to explore Cx43 and ECM alterations as well as susceptibility to inducible VF in hairless versus wild type SHR.

Design and Methods

Six-month-old wild type SHR males and females and hairless SHR housed at standard 22°C were examined. Biometric parameters, including electro- and echocardiography were registered. Left ventricular tissue was used for immunolabeling of Cx43 to reveal its topology and for western blotting to determine protein levels of Cx43 and its function modulating protein kinases, PKCε, PKCδ and PKCα as well as for ECM markers, TGF-β, SMAD2/3, MMP-2, collagen-1 and its histological deposition. Susceptibility of the heart to electrically-inducible VF was tested using isolated perfused heart technique.

Key results

Comparing to wild type males, hairless SHR exhibited less pronounced prolongation of QT and QRS interval as well as an increase of ejection fraction. Myocardial Cx43 and its variant phosphorylated at serine-368, which preserve electrical coupling, were increased and pro-arrhythmic cardiomyocyte Cx43 topology was attenuated in males and females hairless vs wild type SHR. Moreover, the level of β-catenin, which ensures mechano-electrical coupling, was also increased in hairless SHR. On the other hand, collagen-1 and hydroxyproline were lower and the TGF-β1 and SMAD2/3 proteins were suppressed in males hairless vs wild type SHR. Myocardial ECM remodelling was less pronounced in females regardless rat strain. Hairless SHR males were less susceptible to inducible VF compared to wild type SHR, while females were less prone to develop VF regardless the strain.

In conclusion, our findings imply that postnatal acclimation of hairless SHR is accompanied by attenuation of hypertension-induced adverse down-regulation of Cx43 and up-regulation of extracellular matrix proteins. It may contribute, at least in part, to the protection from malignant cardiac arrhythmias and mechanical dysfunction.