Cardiac Cell Function Research Articles

An analysis of the dynamic role of lipids in the progression and development of cardiovascular disease

The main cause of mortality around the globe is cardiovascular disease (CVD). There are several indications followed by experimental validation that dysfunctional lipotoxicity is one of the primary causes of cardiovascular illnesses such as atherosclerosis, “coronary heart disease”, (CHD), and hypertension. Obesity and diabetes raise the number of circulating lipids, which may enhance the production of harmful intermediates and cause issues related to cardiovascular disorders. Effective and affordable medicines to lower cardiovascular risk are desperately required. Lipids or lipoprotein particles play a critical role in atherosclerosis, the pathophysiology behind CVD. They also have an effect on inflammatory processes, vascular or cardiac cell function, leukocyte activity, and ultimately the health of the heart and blood arteries. In this paper, an attempt was made to identify the cause of lipid buildup that led to the pathophysiology of cardiovascular disorders as well as the processes behind lipotoxicity. A summary of current and developing treatment approaches to decrease the incidence of lipid-induced cardiovascular disease is also compiled through previous studies.

Oxygen-generating microparticles downregulate HIF-1α expression, increase cardiac contractility, and mitigate ischemic injury.

Myocardial hypoxia is the low oxygen tension in the heart tissue implicated in many diseases, including ischemia, cardiac dysfunction, or after heart procurement for transplantation. Oxygen-generating microparticles have recently emerged as a potential strategy for supplying oxygen to sustain cell survival, growth, and tissue functionality in hypoxia. Here, we prepared oxygen-generating microparticles with poly D,L-lactic-co-glycolic acid, and calcium peroxide (CPO), which yielded a continuous morphology capable of sustained oxygen release for up to 24h. We demonstrated that CPO microparticles increased primary rat cardiomyocyte metabolic activity while not affecting cell viability during hypoxia. Moreover, hypoxia-inducible factor (HIF)-1α, which is upregulated during hypoxia, can be downregulated by delivering oxygen using CPO microparticles. Single-cell traction force microscopy data demonstrated that the reduced energy generated by hypoxic cells could be restored using CPO microparticles. We engineered cardiac tissues that showed higher contractility in the presence of CPO microparticles compared to hypoxic cells. Finally, we observed reduced myocardial injuries in ex vivo rabbit hearts treated with CPO microparticles. In contrast, an acute early myocardial injury was observed for the hearts treated with control saline solution in hypoxia. In conclusion, CPO microparticles improved cell and tissue contractility and gene expression while reducing hypoxia-induced myocardial injuries in the heart. STATEMENT OF SIGNIFICANCE: Oxygen-releasing microparticles can reduce myocardial ischemia, allograft rejection, or irregular heartbeats after heart transplantation. Here we present biodegradable oxygen-releasing microparticles that are capable of sustained oxygen release for more than 24 hrs. We then studied the impact of sustained oxygen release from microparticles on gene expresseion and cardiac cell and tissue function. Previous studies have not measured cardiac tissue or cell mechanics during hypoxia, which is important for understanding proper cardiac function and beating. Using traction force microscopy and an engineered tissue-on-a-chip, we demonstrated that our oxygen-releasing microparticles improve cell and tissue contractility during hypoxia while downregulating the HIF-1α expression level. Finally, using the microparticles, we showed reduced myocardial injuries in rabbit heart tissue, confirming the potential of the particles to be used for organ transplantation or tissue engineering.

A novel and cost-effective method for high-throughput 3D culturing and rhythmic assessment of hiPSC-derived cardiomyocytes using retroreflective Janus microparticles

BackgroundHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) gain attention as a potent cell source in regenerative medicine and drug discovery. With the necessity of the demands for experimental models to create a more physiologically relevant model of the heart in vitro we herein investigate a 3D culturing platform and a method for assessing rhythm in hiPSC-CMs.MethodsThe 3D cell culture PAMCELL™ plate is designed to enable cells to attach exclusively to adhesive patterned areas. These cell adhesive zones, named as micro-patterned pads, feature micron silica beads that are surface-modified with the well-known arginyl-glycyl-aspartic acid (RGD) peptide. RGD binding to the surface of hiPSC-CMs facilitates cell–cell attachment and the formation of uniform-size spheroids, which is controlled by the diameter of the micro-patterned pads. The assessment and evaluation of 3D hiPSC-CMs beating pattern are carried out using reflective properties of retroreflective Janus micro-particle (RJP). These RJPs are modified with an antibody targeting the gap junction protein found on the surface of hiPSC-CM spheroids. The signal assessment system comprises a camera attached to an optical microscope and a white light source.ResultsThe 3D PAMCELL™ R100 culture plate efficiently generate approximately 350 uniform-sized hiPSC-CM spheroids in each well of a 96-well plate and supported a 20-day culture. Analysis of genes and protein expression levels reveal that iPSC-CM spheroids grown on PAMCELL™ R100 retain cardiac stem cell characteristics and functions, outperforming traditional 2D culture platform. Additionally, the RJPs enable monitoring and evaluation of in vitro beating properties of cardiomyocytes without using complex monitoring setup. The system demonstrates its capability to identify alteration in the rhythmic activity of cardiac cells when exposed to ion channel blockers, nifedipine and E4031.ConclusionsThe integration of the 3D culture method and RJPs in this study establishes a platform for evaluating the rhythmic properties of 3D hiPSC-CMs. This approach holds significant potential for identifying arrhythmias or other cardiac abnormalities, ultimately contributing to the development of more effective therapies for heart diseases.Graphical

Endocannabinoid regulation of inward rectifier (Kir2.1) channels.

The inward rectifier potassium channel Kir2.1 (KCNJ2) is an important regulator of resting membrane potential in both excitable and non-excitable cells. The function of Kir2.1 channels are dependent on their lipid environment, including the availability of PI(4,5)P2, secondary anionic lipids, cholesterol and long-chain fatty acids acyl coenzyme A (LC-CoA). Endocannabinoids are a class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. While these lipids are identified as ligands for cannabinoid receptors (CBRs), there is a growing body of evidence that they can directly regulate the function of numerous ion channels independently of CBRs. Here we examine the effects of a panel of endocannabinoids on Kir2.1 function, and demonstrate that a subset of endocannabinoids can alter Kir2.1 conductance to varying degrees independently of CBRs. These findings may have broader implications on the function of cardiac, neuronal and/or immune cells.

Substrate stiffness induces a chronic adaptation in contractile work and electrophysiology of cardiomyocytes.

The mechanical function or failure of the heart is defined by the adaptability of its contractility under different conditions. This, coupled with dynamic changes of heart mechanics, via hemodynamics or pathological stiffening, have driven decades of research to understand how external mechanical cues affect function of cardiac muscle cells (CMs). Despite a growing body of research on the effect of microenvironment mechanics (often stiffness) on contractility of CMs, there has been little appreciation of how this adaptation evolves over time. In vivo, CMs experience a gradual increase in the stiffness of their microenvironment, from ∼1 kPa in neonatal heart, to ∼10 kPa for adult heart to ∼50 kPa in fibrotic myocardium (e.g post myocardial infarction). While such mechanical changes have shown to affect CMs behaviour, the role of chronic adaptation over time is often ignored. Here we quantified the evolution of contractile work of CMs over six days. Primary CMs were cultured on substrates of neonatal, adult, and fibrotic myocardium and we measured their contractility using Traction Force Microsocpy (TFM) on days 6, 8, 10, and 12. We observed a gradual increase in contractility in all conditions, suggesting an adaptation mechanism controlling the acute response over longer time scales. To study their electrical response, we used dye-free diffraction imaging. We observed that monolayers on 1 kPa substrates show higher autonomous beating rates, and while substrates with stiffnesses of 10 and 50 kPa show similar beating rates, the monolayers on the 50 kPa substrate have a smaller contraction magnitude. We verified these results with fluorescent imaging of a calcium indicator (FlouO-4). CMs on neonatal stiffness showed significantly faster action potentials (smaller duration) than their counterparts on stiffer substrates. These results indicate the importance of the time-dependent response of CMs to the mechanics of their microenvironment.

Immune mechanisms of cardiac aging.

Advances in healthcare and improvements in living conditions have led to rising life expectancy worldwide. Aging is associated with excessive oxidative stress, a chronic inflammatory state, and limited tissue healing, all of which result in an increased risk of heart failure. In fact, the prevalence of heart failure approaches 40% in the ninth decade of life, with the majority of these cases suffering from heart failure with preserved ejection fraction (HFpEF). In cardiomyocytes (CMs), age-related mitochondrial dysfunction results in disrupted calcium signaling and covalent protein-linked aggregates, which cause cardiomyocyte functional disturbances, resulting in increased stiffness and diastolic dysfunction. Importantly, aging is also associated with chronic low-grade, sterile inflammation, which alters the function of interstitial cardiac cells and leads to cardiac fibrosis. Taken together, cardiac aging is associated with cellular, structural, and functional changes in the heart that contribute to the rising prevalence of heart failure in older people.

545 INVESTIGATING THE RISK OF CARDIAC FIBROSIS IN RESPONSE TO HEAT-NOT-BURN CIGARETTES THROUGH HUMAN CARDIAC STROMAL CELLS

Abstract Background The use of alternative smoking devices, such as heat-not-burn cigarettes (HNBC), is increasing on a global scale, and their impact on health is still uncertain. Objective To investigate the effects of circulating molecules in HNBC chronic smokers on the fibrotic specification and paracrine function of cardiac stromal cells (CSCs). Methods Resident CSCs were isolated from the atrial tissue of non-smokers patients with cardiovascular diseases, and exposed to the serum pools derived from 60 young healthy subjects, stratified in exclusive HNBC smokers, traditional combustion cigarette (TCC) smokers, or non-smokers (NS) as reference. Results CSCs treated with TCC serum versus NS displayed impaired 3D growth (NS 568±24 vs. TCC 442±27 spheroids/well, p≤0,01) and reduced migration after 6h (NS 0,62±0,04 vs. TCC 0,80±0,03 normalized scratch area versus t0, p≤0,01) and 10h (NS 0,49±0,03 vs. TCC 0,36±0,03 normalized scratch area versus t0, p≤0,05), as well as significantly increased expression (IL-6, IL-8) and/or release of pro-inflammatory (e.g CRP, PAI-1, CD40L, CXCL4) and pro-fibrotic cytokines (PDGFA, MMP1). CSCs cultured with HNBC serum showed significantly increased mRNA levels of pro-fibrotic genes (PDGFA, THY1, COL1A1, p<0,01), and significantly reduced expression of the gap junction protein CX43 (p<0,05). Nonetheless, both TCC and HNBC sera reduced the release of angiogenic and protective factors (e.g. VEGF, Adiponectin, CHI3L1) from CSCs, as assessed by protein arrays and ELISA. In fact, their paracrine support to tube-formation by endothelial cells (NS 63,29±4,72 vs. TCC 41,43±6,64 vs. HNBC 37,29±5,67 mesh number per well, p≤0,05) and to preserved cell viability of neonatal rat cardiomyocytes (NS 0,60±0,04 vs. TCC 0,40±0,01 vs. HNBC 0,47±0,01 OD490 normalized OD versus t0, p≤0,001) were significantly impaired. Treatment with the sera of both types of smokers also increased the expression of NOX isoforms and the release of H2O2 (NS 1,76±0,09 vs. TCC 3,84±0,25 vs. HNBC 2,90±0,35 µM, p≤0,05) and significantly reduced the release of NO (NS 6,98±0,43 vs. TCC 4,65±0,07 vs. HNBC 5,06±0,13 µM, p≤0,01) by CSCs. Conclusion The circulating molecules in the serum of chronic HNBC smokers induce fibrotic specification in CSCs. They also reduce the beneficial paracrine effects of stromal cells on endothelial cells and cardiomyocytes, albeit to a reduced extent for some features. These results point to a potential risk for atrial fibrosis development triggered by chronic HNBC use.

Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes.

IntroductionGene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery.MethodsHere, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency.ResultsBy varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment.ConclusionCollectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.

Resveratrol regulates paracrine function of cardiac microvascular endothelial cells under hypoxia/reoxygenation condition.

The secreted factors from cardiac microvascular endothelial cells (CMECs) regulate the physiological activity of adjacent tissues and could be modulated by myocardial ischemia/reperfusion injury (MIRI). How this paracrine function of CMECs is regulated by MIRI and resveratrol remains to be elucidated. CMECs pretreated with/ without resveratrol were subjected to hypoxia/reoxygenation (H/R). Apoptosis was measured by flowcytometry. Protein antibody arrays were performed to find the alteration of cytokine secreted by CMECs. The Gene Ontology analysis was applied to interpret the function of modulated factors. We revealed resveratrol inhibited apoptosis of CMECs dose-dependently after H/R and reached its peak effect at the concentration of 100 μM. 29 factors were significantly changed by H/R, and resveratrol at 100 μM changed 98 types of factors compared with the H/R group. Among these factors, eight were increased by H/R and then were decreased by resveratrol. Eleven were attenuated by H/R and further decreased by resveratrol. Insulin-like growth factor binding protein-1 was upregulated by H/R and it was further increased by resveratrol. The altered factors were involved in cell proliferation, cell growth, cell motility, chemotaxis, angiogenesis and vasculogenesis. The study suggests that resveratrol inhibits the apoptosis and modulates the paracrine function of CMECs under ischemia/reperfusion condition.

The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro

Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coronavirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6–3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24–48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease.

Fraction of the T-Tubular Membrane as an Important Parameter in Cardiac Cellular Electrophysiology: A New Way of Estimation.

The transverse-axial tubular system (t-tubules) plays an essential role in excitation-contraction coupling in cardiomyocytes. Its remodelling is associated with various cardiac diseases. Numerous attempts were made to analyse characteristics essential for proper understanding of the t-tubules and their impact on cardiac cell function in health and disease. The currently available methodical approaches related to the fraction of the t-tubular membrane area produce diverse data. The widely used detubulation techniques cause irreversible cell impairment, thus, distinct cell samples have to be used for estimation of t-tubular parameters in untreated and detubulated cells. Our proposed alternative method is reversible and allows repetitive estimation of the fraction of t-tubular membrane (f t) in cardiomyocytes using short-term perfusion of the measured cell with a low-conductive isotonic sucrose solution. It results in a substantial increase in the electrical resistance of t-tubular lumen, thus, electrically separating the surface and t-tubular membranes. Using the whole-cell patch-clamp measurement and the new approach in enzymatically isolated rat atrial and ventricular myocytes, a set of data was measured and evaluated. The analysis of the electrical equivalent circuit resulted in the establishment of criteria for excluding measurements in which perfusion with a low conductivity solution did not affect the entire cell surface. As expected, the final average f t in ventricular myocytes (0.337 ± 0.017) was significantly higher than that in atrial myocytes (0.144 ± 0.015). The parameter f t could be estimated repetitively in a particular cell (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method is fast, simple, and leaves the measured cell intact. It can be applied in the course of experiments for which it is useful to estimate both the surface and t-tubular capacitance/area in a particular cell.

Characterization of Human Cardiac Progenitor Cell Secretome

Heart Disease (HD) remains the greatest cause of death worldwide. Due to the heart’s poor capacity to regenerate after damage, there is a critical need for treatments that can restore cardiac cell integrity and function in HD patients. Preclinical and clinical evidence has highlighted the potential role of Cardiac Progenitor Cells (CPCs) in regulation of cardiac repair after injury, with an emerging role of CPC secreted proteins in this process. We have been studying CPCs isolated from human left ventricular myocardium characterized by their high proliferative capacity in vitro. These cells (human highly proliferative clones, hHiPC) are characterized by high expression of CD105/Endoglin and show varying potential for differentiating into endothelial cells in vitro. When injected into the mouse myocardium after surgically induced myocardial infarction (MI), individual hHiPC clones show variable effects on heart remodeling. For instance, when clone 11 was injected into the infarct zone post‐MI we saw a trend towards improvement in the fractional shortening (%) compared to clone 22 and saline injected controls. We hypothesized that hHiPCs associated with improvement in cardiac remodeling after MI will have a secretome characterized by higher expression of pro‐angiogenic and pro‐proliferative proteins, and these are regulated by the BMP/CD105 pathway. An unbiased and global proteomics approach (LC‐MS/MS) was used in our lab to identify proteins secreted from hHiPCs pre‐treated with the CD105 ligand BMP9 (5 ng/mL) compared to non‐treated controls. Our secretome data identified ~700 proteins in which 57% were characterized from UniProt as having a signal peptide sequence. Several pro‐angiogenic secreted proteins were identified in clone 11 including CCN2, CXCL6, and TGFB2. We found a >2.5‐fold significant increase in the secretion of Sclerostin (SOST) in BMP9 treated hHiPC compared to non‐treated controls. SOST is a known BMP‐target protein and WNT inhibitor. Previous evidence has highlighted the role of WNT‐signaling in heart remodeling after myocardial induced injury, but the role of Sclerostin in this process has yet to be elucidated. To further characterize the relationship between BMP9 and the secretion of SOST we performed a Real‐Time q‐PCR and ELISA assay of cell lysates and conditioned media between BMP9 treated and non‐treated controls and found a dramatic increase in the expression of SOST in BMP9 treated clone 11. We also found an increase (4.5‐fold) in CD105 mRNA expression after BMP9 treatment. Similarly, using LC‐MS/MS we found a significant increase in CD105 expression (2.5‐fold, p‐value = 7.39x10‐6) in the membrane fraction of clone 11 compared to clone 22 indicating its potential role in positive heart remodeling effects. Future studies will be conducted in vivo to determine the role SOST/WNT signaling has in heart remodeling after ischemic injury. Further, the increase in pro‐angiogenic factors after BMP9 treatment may suggest the potential relevance of BMP9 treatment in cardiac progenitor cell secretome mediated repair and we will test this hypothesis in the future.

Empagliflozin attenuates cardiac microvascular ischemia/reperfusion through activating the AMPKα1/ULK1/FUNDC1/mitophagy pathway

Mitophagy preserves microvascular structure and function during myocardial ischemia/reperfusion (I/R) injury. Empagliflozin, an anti-diabetes drug, may also protect mitochondria. We explored whether empagliflozin could reduce cardiac microvascular I/R injury by enhancing mitophagy. In mice, I/R injury induced luminal stenosis, microvessel wall damage, erythrocyte accumulation and perfusion defects in the myocardial microcirculation. Additionally, I/R triggered endothelial hyperpermeability and myocardial neutrophil infiltration, which upregulated adhesive factors and endothelin-1 but downregulated vascular endothelial cadherin and endothelial nitric oxide synthase in heart tissue. In vitro, I/R impaired the endothelial barrier function and integrity of cardiac microvascular endothelial cells (CMECs), while empagliflozin preserved CMEC homeostasis and thus maintained cardiac microvascular structure and function. I/R activated mitochondrial fission, oxidative stress and apoptotic signaling in CMECs, whereas empagliflozin normalized mitochondrial fission and fusion, neutralized supraphysiologic reactive oxygen species concentrations and suppressed mitochondrial apoptosis. Empagliflozin exerted these protective effects by activating FUNDC1-dependent mitophagy through the AMPKα1/ULK1 pathway. Both in vitro and in vivo, genetic ablation of AMPKα1 or FUNDC1 abolished the beneficial effects of empagliflozin on the myocardial microvasculature and CMECs. Taken together, the preservation of mitochondrial function through an activation of the AMPKα1/ULK1/FUNDC1/mitophagy pathway is the working mechanism of empagliflozin in attenuating cardiac microvascular I/R injury.

Tough Decoy-Mediated Cardiac Gene Suppression.

MicroRNA (miRNA) is a small, non-coding RNA molecule (~22 nucleotides) that acts as a post-transcriptional gene regulator, primarily by inhibiting the translation of target mRNA transcripts or affecting cell mRNA stability. Since miRNAs are comprehensively involved in gene regulation, their abnormalities are associated with various human diseases, including cardiovascular disease. Additionally, targeted inhibition of disease-related miRNAs and their targets should have therapeutic potential. Therefore, this chapter describes the experimental steps for targeted inhibition of specific miRNAs using adenoviral vectorized tough decoys that efficiently silence miRNA function in cardiac cells.

Graphene-Based Scaffolds: Fundamentals and Applications for Cardiovascular Tissue Engineering.

Cardiac tissue engineering requires materials that can faithfully recapitulate and support the native in vivo microenvironment while providing a seamless bioelectronic interface. Current limitations of cell scaffolds include the lack of electrical conductivity and suboptimal mechanical properties. Here we discuss how the incorporation of graphene into cellular scaffolds, either alone or in combination with other materials, can affect morphology, function, and maturation of cardiac cells. We conclude that graphene-based scaffolds hold great promise for cardiac tissue engineering.

Aging influences the cardiac macrophage phenotype and function during steady state and during inflammation.

Aging‐mediated immune dysregulation affects the normal cardiac immune cell phenotypes and functions, resulting in cardiac distress. During cardiac inflammation, immune activation is critical for mounting the regenerative responses to maintain normal heart function. We investigated the impact of aging on myeloid cell phenotype and function during cardiac inflammation induced by a sub‐lethal dose of LPS. Our data show that hearts of old mice contain more myeloid cells than the hearts of young mice. However, while the number of monocytic‐derived suppressor cells did not differ between young and old mice, monocytic‐derived suppressor cells from old mice were less able to suppress T‐cell proliferation. Since cardiac resident macrophages (CRMs) are important for immune surveillance, clearance of dead cells, and tissue repair, we focused our studies on CRMs phenotype and function during steady state and LPS treatment. In the steady state, we observed significantly more MHC‐IIlow and MHC‐IIhigh CRMs in the hearts of old mice; however, these populations were decreased in both young and aged mice upon LPS treatment and the decrease in CRM populations correlated with defects in cardiac electrical activity. Notably, mice treated with a liver X receptor (LXR) agonist showed an increase in MerTK expression in CRMs of both young and old mice, which resulted in the reversal of cardiac electrical dysfunction caused by lipopolysaccharide (LPS). We conclude that aging alters the phenotype of CRMs, which contributes to the dysregulation of cardiac electrical dysfunction during infection in aged mice.

Stem cells from the amniotic fluid: a promising tool to face the cytokine storm associated to SARS‐CoV‐2 infections

Infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the etiological agent of coronavirus disease 19 (COVID‐19) is associated with significant morbidity and mortality. Despite significant progress since the emergence of this disease, efficacious therapeutics are still urgently required. Infection is associated with a broad spectrum of symptoms and varying disease severity. In severe cases, a multi‐organ disease is apparent, involving the respiratory, coagulation, immune and cardiac systems. A central tenant of severe disease is an excessive immune response to infection leading to a cytokine storm and associated organ and tissue damage. Immunological therapies capable of attenuating the cytokine storm may therefore provide novel treatment options, and the present study explored whether mesenchymal stem cells (MSCs) from the amniotic fluid (AF) and their derivatives, through their immunomodulatory, anti‐oxidant, and regenerative functions, could provide such an option.Next Generation Sequencing analysis of MSCs isolated from human AF identified a plethora of molecules with the potential to positively influence the prognosis in SARS‐Cov‐2 infection patients. These included mRNAs involved in the regulation of several immune pathways that are basis of the impaired immune response and cytokine storm caused seen in SARS‐CoV‐2‐positive patients, such as: HIF‐1, IL‐17, Toll‐like receptors, RAP1, TNF, WNT, PI3K‐Akt and NF‐kappa B signalling. AF‐MSCs also contained bioactive molecules involved in respiratory endothelial protection and repair, such as VEGF, IL‐1, TGF‐β1, EGFR, in extracellular matrix re‐organization or associated with cardiac muscle cell function. Analysis of non‐coding RNAs identified the presence of microRNAs that regulate macrophage polarization (towards an anti‐inflammatory phenotype) and attenuate inflammation, as well as regulate the host response to viral infection. Among those, exosomal miRNAs have been also identified.Building upon these observations, further efforts will be made to elucidate the potential therapeutic use of AF‐MSCs and their derivatives in combating the immune‐related side effects of SARS‐CoV‐2. The successful incorporation of such an MSC‐based therapy into the treatment regimen of SARS‐CoV‐2 infected patients has significant potential to reduce the morbidity and mortality associated with COVID‐19.

MtDNA Heteroplasmy at the Core of Aging-Associated Heart Failure. An Integrative View of OXPHOS and Mitochondrial Life Cycle in Cardiac Mitochondrial Physiology

The most common aging-associated diseases are cardiovascular diseases which affect 40% of elderly people. Elderly people are prone to suffer aging-associated diseases which are not only related to health and medical cost but also to labor, household productivity and mortality cost. Aging is becoming a world problem and it is estimated that 21.8% of global population will be older than 65 years old in 2050; and for the first time in human history, there will be more elderly people than children. It is well accepted that the origin of aging-associated cardiovascular diseases is mitochondrial dysfunction. Mitochondria have their own genome (mtDNA) that is circular, double-stranded, and 16,569 bp long in humans. There are between 500 to 6000 mtDNA copies per cell which are tissue-specific. As a by-product of ATP production, reactive oxygen species (ROS) are generated which damage proteins, lipids, and mtDNA. ROS-mutated mtDNA co-existing with wild type mtDNA is called mtDNA heteroplasmy. The progressive increase in mtDNA heteroplasmy causes progressive mitochondrial dysfunction leading to a loss in their bioenergetic capacity, disruption in the balance of mitochondrial fusion and fission events (mitochondrial dynamics, MtDy) and decreased mitophagy. This failure in mitochondrial physiology leads to the accumulation of depolarized and ROS-generating mitochondria. Thus, besides attenuated ATP production, dysfunctional mitochondria interfere with proper cellular metabolism and signaling pathways in cardiac cells, contributing to the development of aging-associated cardiovascular diseases. In this context, there is a growing interest to enhance mitochondrial function by decreasing mtDNA heteroplasmy. Reduction in mtDNA heteroplasmy is associated with increased mitophagy, proper MtDy balance and mitochondrial biogenesis; and those processes can delay the onset or progression of cardiovascular diseases. This has led to the development of mitochondrial therapies based on the application of nutritional, pharmacological and genetic treatments. Those seeking to have a positive impact on mtDNA integrity, mitochondrial biogenesis, dynamics and mitophagy in old and sick hearts. This review covers the current knowledge of mitochondrial physiopathology in aging, how disruption of OXPHOS or mitochondrial life cycle alter mtDNA and cardiac cell function; and novel mitochondrial therapies to protect and rescue our heart from cardiovascular diseases.

The Influence of the LINC00961/SPAAR Locus Loss on Murine Development, Myocardial Dynamics, and Cardiac Response to Myocardial Infarction.

Long non-coding RNAs (lncRNAs) have structural and functional roles in development and disease. We have previously shown that the LINC00961/SPAAR (small regulatory polypeptide of amino acid response) locus regulates endothelial cell function, and that both the lncRNA and micropeptide counter-regulate angiogenesis. To assess human cardiac cell SPAAR expression, we mined a publicly available scRNSeq dataset and confirmed LINC00961 locus expression and hypoxic response in a murine endothelial cell line. We investigated post-natal growth and development, basal cardiac function, the cardiac functional response, and tissue-specific response to myocardial infarction. To investigate the influence of the LINC00961/SPAAR locus on longitudinal growth, cardiac function, and response to myocardial infarction, we used a novel CRISPR/Cas9 locus knockout mouse line. Data mining suggested that SPAAR is predominantly expressed in human cardiac endothelial cells and fibroblasts, while murine LINC00961 expression is hypoxia-responsive in mouse endothelial cells. LINC00961–/– mice displayed a sex-specific delay in longitudinal growth and development, smaller left ventricular systolic and diastolic areas and volumes, and greater risk area following myocardial infarction compared with wildtype littermates. These data suggest the LINC00961/SPAAR locus contributes to cardiac endothelial cell and fibroblast function and hypoxic response, growth and development, and basal cardiovascular function in adulthood.

Short-term Effects of Metformin on Cardiac and Peripheral Blood Cells Following Cecal Ligation and Puncture-induced Sepsis.

Sepsis has well-documented inflammatory effects on cardiovascular and blood cells. This study is designed to investigate potential anti-inflammatory effects of metformin on cardiac and blood cells 12 and 24 h following cecal ligation and puncture (CLP)-induced sepsis. For the purpose of this study, 36 male Wistar rats were divided into six groups: two groups underwent CLP, two groups underwent CLP and received metformin, and two groups only received sham operations. 12 h later, 18 rats (half of rats in each of the three aforementioned groups) were sacrificed and cardiac and blood cells were harvested. Subsequently, 12 h later, the rest of the rats were euthanatized. In all harvested blood and cardiac cells, oxidative stress indicators, antioxidant properties, count of blood cells, neutrophil infiltration, percentage of weight loss and pathological assessment were conducted. In our experiment, metformin elevated antioxidant levels, improved function of blood cells and percentage of weight loss. Moreover, in the groups which received metformin, oxidative stress and neutrophil infiltration markers were decreased significantly. Moreover, pathological investigations of cardiac cell injury were reduced in the metformin group. Our findings suggest that in CLP induced sepsis model, metformin can improve the function of blood and cardiac cells through alleviating inflammation, improvement of anti-inflammation properties, and enhancement of blood profile, and all these effects are more pronounced after 24 h in comparison with 12 h after induction of sepsis.