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Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC EVICARE, Van Herk Foundation Introduction The use of cardiac progenitor cell (CPC)-derived small Extracellular Vesicles (sEVs) has shown great potential to stimulate cardiac repair post myocardial infarction. sEVs are released by cells and play a role in intercellular communication through transfer of their bioactive content, like RNAs and proteins. Increasing evidence indicates that sEVs present a heterogeneous mixture of vesicle populations, depicting different functional aspects, which hampers clinical translation. Studying sEV heterogeneity could provide new insights into contributing therapeutic mechanisms underlying sEV-mediated cardiac repair. Methods CPC-derived sEVs were purified by flow through chromatography followed by size-exclusion chromatography (SEC) and asymmetric flow field flow fractionation (A4F) for fractionation of different sEV-subfractions. Based on the differential expression of common sEV markers, different subpopulations were determined. sEV subpopulations were further characterized using western blot, nanoparticle tracking analysis, bicinchoninic acid protein assay, transmission electron microscopy and mass spectrometry (MS). Functional differences were studied using different cellular assays to determine AKT phosphorylation, wound healing, angiogenesis and proteasome activity. Results SEC or A4F were used to separate distinct subpopulations of CPC-derived sEVs with a different appearance, size and function. MS analysis confirmed the differences in proteomic expression levels of classical sEV markers, as well as annexins, rab proteins, integrins, histones and proteasomal proteins. Furthermore, differences in cellular components linked to their cellular origin were found. Exposure of recipient endothelial cells to sEV subpopulations demonstrated clear functional differences. Additionally, differences in proteolytic activity of the sEV subpopulations were identified. Conclusions SEC and AF4 allow for isolation and in-depth study of the functional heterogeneity of sEVs. In our study, we observed the presence of different subpopulations based on size, with a different composition and biological function. Increased knowledge of sEV heterogeneity will contribute to a better understanding of the mechanisms of action of sEVs and will improve translation to the clinic and potentially an off-the-shelf approach to stimulate cardiac repair. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO - The Dutch Research Council Background Current in vivo preclinical models lack the predictability of the clinical efficacy resulting in a high dropout rate of therapeutic candidates. Advanced human-based models are required to bridge this gap and indicate the potential of novel therapeutic approaches[1].  Myocardial tissue slices are an in vitro model that recapitulates the native multicellular architecture of the heart. This allows for modeling cellular processes in a macroscopic context entailing great promise. The most common cardiomyopathy-related mutation in the Netherlands is the loss of arginine at position 14 (p.Arg14del) in the phospholamban protein (PLN). PLN is a critical regulator of calcium cycling and contractility in the heart. The p.Arg14del mutation results in a super inhibition of SERCA2a and thus aberrant calcium handling and reduced contractile force. Mice models harbouring the PLN p.Arg14del do not completely recapitulate the human manifestation mainly due to the difference between species; heart rate, Calcium-cycling and ion properties, and different myosin heavy chain isoforms, showing the unmet need for a human-based model[2,3]. Methods & Results 300 µm thick viable myocardial tissue were sectioned from a PLN p.Arg14del patient's left ventricle. Although the myocardial tissue slices were kept alive for eight days in static culture, these conditions initiated cell death and dedifferentiation. The tissue slices show the greatest resemblance to the intact architecture of the in vivo human heart, it is the most relevant model for viral transduction in the human heart, and proof-of-principle of this is performed. Myocardial tissue slices of a PLN p.Arg14del patient retain the structural phenotype shown by the fibrofatty deposition. Similarly, functional patient characteristics, aberrant calcium handling, and reduced contractile force are preserved. Conclusion Myocardial tissue slices recapitulate the (patho)physiology of the heart, as shown here with the PLN p.Arg14del case. However, the static culture conditions induce remodeling of the heart and thus only allow for acute measures in the native heart. To prolong the period that the slices recapitulate the native heart, culture conditions should mimic the environment of the heart. The tissue slices allow for a currently unmet need to modulate the complex architecture of the human heart with e.g. novel delivery tools or therapeutic interventions. All in all, myocardial tissue slices are a promising model that can give novel insights into the physiology of the human heart, and therapeutic intervention on induced or genetic cardiomyopathies. Open in new tab Download slide Tissue Slices PLN p.Arg14del patient [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.M.C.P. is supported by a Netherlands Cardiovascular Research Initiative (CVON) grant (REMAIN 2014B27) and J.P.G.S is supported by Horizon2020 ERC-2016-COG-EVICARE [725229] and BRAVE (grant number 874827. Purpose Restoring perfusion, either by coronary angioplasty or bypass anastomosis, is performed around 2 million times per year throughout Europe to treat patients after myocardial infarction. Although this treatment is highly efficient to resolve myocardial ischemia, reperfusion is accompanied by damage through the release of reactive oxygen species (ROS). This ischemia/reperfusion (I/R) injury is manifested by cardiomyocyte death, caused to a certain extent by necroptosis. As a potential means of cardioprotection, we applied the small molecule compound 547 inhibiting RIP1-kinase, a central mediator of the necroptosis pathway, in an in vitro model of human cardiac I/R injury. Methods Human foetal cardiomyocyte progenitor cells (hfCPCs) were cultured and characterized for cell type defining markers before being subjected to simulated I/R damage using hydrogen peroxide (H2O2). Cardioprotective effects of 547 were assessed 24 hours after induction of damage and application of the compound. Cell viability and necroptosis pathway activation were assessed by flow cytometry, immunocytochemistry, and western blotting. Furthermore, mitochondrial damage was determined by JC-1 staining. Results hfCPCs expressed indicative markers SCA1, GATA4, PECAM-1, VEGF and C-KIT. Treatment with compound 547 significantly protected hfCPCs from H2O2 induced damage (viable cells: 84,3 ± 4,3% vs 1,6 ± 0,31%; P<0,0001). 547 decreased the necrotic cell population (DAPI+ AnnV-) (3,0 ± 1,1% vs 10,1 ± 1,5%, P<0,0001) and reduced phosphorylation of necrosome components RIP1 (1,3 ±  0,35 fold vs 3,7 ±  0,78 fold; P< 0,01), RIP3 (1,0 ± 0,46 fold vs 4,3 ± 1,5 fold; P< 0,05), and MLKL (1,1 ± 0,15 fold vs 5,7 ± 1,7 fold; P< 0,01) without decreasing unphosphorylated protein or mRNA levels. Interestingly, 547 treatment prevented oxidative stress induced loss of nuclear RIP1 and RIP3 as measured by immunocytochemistry and western blotting of cell fractions (nuclear/cytoplasmic ratio RIP1: 0,80 ±  0,0006 [Control] vs 1,5 ±  0,17 fold [547]; P< 0,01; nuclear/cytoplasmic ratio RIP3: 0,76 ±  0,10 [Control] vs 1,6 ± 0,18 fold [547]; P< 0,01). Remarkably, compound 547 also decreased H2O2–induced mitochondrial depolarization (JC-1 staining/membrane depolarization index: 4,5 ± 0,85% vs 1,2 ±  0,38%; P< 0,05)) and expression and activation of CAMKII (0,46 ± 0,13 fold; P<0,01), a protein mediating mitochondria-dependent cell death. Conclusions Administration of compound 547 led to inhibition of necroptosis, increased cell viability and reduced mitochondrial damage in an in vitro human cardiac I/R injury model, emphasizing the potential of necroptosis inhibition for cardioprotection. Assessing clinical applicability will require further studies in relevant in vivo models. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This research is support by the European Research Council (ERC) consolidator grant EVICARE (ERC-2016-COG-725229). Background Repair of damaged heart tissue upon myocardial infarction remains a major challenge. Transplantation of cardiac progenitor cells (CPCs) has been studied as a potential regenerative therapy, but recent studies have shown that the cardioprotective effect of CPCs is mediated by the release of extracellular vesicles (EVs). The benefits of CPC-derived EVs have mostly been associated with stimulation of angiogenesis and inhibition of cell death. Although macrophages have been suggested to be key for cardiac repair, the effect of CPC-EVs on macrophage polarization is poorly explored. Purpose Here, we hypothesized that CPC-derived EVs can modulate the inflammatory response after myocardial infarction by interacting with macrophages. Methods EVs were isolated from serum-starved CPCs by ultrafiltration followed by size exclusion chromatography. Human monocytes were isolated from the blood of healthy donors and differentiated into macrophages with M-CSF. Macrophages were subsequently stimulated with LPS + IFNy or with IL4 in order to induce an inflammatory M1 and reparative M2 phenotype, respectively. A third group of macrophages were cultured in media without LPS, IFNy or IL4 to resemble naive M0 macrophages. The obtained macrophages were exposed to CPC-EVs and analyzed by flow cytometry, bulk RNA sequencing and confocal microscopy to assess macrophage phenotype changes. Gene candidates were selected based on the p-value and fold-change, and validated by RT-PCR. Small molecule inhibitors were used to further investigate the mechanism by which CPC-EVs are taken up by macrophages and the pathways involved in macrophage response. Results Stimulation of macrophages with CPC-EVs enhanced the expression of the pro-inflammatory marker CD80, while slightly decreasing the anti-inflammatory marker CD206, in M0 and M2 macrophages. CPC-EV-stimulated macrophages also adopted a morphology that reflects the inflammatory macrophage. In line with these findings, bulk RNA sequencing on M0 and M2 polarized macrophages revealed upregulation of genes involved in inflammatory response, including cytokine and both type I and II interferon signaling. EV exposure did not significantly affect gene expression in M1 polarized macrophages. Ongoing investigations will provide in-depth insight into the mechanism by which CPC-EVs induce this response in macrophages. Conclusion Our data suggests that CPC-EVs are able to induce macrophage polarization towards an inflammatory phenotype, which might have implications for CPC-EV treatment after myocardial infarction. This underlines an urgent need to understand the molecular mechanisms underlying the immunomodulatory effect of CPC-derived EVs before moving into a clinical setting. [ABSTRACT FROM AUTHOR]

Arteriogenesis is a complicated process induced by local shear stress caused by local occlusion and is enhanced by growth factors such as VEGF, which is secreted by inflammatory cells and endothelial cells in response to hypoxia. To search for microRNAs involved in arteriogenesis, we performed microarray analysis on hindlimb ischemia mice tissue. We observed a dynamic temporal regulation of microRNAs expression , among which miR-132 /212 were significantly upregulated 4 days after occlusion of femoral artery. The aim of our study was to unravel the role of miR-132/212 in arteriogenesis. In a pericyte-endothelial cell in vitro co-culture assay, overexpression of miR-132/212 in endothelial cells resulted in enhanced neovascularization capacity, including more tubular structures and junctions and longer total tubule length. Inhibition of miR-132/212 decreased total tubule length, and the number of junctions and tubule structures. Ex vivo aorta ring assay demonstrated more branches in wild-type than of miR-132/212 knockout mice. In line with these, microRNA132/212 Knockout mice displayed slower perfusion recovery after hind-limb ischemia than wild type mice. Immunohistochemistry studies further demonstrated that knockout mice have similar number of collateral arteries but they are smaller than wild type. Based on microRNA targets prediction and luciferase assay we found that miR-132/212 enhanced Ras-MAPK signaling by directly inhibiting Rasa1, Spred1, and Spry1 expression which are inhibitors of the Ras-MAPK signaling pathway. In summary, our results show that miR-132/212 promote arteriogenesis by prolonging VEGF initiated Ras-MAPK signaling. [ABSTRACT FROM PUBLISHER]

Background and Purposes: Reduced inward rectifier potassium channel (K ir 2.1) functioning is associated with heart failure and may cause Andersen-Tawil Syndrome, among others characterized by ventricular arrhythmias. Most heart failure or Andersen-Tawil Syndrome patients are treated with β-adrenoceptor antagonists (β-blockers) or sodium channel blockers; however, these do not specifically address the inward rectifier current (I K1 ) nor aim to improve resting membrane potential stability. Consequently, additional pharmacotherapy for heart failure and Andersen-Tawil Syndrome treatment would be highly desirable. Acute propafenone treatment at low concentrations enhances I K1 current, but it also exerts many off-target effects. Therefore, discovering and exploring new I K1 -channel openers is necessary., Experimental Approach: Effects of propafenone and 10 additional propafenone analogues were analysed. Currents were measured by single-cell patch-clamp electrophysiology. K ir 2.1 protein expression levels were determined by western blot analysis and action potential characteristics were further validated in human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMCs). Molecular docking was performed to obtain detailed information on drug-channel interactions., Key Results: Analogues GPV0019, GPV0057 and GPV0576 strongly increased the outward component of I K1 while not affecting the K ir 2.1 channel expression levels. GPV0057 did not block I Kr at concentrations below 0.5 μmol L -1 nor Na V 1.5 current below 1 μmol L -1 . Moreover, hiPSC-CMC action potential duration was also not affected by GPV0057 at 0.5 and 1 μmol L -1 . Structure analysis indicates a mechanism by which GPV0057 might enhance K ir 2.1 channel activation., Conclusion and Implications: GPV0057 has a strong efficiency towards increasing I K1 , which makes it a good candidate to address I K1 deficiency-associated diseases., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Cell therapy, gene therapy, and tissue engineering have been explored as potential strategies to repair or regenerate damaged cardiac tissue. Despite the presence of encouraging preclinical data, clinical trials of regenerative cardiac therapies have yielded mixed results. Our study aimed to investigate the fate of all registered clinical trials within regenerative cardiac medicine, with the purpose of exploring the potential role of publication bias (or trial-completion bias), how published and unpublished research affects the field, and to draw lessons and recommendations for future clinical trials. In this analysis, we show that only a third of all registered trials has yielded results and that a significant number of trials are not completed. Furthermore, we identified significant heterogeneity in study design, study phase, funding, specific therapies used, primary outcome measures and methods of outcome assessment. These observations might hinder the successful translation of cardiac regenerative therapies into clinical practice., (© 2024. The Author(s).)

A newly identified pathogenic variant (A527G) in alpha B-crystallin (αB-crystallin) has been linked to congenital cataract and young-onset dilated cardiomyopathy (DCM) within a Dutch family, although the disease mechanism remains unclear. Four human induced pluripotent stem cell (hiPSC) clones were generated from three symptomatic patients carrying the A527G variant, and one healthy proband. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai viral pluripotency vectors. The established hiPSCs clones exhibited regular ESC-like morphology, expression of pluripotency markers, and normal karyotyping. These hiPSC lines can facilitate future studies to understand the chaperone function and its role in DCM disease progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose of Review: This review aims to evaluate the potential of CRISPR-based gene editing tools, particularly prime editors (PE), in treating genetic cardiac diseases. It seeks to answer how these tools can overcome current therapeutic limitations and explore the synergy between PE and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for personalized medicine., Recent Findings: Recent advancements in CRISPR technology, including CRISPR-Cas9, base editors, and PE, have demonstrated precise genome correction capabilities. Notably, PE has shown exceptional precision in correcting genetic mutations. Combining PE with iPSC-CMs has emerged as a robust platform for disease modeling and developing innovative treatments for genetic cardiac diseases. The review finds that PE, when combined with iPSC-CMs, holds significant promise for treating genetic cardiac diseases by addressing their root causes. This approach could revolutionize personalized medicine, offering more effective and precise treatments. Future research should focus on refining these technologies and their clinical applications., (© 2024. The Author(s).)

Background: Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored., Methods: We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo., Results: We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury., Conclusions: Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury., Competing Interests: None.

Exercise improves cardiac function and metabolism. Although long-term exercise leads to circulating and micro-environmental metabolic changes, the effect of exercise on protein post-translational lactylation modifications as well as its functional relevance is unclear. Here, we report that lactate can regulate cardiomyocyte changes by improving protein lactylation levels and elevating intracellular N 6 -methyladenosine RNA-binding protein YTHDF2. The intrinsic disorder region of YTHDF2 but not the RNA m 6 A-binding activity is indispensable for its regulatory function in influencing cardiomyocyte cell size changes and oxygen glucose deprivation/re-oxygenation (OGD/R)-stimulated apoptosis via upregulating Ras GTPase-activating protein-binding protein 1 (G3BP1). Downregulation of YTHDF2 is required for exercise-induced physiological cardiac hypertrophy. Moreover, myocardial YTHDF2 inhibition alleviated ischemia/reperfusion-induced acute injury and pathological remodeling. Our results here link lactate and lactylation modifications with RNA m 6 A reader YTHDF2 and highlight the physiological importance of this innovative post-transcriptional intrinsic regulation mechanism of cardiomyocyte responses to exercise. Decreasing lactylation or inhibiting YTHDF2/G3BP1 might represent a promising therapeutic strategy for cardiac diseases., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Cardiac tissue engineering (cTE) has already advanced towards the first clinical trials, investigating safety and feasibility of cTE construct transplantation in failing hearts. However, the lack of well-established preservation methods poses a hindrance to further scalability, commercialization, and transportation, thereby reducing their clinical implementation. In this study, hypothermic preservation (4 °C) and two methods for cryopreservation ( i.e. , a slow and fast cooling approach to -196 °C and -150 °C, respectively) were investigated as potential solutions to extend the cTE construct implantation window. The cTE model used consisted of human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts embedded in a natural-derived hydrogel and supported by a polymeric melt electrowritten hexagonal scaffold. Constructs, composed of cardiomyocytes of different maturity, were preserved for three days, using several commercially available preservation protocols and solutions. Cardiomyocyte viability, function (beat rate and calcium handling), and metabolic activity were investigated after rewarming. Our observations show that cardiomyocytes' age did not influence post-rewarming viability, however, it influenced construct function. Hypothermic preservation with HypoThermosol® ensured cardiomyocyte viability and function. Furthermore, fast freezing outperformed slow freezing, but both viability and function were severely reduced after rewarming. In conclusion, whereas long-term preservation remains a challenge, hypothermic preservation with HypoThermosol® represents a promising solution for cTE construct short-term preservation and potential transportation, aiding in off-the-shelf availability, ultimately increasing their clinical applicability.

Despite research efforts being made towards preserving (or even regenerating) heart tissue after an ischemic event, there is a lack of resources in current clinical treatment modalities for patients with acute myocardial infarction that specifically address cardiac tissue impairment. Modified messenger RNA (modRNA) presents compelling properties that could allow new therapeutic strategies to tackle the underlying molecular pathways that ultimately lead to development of chronic heart failure. However, clinical application of modRNA for the heart is challenged by the lack of effective and safe delivery systems. Lipid nanoparticles (LNPs) represent a well characterized class of RNA delivery systems, which were recently approved for clinical usage in mRNA-based COVID-19 vaccines. In this study, we evaluated the potential of LNPs for cardiac delivery of modRNA. We tested how variations in C12-200 modRNA-LNP composition affect transfection levels and biodistribution after intramyocardial administration in both healthy and myocardial-infarcted mice, and determined the targeted cardiac cell types. Our data revealed that LNP-mediated modRNA delivery outperforms the current state of the art (modRNA in citrate buffer) upon intramyocardial administration in mice, with only minor differences among the formulations tested. Furthermore, we determined both in vitro and in vivo that the cardiac cells targeted by modRNA-LNPs include fibroblasts, endothelial cells and epicardial cells, suggesting that these cell types could represent targets for therapeutic interference with these LNP formulations. These outcomes may serve as a starting point for LNP development specifically for therapeutic mRNA cardiac delivery applications., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Statins are effective drugs in reducing cardiovascular morbidity and mortality by inhibiting cholesterol synthesis. These effects are primarily beneficial for the patient's vascular system. A significant number of statin users suffer from muscle complaints probably due to mitochondrial dysfunction, a mechanism that has recently been elucidated. This has raised our interest in exploring the effects of statins on cardiac muscle cells in an era where the elderly and patients with poorer functioning hearts and less metabolic spare capacity start dominating our patient population. Here, we investigated the effects of statins on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs). hiPSC-derived CMs were exposed to simvastatin, atorvastatin, rosuvastatin, and cerivastatin at increasing concentrations. Metabolic assays and fluorescent microscopy were employed to evaluate cellular viability, metabolic capacity, respiration, intracellular acidity, and mitochondrial membrane potential and morphology. Over a concentration range of 0.3-100 µM, simvastatin lactone and atorvastatin acid showed a significant reduction in cellular viability by 42-64%. Simvastatin lactone was the most potent inhibitor of basal and maximal respiration by 56% and 73%, respectively, whereas simvastatin acid and cerivastatin acid only reduced maximal respiration by 50% and 42%, respectively. Simvastatin acid and lactone and atorvastatin acid significantly decreased mitochondrial membrane potential by 20%, 6% and 3%, respectively. The more hydrophilic atorvastatin acid did not seem to affect cardiomyocyte metabolism. This calls for further research on the translatability to the clinical setting, in which a more conscientious approach to statin prescribing might be considered, especially regarding the current shift in population toward older patients with poor cardiac function., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell-cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co-isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co-isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC., Competing Interests: PV serves on the scientific advisory board of Evox Therapeutics Ltd. SEB has equity and interests in Evox Therapeutics Ltd, and has held an employment contract with Evox Therapeutics Ltd., Oxford, UK, during the completion of this article. MJAW is a cofounder of Evox Therapeutics, Pepgen, Orfonyx and Isogenix. MJAW is a scientific advisory board member of Evox Therapeutics and Pepgen, and is a Director of Oxford University Innovation. The University of Oxford has patents issued and/or pending for oligonucleotide technologies and delivery technologies (on which MJAW is an inventor). Evox Therapeutics, Pepgen, Orfonyx and Isogenix had no influence on the conception or realisation of this article., (© 2024 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge., Methods: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming., Results: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties., Conclusions: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT., (© 2024. The Author(s).)

The aim was to optimize the perfusate composition by including a hemofiltrator to the PhysioHeartplatform for ex situ heart perfusion of porcine slaughterhouse hearts. Fourteen hearts were harvested from Dutch Landrace pigs and slaughtered for human consumption. All hearts were preserved for 4 hours using static cold storage before reperfusion for 4 hours on the PhysioHeart platform. Seven hearts were assigned to the hemofiltration group, where a hemofiltrator was added to the perfusion circuit, while the control group did not receive hemofiltration. In the hemofiltration group, the perfusion fluid was filtrated for 1 hour with a flow of 1 L/hour before reperfusion. After mounting the heart, hemofiltration was maintained at 1 L/hour, and cardiac function and blood samples were analyzed at multiple time points. Preserved cardiac function was defined as a cardiac output >3.0 L/min with a mean aortic pressure >60 mm Hg and a left atrial pressure <15 mm Hg. Hemofiltration resulted in a significantly reduced potassium concentration at all time points ( p < 0.001), while sodium levels remained at baseline values ( p < 0.004). Furthermore, creatinine and ammonia levels decreased over time. Functional assessment demonstrated a reduced left atrial pressure ( p < 0.04) and a reduction of the required dobutamine dose to support myocardial function ( p < 0.003) in the hemofiltration group. Preserved cardiac function did not differ between groups. Hemofiltration results in an improved biochemical composition of the whole blood perfusate and preserves cardiac function better during normothermic perfusion based on a reduced left atrial pressure (LAP) and dobutamine requirement to support function., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2023.)

The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)