Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.

Consistent success in life-supporting porcine cardiac xenotransplantation.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.

Correction: Samak et al. Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes. Int. J. Mol. Sci. 2023, 24, 2482.

In the original publication [...].

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function.

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

Free-breathing myocardial T1 mapping using inversion-recovery radial FLASH and motion-resolved model-based reconstruction.

PURPOSE: To develop a free-breathing myocardial T 1 $$ {\mathrm{T}}_1 $$ mapping technique using inversion-recovery (IR) radial fast low-angle shot (FLASH) and calibrationless motion-resolved model-based reconstruction. METHODS: Free-running (free-breathing, retrospective cardiac gating) IR radial FLASH is used for data acquisition at 3T. First, to reduce the waiting time between inversions, an analytical formula is derived that takes the incomplete T 1 $$ {\mathrm{T}}_1 $$ recovery into account for an accurate T 1 $$ {\mathrm{T}}_1 $$ calculation. Second, the respiratory motion signal is estimated from the k-space center of the contrast varying acquisition using an adapted singular spectrum analysis (SSA-FARY) technique. Third, a motion-resolved model-based reconstruction is used to estimate both parameter and coil sensitivity maps directly from the sorted k-space data. Thus, spatiotemporal total variation, in addition to the spatial sparsity constraints, can be directly applied to the parameter maps. Validations are performed on an experimental phantom, 11 human subjects, and a young landrace pig with myocardial infarction. RESULTS: In comparison to an IR spin-echo reference, phantom results confirm good T 1 $$ {\mathrm{T}}_1 $$ accuracy, when reducing the waiting time from 5 s to 1 s using the new correction. The motion-resolved model-based reconstruction further improves T 1 $$ {\mathrm{T}}_1 $$ precision compared to the spatial regularization-only reconstruction. Aside from showing that a reliable respiratory motion signal can be estimated using modified SSA-FARY, in vivo studies demonstrate that dynamic myocardial T 1 $$ {\mathrm{T}}_1 $$ maps can be obtained within 2 min with good precision and repeatability. CONCLUSION: Motion-resolved myocardial T 1 $$ {\mathrm{T}}_1 $$ mapping during free-breathing with good accuracy, precision and repeatability can be achieved by combining inversion-recovery radial FLASH, self-gating and a calibrationless motion-resolved model-based reconstruction.

Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes.

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease.

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.

AIMS: Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. METHODS AND RESULTS: Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. CONCLUSION: We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Agrin Promotes Coordinated Therapeutic Processes Leading to Improved Cardiac Repair in Pigs.

BACKGROUND: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. METHODS: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia-reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. RESULTS: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin's mechanisms of action. CONCLUSIONS: A single dose of agrin is capable of reducing ischemia-reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.

"Empowering" Cardiac Cells via Stem Cell Derived Mitochondrial Transplantation- Does Age Matter?

With cardiovascular diseases affecting millions of patients, new treatment strategies are urgently needed. The use of stem cell based approaches has been investigated during the last decades and promising effects have been achieved. However, the beneficial effect of stem cells has been found to being partly due to paracrine functions by alterations of their microenvironment and so an interesting field of research, the "stem- less" approaches has emerged over the last years using or altering the microenvironment, for example, via deletion of senescent cells, application of micro RNAs or by modifying the cellular energy metabolism via targeting mitochondria. Using autologous muscle-derived mitochondria for transplantations into the affected tissues has resulted in promising reports of improvements of cardiac functions in vitro and in vivo. However, since the targeted treatment group represents mainly elderly or otherwise sick patients, it is unclear whether and to what extent autologous mitochondria would exert their beneficial effects in these cases. Stem cells might represent better sources for mitochondria and could enhance the effect of mitochondrial transplantations. Therefore in this review we aim to provide an overview on aging effects of stem cells and mitochondria which might be important for mitochondrial transplantation and to give an overview on the current state in this field together with considerations worthwhile for further investigations.

Tß4 Increases Neovascularization and Cardiac Function in Chronic Myocardial Ischemia of Normo- and Hypercholesterolemic Pigs.

Translations of new therapeutic options for cardiovascular disease from animal studies into a clinical setting have been hampered, in part by an improper reflection of a relevant patient population in animal models. In this study, we investigated the impact of thymosin ß4 (Tß4), which promotes collateralization and capillarization, during hypercholesterolemia, a known risk factor of coronary artery disease. Initial in vitro results highlighted an improved endothelial cell function upon Tß4 treatment under control conditions and during hypercholesterolemic stress (scratch area [pixels]: oxidized low-density lipoprotein [oxLDL], 191,924 ± 7,717; and oxLDL + Tß4, 105,621 ± 11,245). To mimic the common risk factor of hypercholesterolemia in vivo, pigs on regular (NC) or high-fat (HC) diet underwent chronic myocardial ischemia followed by recombinant adeno-associated virus (rAAV)-mediated transduction of Tß4 or LacZ as a control. We show that Tß4 overexpression improves capillarization and collateralization (collaterals: NC + rAAV.LacZ, 2.1 ± 0.5; NC + rAAV.Tß4, 6.7 ± 0.5; HC + rAAV.LacZ, 3.0 ± 0.3; and HC + rAAV.Tß4, 6.0 ± 0.4), ultimately leading to an improved myocardial function in both diet groups (ejection fraction [EF] at day 56 [%]: NC + rAAV.LacZ, 26 ± 1.1; NC + rAAV.Tß4, 45 ± 1.5; HC + rAAV.LacZ, 26 ± 2.5; and HC + rAAV.Tß4, 41 ± 2.6). These results demonstrate the potency of Tß4 in a patient-relevant large animal model of chronic myocardial ischemia.

Annexin A1 counteracts chemokine-induced arterial myeloid cell recruitment.

RATIONALE: Chemokine-controlled arterial leukocyte recruitment is a crucial process in atherosclerosis. Formyl peptide receptor 2 (FPR2) is a chemoattractant receptor that recognizes proinflammatory and proresolving ligands. The contribution of FPR2 and its proresolving ligand annexin A1 to atherosclerotic lesion formation is largely undefined. OBJECTIVE: Because of the ambivalence of FPR2 ligands, we here investigate the role of FPR2 and its resolving ligand annexin A1 in atherogenesis. METHODS AND RESULTS: Deletion of FPR2 or its ligand annexin A1 enhances atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment. Mechanistically, we identify annexin A1 as an endogenous inhibitor of integrin activation evoked by the chemokines CCL5, CCL2, and CXCL1. Specifically, the annexin A1 fragment Ac2-26 counteracts conformational activation and clustering of integrins on myeloid cells evoked by CCL5, CCL2, and CXCL1 through inhibiting activation of the small GTPase Rap1. In vivo administration of Ac2-26 largely diminishes arterial recruitment of myeloid cells in a FPR2-dependent fashion. This effect is also observed in the presence of selective antagonists to CCR5, CCR2, or CXCR2, whereas Ac2-26 was without effect when all 3 chemokine receptors were antagonized simultaneously. Finally, repeated treatment with Ac2-26 reduces atherosclerotic lesion sizes and lesional macrophage accumulation. CONCLUSIONS: Instructing the annexin A1-FPR2 axis harbors a novel approach to target arterial leukocyte recruitment. With the ability of Ac2-26 to counteract integrin activation exerted by various chemokines, delivery of Ac2-26 may be superior in inhibition of arterial leukocyte recruitment when compared with blocking individual chemokine receptors.

C-terminal variable AGES domain of Thymosin ß4: the molecule's primary contribution in support of post-ischemic cardiac function and repair.

Repairing defective cardiac cells is important towards improving heart function. Due to the frequency and severity of ischemic heart disease, management of patients featuring this type of cardiac failure receives significant interest. Previously we discovered that Thymosin ß4 (TB4), a 43 amino-acid secreted actin sequestering peptide, is beneficial for myocardial cell survival and coronary re-growth after infarction in adult mammals. Considering the regenerative potential of full-length TB4 in the heart, and that minimal structural variations alter TB4's influence on actin assembly and cell movement, we investigated how various TB4 domains affect cardiac cell behavior and post-ischemic mammalian heart function. We synthesized 17 domain combinations of full-length TB4 and analyzed their impact on embryonic cardiac cells in vitro, and after cardiac infarction in vivo. We discovered the domains of TB4 affect cardiac cell behavior distinctly. We revealed TB4 specific C-terminal tetrapeptide, AGES, increases embryonic cardiac cell migration and myocyte beating in culture, and improves adult mammalian heart function following ischemia. Investigating the molecular background and mechanism we discovered systemic injection of AGES enhances early myocyte survival by activating Akt-mediated signaling mechanisms, increases coronary vessel growth and inhibits inflammation in mice and pigs. Biodistribution analyses revealed cardiomyocytes uptake AGES efficiently in vitro and in vivo projecting a potential independent clinical utilization for the tetrapeptide. Our comprehensive domain investigations also suggest, preservation and/or restoration of cardiomyocyte communication is a target of TB4 and AGES, and critical to improve post-ischemic heart function in pigs. In summary, we identified the C-terminal four amino-acid variable end of TB4 as the essential and responsible domain for the molecule's full benefits in the hypoxic heart. Additionally, we introduced AGES as a novel, systemically applicable drug candidate to aid cardiac infarction in adult mammals.

Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model.

BACKGROUND: MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. METHODS AND RESULTS: Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid-modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly (P<0.01) reduced miR-92a expression in the infarct zone regardless of the application venue. However, catheter-based delivery, but not intravenous infusion, of LNA-92a significantly (P<0.05) reduced the infarct size compared with control LNA-treated pigs, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure (P<0.05). Histochemistry revealed that LNA-92a increased capillary density but decreased leukocyte influx and cardiac cell death. Complete loss of miR-92a in mice attenuated the infarct-related myocardial dysfunction to a larger extent than cardiomyocyte-specific miR-92a deletion. In vitro, LNA-92a protected against hypoxia/reoxygenation-induced cardiomyocyte cell death. CONCLUSIONS: Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia.

SDF-1 fused to a fractalkine stalk and a GPI anchor enables functional neovascularization.

The facilitated recruitment of vascular progenitor cells (VPCs) to ischemic areas might be a therapeutic target for neovascularization and repair. However, efficient and directed attraction of VPCs remains a major challenge in clinical application. To enhance VPC homing, we developed a fusion protein (S1FG), based on the biology of stroma-derived factor-1/CXCL12 and the mucin backbone taken from fractalkine/CXCL12. A GPI-anchor was included to link the fusion-protein to the cell surface. HUVECs transfected with S1FG were capable of increasing firm adhesion of CXCR4+-mononuclear cells (THP-1) under shear stress conditions in vitro. In an in vivo rabbit model of chronic hind limb ischemia, local S1FG application enhanced the recruitment of adoptively transferred embryonic EPCs (eEPCs) to the ischemic muscles 2.5-fold. S1FG combined with eEPC(low) (2 × 10(6)) yielded similar capillary growth as eEPC(high) (5 × 10(6)) alone. Compared to controls, collateral formation was increased in the S1FG eEPC(low) group, but not the eEPC(high) group without S1FG, whereas perfusion was found enhanced in both groups. In addition, S1FG also increased collateral formation and flow when combined with AMD3100 treatment, to increase circulating levels of endogenous VPC. These data demonstrate that the fusion protein S1FG is capable of enhancing the recruitment of exogenously applied or endogenously mobilized progenitor cells to sites of injury. Recombinant versions of S1FG applied via catheters in combination with progenitor cell mobilization may be useful in the treatment of chronic ischemic syndromes requiring improved perfusion.

Targeting microRNAs for cardiovascular therapeutics in coronary artery disease.

PURPOSE OF REVIEW: To discuss the possible therapeutic options for miRNA as targets in coronary heart disease. Noncoding RNAs regulate gene expression at a posttranscriptional state. Modulation of miRNAs might be a new therapeutic option in coronary heart disease. RECENT FINDINGS: Noncoding RNAs (long and short noncoding RNAs) might be used as biomarkers in cardiovascular disease, as they are differentially regulated and released in the pathophysiological situation of coronary heart disease. In acute myocardial infarction already a lot of miRNAs are investigated as biomarkers, still not superior to high-sensitive Troponin T. In rare cardiovascular diseases such as Tako-Tsubo cardiomyopathy or the different stages of heart failure, development of new biomarkers is even more important. In addition, miRNA inhibition via antimiRs is capable of attenuating cardiovascular disease in small and large animal models. Over-expression of 'protective' miRNAs (miR mimics) improved the outcome of cardiovascular disease in vitro and in first small animal models. SUMMARY: Noncoding RNAs are promising new biomarkers for cardiovascular disease. Directly targeting miRNA for disease modulation is possible for the specific inhibition, as well as for overexpression of 'protective' miRNAs. However, additional preclinical and clinical testing has to be performed before this therapy will enter clinical routine.

Non-human primate studies for cardiomyocyte transplantation-ready for translation?

Non-human primates (NHP) are valuable models for late translational pre-clinical studies, often seen as a last step before clinical application. The unique similarity between NHPs and humans is often the subject of ethical concerns. However, it is precisely this analogy in anatomy, physiology, and the immune system that narrows the translational gap to other animal models in the cardiovascular field. Cell and gene therapy approaches are two dominant strategies investigated in the research field of cardiac regeneration. Focusing on the cell therapy approach, several xeno- and allogeneic cell transplantation studies with a translational motivation have been realized in macaque species. This is based on the pressing need for novel therapeutic options for heart failure patients. Stem cell-based remuscularization of the injured heart can be achieved via direct injection of cardiomyocytes (CMs) or patch application. Both CM delivery approaches are in the late preclinical stage, and the first clinical trials have started. However, are we already ready for the clinical area? The present review concentrates on CM transplantation studies conducted in NHPs, discusses the main sources and discoveries, and provides a perspective about human translation.

A Novel Artificial Coronary Plaque to Model Coronary Heart Disease.

BACKGROUND: Experimental coronary artery interventions are currently being performed on non-diseased blood vessels in healthy animals. To provide a more realistic pathoanatomical scenario for investigations on novel interventional and surgical therapies, we aimed to fabricate a stenotic lesion, mimicking the morphology and structure of a human atherosclerotic plaque. METHODS: In an interdisciplinary setting, we engineered a casting mold to create an atherosclerotic plaque with the dimensions to fit in a porcine coronary artery. Oscillatory rheology experiments took place along with long-term stability tests assessed by microscopic examination and weight monitoring. For the implantability in future in vivo setups, we performed a cytotoxicity assessment, inserted the plaque in resected pig hearts, and performed diagnostic imaging to visualize the plaque in its final position. RESULTS: The most promising composition consists of gelatin, cholesterol, phospholipids, hydroxyapatite, and fine-grained calcium carbonate. It can be inserted in the coronary artery of human-sized pig hearts, producing a local partial stenosis and interacting like the atherosclerotic plaque by stretching and shrinking with the vessel wall and surrounding tissue. CONCLUSION: This artificial atherosclerotic plaque model works as a simulating tool for future medical testing and could be crucial for further specified research on coronary artery disease and is going to help to provide information about the optimal interventional and surgical care of the disease.

Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models.

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.