Large animal models are essential for the development of novel therapeutics for myocardial infarction. To optimize translation, we need to assess the effect of experimental design on disease outcome and model experimental design to resemble the clinical course of MI. The aim of this study is therefore to systematically investigate how experimental decisions affect outcome measurements in large animal MI models. We used control animal-data from two independent meta-analyses of large animal MI models. All variables of interest were pre-defined. We performed univariable and multivariable meta-regression to analyze whether these variables influenced infarct size and ejection fraction. Our analyses incorporated 246 relevant studies. Multivariable meta-regression revealed that infarct size and cardiac function were influenced independently by choice of species, sex, co-medication, occlusion type, occluded vessel, quantification method, ischemia duration and follow-up duration. We provide strong systematic evidence that commonly used endpoints significantly depend on study design and biological variation. This makes direct comparison of different study-results difficult and calls for standardized models. Researchers should take this into account when designing large animal studies to most closely mimic the clinical course of MI and enable translational success.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch heart foundation Dekker Senior Scientist grant Myocardial infarction is a global health burden for which there is no treatment available that aims to recover the damaged tissue after the ischemic event. Lipid nanoparticles (LNPs) represent a well characterized class of mRNA delivery systems, which were recently approved for clinical usage in their application for mRNA-based covid-19 vaccines. After myocardial infarction, endogenous mechanisms that enable repair of the functional damaged tissue can be triggered by modified mRNA (modRNA) delivery, locally in the infarcted area. As a first step, in order to optimize the LNP formulation for effective myocardial delivery and study cellular tropism of the LNPs in the heart, different LNPs formulations will be evaluated as delivery systems in a murine healthy heart model. Different LNP formulations varying in type and amount of helper lipid were used as delivery systems for modRNA encoding the reporter genes luciferase or eGFP. In vitro, LNPs were evaluated for modRNA delivery in a human endothelial cell line (HMEC-1), induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and induced pluripotent stem cell -derived fibroblasts (iPS-FBs). In vivo, modRNA delivery was evaluated in C57BL-6 mice, undergoing open chest heart surgery under general anaesthesia in order to infuse LNPs into the left ventricular wall. For determination of luciferase expression levels, animals were infused with luciferin substrate intraperitoneally 24 hrs after injection. Heart, liver, lungs, spleen and kidneys were extracted for imaging in a bioluminescence imaging system. The organs were then stored in liquid nitrogen for further ex-vivo modRNA delivery analysis. For determining cellular tropism, histology was performed on mice treated with eGFP modRNA. Both bioluminescence imaging and luminescence analysis in tissue lysates showed that mRNA transfection is achieved in the myocardium 24 hours after LNP intramyocardial administration. However, all LNP formulations also resulted in high expression levels in other organs, including liver and spleen.  Changes in type or amount of helper lipid in LNPs strongly affected transfection levels. Histology of the treated hearts revealed a distinct transfection pattern. The targeted, interstitial cells were negative for CD31 (marker for endothelial cells and monocytes) and Troponin I3 (marker for cardiomyocytes) (Figure 1). We show that, using an optimized LNP formulation, a significant degree of modRNA local transfection of the heart can be achieved. However, despite the local route of administration (into the left ventricular wall), the highest LNP transfection is shown in remote organs such as liver and spleen. More improvements of the LNP formulations must be done to increase their tropism towards the heart tissue for their optimization as cardiac delivery systems. Determining which cell types are being targeted is also important in order to establish a therapeutic target when applying the LNPs for cardiac therapy.

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC Consolidator Grant: EVICARE Background Extracellular vesicle (EVs) are small, cell-derived lipid bilayer enclosed particles that play a role in intercellular communication through the delivery of their content which includes nucleic acids and proteins. Cardiac progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against ischemia/reperfusion injury via proangiogenic effects. However, the underlying mechanisms for CPC-EV-mediated angiogenesis remain elusive. Here, we investigated protein-mediated effects of CPC-EVs on the endothelium, and explored EV-dependent and –independent recipient cell activation. Methods CPCs were stimulated with calcium ionophore (Ca ion-EVs), previously shown to influence EV release, or vehicle (veh-EVs) for 24 hours and crude EVs were isolated using size exclusion chromatography (SEC). EV concentration and size was assessed using nanoparticle tracking analysis and proteomic composition was profiled using mass spectrometry. Following SEC, iodixanol gradient ultracentrifugation was used to separate EVs from free proteins. CPC-EVs deficient in individual proteins were generated using CRISPR/Cas9 machinery. EV- and protein fractions were functionally characterized based on their potency to activate human microvascular endothelial cells (HMEC-1) and induce wound closure. HMEC-1 activation upon EV-delivery was determined by phosphoproteomics. Results HMEC-1 displayed increased wound closure and activation of AKT-mTOR and (Insulin/IGF-) MAPK signaling pathways upon stimulation with veh-EVs but not with Ca ion-EVs, confirmed by phosphoproteomic analysis. MS-proteomic analysis identified multiple proteins strongly enriched in veh-EVs compared with Ca ion-EVs. GO analysis of these candidate proteins revealed their involvement cell migration and –adhesion. This raised the question whether these identified proteins were truly associated to CPC-EVs, or merely co-isolated. Pure EVs isolated using iodixanol gradients lost part of their ability to activate HMEC-1 compared to crude EV preparations. This hints towards a co-stimulatory role of co-isolated proteins in recipient cell activation. When investigating the contribution of individual candidate proteins to CPC-EV functionality, knock-out of NID1 did not affect EV function, while knock-out of PAPP-A resulted in CPC-EVs with reduced functionality. The IGF-receptor inhibitor PPP abrogated CPC-EV-induced HMEC-1 activation, supporting the association of EV-associated PAPP-A with the activation of intracellular IGF-1-MAPK signaling. Conclusions A specific set of EV proteins including PAPP-A is identified that may be functionally responsible for the activation of endothelial cells upon exposure to CPC-EVs. It is important to identify if these proteins are EV-associated or represent co-isolated factors that contribute to endothelial cell activation. This may lead to a better mechanistic understanding of CPC-EV-mediated cell activation and translation of EV-mediated therapeutics.

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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.