Functional and Mechanistic Analysis of Mesenchymal Stem Cell Secretome to Ameliorate Ischemic Damage of Porcine Heart ex vivo and Human Myocardium

Veterans Health Administration, Decatur PA

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Abstract

Heart transplantation is a key life-saving therapy for patients with end-stage heart failure. However, donor hearts are in severely short supply, resulting in death of many transplantation candidates before a suitable donor heart becomes available. Despite this, approximately 70% of cardiac allografts were rejected for transplantation during 2019-2021; the majority do not meet acceptance criteria, including the limited acceptable time that the heart experiences warm ischemia, which is longer with donation after circulatory death (DCD) compared to donation after brain death, the traditional method. This time correlates with progression of myocardial ischemia/reperfusion (I/R) injury. Ameliorating I/R injury, that occurs both during heart procurement and transport, would improve preservation of graft function, expand the donor pool and increase access to heart transplantation. Human adipose-derived stromal cells (hASC) are abundant, simple to isolate and expand rapidly in vitro, and produce paracrine factors that improve many pathologies, including ischemia/reperfusion injury. In the context of myocardial infarction, they improve myocardial function, inhibit apoptosis, and stimulate angiogenesis. Pre-treatment of explanted hearts with hASC improves myocardial functional recovery following acute I/R injury in an ex-vivo heart perfusion system. Similarly, pre-ischemic infusion of ASC-derived factors (secretome, ASC-S) improved myocardial function during recovery from cold ischemia, and almost completely preserved normal expression of myocardial genes governing mitochondrial processes. A media supplement, B27, with many similarities to hASC-S, ameliorated mitochondrial metabolic problems associated with cold ischemic exposure of both rodent hearts and human iPS-derived cardiomyocytes (hiPS-CM). Finally, both hASC- S and B27 enhanced recovery of beating rate and velocity in hiPS-CM after DCD-like ischemia/reperfusion. Accordingly, the hypothesis that hASC-secretome will ameliorate warm ischemia/reperfusion-induced deterioration of adult human and pig heart slice cultures in vitro, and model donor hearts ex vivo, by mechanisms mediated by soluble factors as well as exosomes, which limit damage to cardiomyocytes by preserving mitochondrial energetics and mitigating reactive oxygen species production and apoptosis will be tested. To do that, three specific aims will be employed: Aim 1. Evaluate the protective effect of ASC-S on pig and human adult heart slices in long-term culture in conditions modeling procurement and transport conditions of DCD hearts, and whether this is best mediated by secretome, its components or B27. Slices of adult human or pig hearts exposed to warm ischemia in vitro in the context of cardiac procurement will be incubated with either ASC-S, or ASC-S derived exosomes, the exosome-free fraction or B27. Time and dose-dependent effects of treatments will be evaluated on apoptosis, infarct area, beat recovery and markers of cell damage. The optimally performing preparation will be studied in Aims 2 and 3. Aim 2. Evaluate the protective effects of extracorporeal infusion of ASC-S or its component on normal pig donor heart preservation during cold static storage following donation after circulatory death (DCD). ASC-S or its components preservation of normal pig hearts ex vivo will be tested as a preclinical model of DCD organ procurement and transport. Heart function will be assessed in a Langendorff preparation. Aim 3. Establishment of the biochemical mechanisms by which ACS-S preserves cardiomyocyte bioenergetics Using a comprehensive suite of biochemical diagnostics (physiological assessment of OXPHOS conductance via high-resolution respirometry, dehydrogenase activity, mitochondrial membrane potential, hydrogen peroxide emission and scavenging and activity of mitochondrial enzymes), we will define the biochemical mechanisms for improved cardiomyocyte energetics with ASC-S treatment in human and pig heart slice cultures and porcine hearts.