Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during the stressful events surrounding early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.

Project description:The first macrophages that seed the developing heart originate from the yolk sac during fetal life. While murine studies reveal important homeostatic and reparative functions in adults, we know little about their roles in the earliest stages of human heart development due to a lack of accessible tissue. Generation of bioengineered human cardiac microtissues from pluripotent stem cells models these first steps in cardiac tissue development, however macrophages have not been included in these studies. To bridge these gaps, we differentiated human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages; akin to yolk sac macrophages) that stably engrafted within cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts to study reciprocal interactions. Engraftment induced a tissue resident macrophage gene program resembling human fetal cardiac macrophages, enriched in efferocytic pathways. Functionally, hESC-macrophages induced production and maturation of cardiomyocyte sarcomeric proteins, and enhanced contractile force, relaxation kinetics, and electrical properties. Mechanistically, the primary effect of hESC-macrophages was during early microtissue formation, where they engaged in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reinforced core resident macrophage identity, reduced microtissue stress and drove hESC-cardiomyocytes to become more similar to human ventricular cardiomyocytes found in early development, both transcriptionally and metabolically. Inhibiting efferocytosis of hESC-cardiomyocytes by hESC-macrophages led to increased cell stress, impaired sarcomeric protein maturation and reduced cardiac microtissue function (contraction and relaxation). Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial, yet previously unappreciated role for human primitive macrophages in enhancing cardiac tissue function.

Project description:Disruption of peripheral circadian rhyme pathways dominantly leads to metabolic disorders. Studies on circadian rhythm proteins in the heart indicated a role for Clock or Per2 in cardiac metabolism. In fact, Per2-/- mice have larger infarct sizes with a deficient lactate production during myocardial ischemia. To test the hypothesis that cardiac Per2 represents an important regulator of cardiac metabolism during myocardial ischemia, we performed lactate measurements during reperfusion in Per1-/-, Per2-/- or wildtype mice followed by gene array studies using various ischemia-reperfusion protocols comparing wildtype and Per2-/- mice. Lactate measurements in whole blood confirmed a dominant role of Per2 for lactate production during myocardial ischemia. Surprisingly, high-throughput gene array analysis of eight different conditions on one 24-microarray plate revealed dominantly lipid metabolism as differentially regulated pathway in wildtype mice when compared to Per2-/-. In all treatment groups, the enzyme enoyl-CoA hydratase, which is essential in fatty acid beta-oxidation, was regulated in wildtype animals only. Studies using nuclear magnet resonance imaging (NMRI) confirmed altered fatty acid populations with higher mono-unsaturated fatty acid levels in hearts from Per2-/- mice. Unexpectedly, studies on gene regulation during reperfusion revealed solely pro inflammatory genes as differentially regulated 'Per2-genes'. Subsequent studies on inflammatory markers showed increasing IL6 or TNFa levels during reperfusion in Per2-/- mice. In summary, these studies reveal a novel role of cardiac Per2 for fatty acid metabolism or inflammation during myocardial ischemia and reperfusion. We pursued studies on Per2 dependent gene expression during myocardial ischemia or reperfusion to understand its impact on cardiac metabolism. We designed different ischemia and reperfusion protocols and performed a high-throughput expression profiling of 24 samples at a time using an industry-standard whole mouse gene array (Affymetrix, Mouse Gene 2.1 ST 24-Array). To understand differential gene regulation during different conditions we performed 1) 30 minutes of ischemia without reperfusion, 2) ischemic preconditioning (IP, 4 x 5 minutes ischemia and reperfusion), as known cardioprotective mechanism, and 3) 30 minutes of ischemia followed by 60 minutes of reperfusion. Based on three arrays per condition the total number of arrays was 24, which we analyzed at the same time on a multi plate array to avoid inter-array variations. Quality analysis using Partek Genomics Suite 6.6 revealed high confidence in the quality of the microarray data and all samples met 'Quality Assurance/Quality Control' (QA/QC) criteria.

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Modeling myocardial physiological growth using human pluripotent stem cell derived cardiomyocytes and 3D cardiac microtissues

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