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:Rationale Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and is associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with findings that microtubule detyrosination (dTyr-MTs) is markedly elevated in heart failure. Acute reduction of dTyr-MTs by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, presenting a new perspective for HCM treatment. Objective In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model (Mybpc3-knock-in; KI), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents, in 3-week-old KI mice, and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show: i) TTL for 6 weeks dose-dependently reduced dTyr-MTs and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes. ii) TTL for 12 weeks reduced the abundance of dTyr-MTs in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in KI mice. iii) TTL for 10 days normalized cell area in HCM hiPSC-cardiomyocytes. iv) TTL overexpression activates transcription of several tubulin isoforms, and omics GO analysis revealed enrichment of components of the cytoskeleton, sarcomere Z-disc, mitochondria, and intercalated disc in KI mice. v) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

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Primitive macrophages induce sarcomeric maturation and functional enhancement of contractile human cardiac microtissues via efferocytic pathways

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