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Fetal Sarcomere Development

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Goal: This project aims to better understand the link between fetal sarcomeric protein function, the force production and mechanical functions of fetal muscle, and the ongoing growth of the muscle tissue.

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This project aims to better understand the link between fetal sarcomeric protein function, the force production and mechanical functions of fetal muscle, and the ongoing growth of the muscle tissue.
 
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Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.
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Congenital contractures such as clubfoot are present in ∼1/250 live births. Several congenital contractures syndromes are caused by mutations in genes that code for skeletal myofilament proteins, including MYH3 and MYH8. Because embryonic (MYH3) and perinatal (MYH8) myosin heavy chains are unique to the prenatal development of muscle, it is important to understand the contractile properties of human embryonic (HE) myosin and human fetal (HF) muscle to determine how mutations affect performance and development. However, information on HF skeletal muscle function is lacking. We previously reported HE myosin crossbridge cycling (measured by in vitro motility) was much slower that rabbit psoas (RP) myosin (Biophys. J. (2010), 98:542a). Here we characterized the contraction and relaxation properties of HF muscle using myofibril mechanics techniques. HF skeletal muscle and myofibrils were isolated from a 15.4 week gestation fetus. During maximal calcium activation (15°C) HF myofibrils produced much lower force (FMAX=5.9±1.2mN/mm2) as compared to myofibrils from human adult (HA) skeletal muscle (84±34mN/mm2) or RP muscle (220±40mN/mm2). Unlike control HA and RP fibrils, no striation pattern was apparent for HF myofibrils, suggesting that immature sarcomeres could explain the lower force production. HF myofibrils had slower kinetics of force development (kACT=0.66±0.1s−1) vs. HA (7.5±6.3s−1) and RP (5.7±0.5s−1) myofibrils. The initial (slow) phase of relaxation upon return to low calcium solution was slower and prolonged (kREL,SLOW=0.59±0.22s−1; tREL,SLOW=174±13ms) vs. HA (2.9±1.7s−1; 71±18ms) or RP (2.1±0.4s−1; 73±14ms) myofibrils. The larger, faster phase of relaxation was also slower (HF=1.5±0.2s−1 vs. HA=12±5s−1 and RP=21±4s−1). Our previous in vitro motility experiments indicated a similar inhibition of filament speed by increasing [ADP] for HE and RP myosin, but this was under low load, thus ongoing experiments will determine if ADP release is responsible for the slower kinetics of HF muscle. Funded by F31AR06300(A.R.), 5K23HD057331(A.B.), HD048895(M.B., M.R.).
Little is known about the contraction and relaxation properties of fetal skeletal muscle, and measurements thus far have been made with non-human mammalian muscle. Data on human fetal skeletal muscle contraction is lacking, and there are no published reports on the kinetics of either fetal or adult human skeletal muscle myofibrils. Understanding the contractile properties of human fetal muscle would be valuable in understanding muscle development, as well as a variety of muscle diseases that are associated with mutations in fetal muscle sarcomere proteins. Therefore, we characterized the contractile properties of developing human fetal skeletal muscle and compared them to adult human skeletal muscle and rabbit psoas muscle. Electron micrographs showed human fetal muscle sarcomeres are not fully formed but myofibril formation is visible. Isolated myofibril mechanical measurements revealed much lower specific force, and slower rates of isometric force development, slow phase relaxation, and fast phase relaxation. The duration of slow phase relaxation was also significantly longer compared to both adult groups, but was similarly affected by elevated ADP. F-actin sliding on human fetal skeletal myosin coated surfaces in in vitro motility (IVM) assays was much slower compared with adult rabbit skeletal myosin, though the Km(app) of F-actin speed with ATP titration suggests a greater affinity of human fetal myosin for nucleotide binding. Replacing ATP with 2 deoxy-ATP (dATP) increased F-actin speed for both groups by a similar amount. Titrations of ADP into IVM assays produced a similar inhibitory affect for both groups, suggesting ADP binding may be similar, at least under low load. Together, our results suggest slower but similar mechanisms of myosin chemomechanical transduction for human fetal muscle that may also be limited by immature myofilament structure.