Rationale The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood. Objective We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches. Methods and Results Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a three dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic strain markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) vs. unstrained constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ~10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation. Conclusions Our results indicate that both mechanical load and vascular cell co-culture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Background Tissue engineering enables the generation of functional human cardiac tissue using cells derived in vitro in combination with biocompatible materials. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electrical pacing on the maturation of hiPSC-derived cardiac tissues. Methods Cardiomyocytes derived from hiPSCs were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electrical pacing conditions. Results The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm2 vs 0.055±0.009 mN/mm2 for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 expression, which correlated with a less negative force-frequency relationship. When electrical pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm2), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 and SERCA2 further increased by combined static stress and electrical stimulation. Conclusions These studies demonstrate that electrical pacing and mechanical stimulation promote maturation of the structural, mechanical and force generation properties of hiPSC-derived cardiac tissues.

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of ϳ110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where fila- Myosin binding protein-C (MyBP-C)2 is a sarcomeric protein associated with the thick filaments of vertebrate striated muscle (1). Although the precise function of MyBP-C within the sarcomere is not well understood, evidence from MyBP-C knock-out mice that lack cardiac MyBP-C (2) indicate cMyBP-C slows cross-bridge cycling and rates of force development, especially at submaximal [Ca 2ϩ ] (3-5). The idea that MyBP-C limits cross-bridge kinetics was initially proposed by Hofmann et al. (6) who suggested that MyBP-C acts as an internal load within the sarcomere based on their observations that partial extraction of MyBP-C from skeletal fibers reversibly accelerated a low velocity phase of shortening at submaximal Ca 2ϩ activation (7). Although the exact structural arrangement of MyBP-C within the sarcomere is not known, MyBP-C could contribute to an internal load by tethering myosin heads to the thick filament and thereby limiting the extension of attached myosin heads as shortening proceeds (6). Consistent with this idea, Calaghan et al. (8) proposed that simultaneous binding of MyBP-C to two positions on myosin, i.e. to myosin S2 (near the S1/S2 junction) and to the light meromyosin segment of myosin rod, could restrict the extension of myosin heads away from the thick filament. The net effect might be to limit myosin interactions with actin. However, a recombinant MyBP-C protein containing only the C1C2 domains and thus a single S2 binding site increased Ca 2ϩ sensitivity of force in myocytes from cMyBP-C knock-out mice (9). Because effects of C1C2 did not depend on a second myosin binding site, the results implied that the C1C2 domains could affect actomyosin interactions independent of tethering myosin heads to thick filaments.The current experiments were performed to investigate mechanisms by which N-terminal domains of MyBP-C influence myosin contractile properties and whether these effects depend on organization of myosin into thick filaments. Results from in vitro motility assays demonstrate that organized thick filaments are not required for recombinant proteins containing N-terminal domains of MyBP-C to affect mechanical properties of myosin and further suggest that effects of MyBP-C to slow cross-bridge kinetics may be due to slow...

Cooperativity in contractile behavior of myofilament systems almost assuredly arises because of interactions between neighboring sites. These interactions may be of different kinds. Tropomyosin thin-filament regulatory units may have neighbors in steric blocking positions (off) or steric permissive positions (on). The position of these neighbors influence the tendency for the regulatory unit to assume the on or off state. Likewise, the tendency of a myosin cross-bridge to achieve a force-bearing state may be influenced by whether neighboring cross-bridges are in force-bearing states. Also, a cross-bridge in the force-bearing state may influence the tendency of a regulatory unit to enter the on state. We used a mathematical model to examine the influence of each of these three kinds of neighbor interactions on the steady-state force-pCa relation and on the dynamic force redevelopment process. Each neighbor interaction was unique in its effects on maximal Ca(2+)-activated force, position, and symmetry of the force-pCa curve and on the Hill coefficient. Also, each neighbor interaction had a distinctive effect on the time course of force development as assessed by its rate coefficient, k(dev). These diverse effects suggest that variations in all three kinds of nearest-neighbor interactions may be responsible for a wide variety of currently unexplained observations of myofilament contractile behavior.