Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening, disease modeling, and heart repair. However, the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation, we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one, whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 μN/μm. After three weeks in culture, optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload, whereas twitch velocities decreased with afterload. Consequently, the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically, the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 μN/μm) and then plateaued. Applied afterload increased sarcomere length, cardiomyocyte area and elongation, which are hallmarks of maturation. Furthermore, progressively increasing the level of afterload led to improved calcium handling, increased expression of several key markers of cardiac maturation, including a shift from fetal to adult ventricular myosin heavy chain isoforms. However, at the highest afterload condition, markers of pathological hypertrophy and fibrosis were also upregulated, although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together, our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs, while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload.

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathycausing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors. myosin filament mechanosensing | heart regulation | small-angle X-ray diffraction | cardiac muscle | Frank-Starling mechanism I n each sarcomere, the structural unit of the skeletal and cardiac muscles, myosin motors arranged in antiparallel arrays in the two halves of the thick myosin-containing filament work cooperatively, generating force and shortening by cyclic ATPdriven interactions with the interdigitating thin actin-containing filaments. The textbook model for the activation of contraction indicates that the binding to actin of myosin motors from the neighboring thick filament is controlled by a calcium-dependent structural change in the thin filament. However, in these muscles at rest, most of the myosin motors are in the off state and packed into helical tracks with 43-nm periodicity on the surface of the thick filaments (1-4), making them unavailable for binding to the thin filament and ATP hydrolysis (5, 6). Recent X-ray diffraction experiments on single fibers from skeletal muscle showed that, in addition to the canonical thin filament activation system, a thick filament mechanosensing mechanism provides a way for selective unlocking of myosin motors during high load contraction (7). This thick filament-based regulation has not yet been shown in cardiac muscle, in which several regulatory systems are significant. In contrast to skeletal muscle, during heart contraction, the internal concentration of Ca 2+ ([Ca 2+ ] i ) may not reach the full activation level, and thus, the mechanical response depends on both [Ca 2+ ] i and the sensitivity of the filaments to Ca 2+ (8, 9). For a given [Ca 2+ ] i , the maximal force i...

Thick filament mechanosensing has been proposed as the mechanism by which myosin motors in cardiac muscle become available to bind actin. Accordingly, Caremani et al., using x-ray diffraction from intact rat trabeculae, show that myosin motors fully return to their OFF state during diastole independently of inotropic interventions.

The maximum velocity at which a skeletal muscle can shorten (i.e. the velocity of sliding between the myosin filament and the actin filament under zero load, V ) is already set at the end of the latency relaxation (LR) preceding isometric force generation, ∼10 ms after the start of electrical stimulation in frog muscle fibres at 4°C. At this time, Ca -induced activation of the actin filament is maximal, while the myosin filament is in the OFF state characterized by most of the myosin motors lying on helical tracks on the filament surface, making them unavailable for actin binding and ATP hydrolysis. Here, the number of actin-attached motors per half-thick filament during V shortening (n) is estimated by imposing, on tetanized single fibres from Rana esculenta (at 4°C and sarcomere length 2.15 μm), small 4 kHz oscillations and determining the relation between half-sarcomere (hs) compliance and force during the force development following V shortening. When V shortening is superimposed on the maximum isometric force T , n decreases progressively with the increase of shortening (range 30-80 nm per hs) and, when V shortening is imposed at the end of LR, n can be as low as 1-4. Reduction of n is accounted for by a constant duty ratio of the myosin motor of ∼0.05 and a parallel switching OFF of the thick filament, providing an explanation for the very low rate of ATP utilization during extended V shortening.