Michael Regnier

Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture

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 3-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 stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned 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 coculture 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.

Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue

Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemic injury. Here, we report the development of scaffold-free prevascularized human heart tissue that survives in vivo transplantation and integrates with the host coronary circulation. Human embryonic stem cells (hESCs) were differentiated to cardiomyocytes by using activin A and BMP-4 and then placed into suspension on a rotating orbital shaker to create human cardiac tissue patches. Optimization of patch culture medium significantly increased cardiomyocyte viability in patch centers. These patches, composed only of enriched cardiomyocytes, did not survive to form significant grafts after implantation in vivo. To test the hypothesis that ischemic injury after transplantation would be attenuated by accelerated angiogenesis, we created “second-generation,” prevascularized, and entirely human patches from cardiomyocytes, endothelial cells (both human umbilical vein and hESC-derived endothelial cells), and fibroblasts. Functionally, vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics more similar to myocardium than patches comprising only cardiomyocytes. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed only of cardiomyocytes. Moreover, the preformed human microvessels anastomosed with the rat host coronary circulation and delivered blood to the grafts. Thus, inclusion of vascular and stromal elements enhanced the in vitro performance of engineered human myocardium and markedly improved viability after transplantation. These studies demonstrate the importance of including vascular and stromal elements when designing human tissues for regenerative therapies.

Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including β-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells

BACKGROUND Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. METHODS AND RESULTS A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. CONCLUSIONS Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

Research Priorities in Hypertrophic Cardiomyopathy Report of a Working Group of the National Heart, Lung, and Blood Institute

Hypertrophic cardiomyopathy (HCM) is a myocardial disorder characterized by left ventricular (LV) hypertrophy without dilatation and without apparent cause (ie, it occurs in the absence of severe hypertension, aortic stenosis, or other cardiac or systemic diseases that might cause LV hypertrophy). Numerous excellent reviews and consensus documents provide a wealth of additional background.1–8 HCM is the leading cause of sudden death in young people and leads to significant disability in survivors. It is caused by mutations in genes that encode components of the sarcomere. Cardiomyocyte and cardiac hypertrophy, myocyte disarray, interstitial and replacement fibrosis, and dysplastic intramyocardial arterioles characterize the pathology of HCM. Clinical manifestations include impaired diastolic function, heart failure, tachyarrhythmia (both atrial and ventricular), and sudden death. At present, there is a lack of understanding of how the mutations in genes encoding sarcomere proteins lead to the phenotypes described above. Current therapeutic approaches have focused on the prevention of sudden death, with implantable cardioverter defibrillator placement in high-risk patients. But medical therapies have largely focused on alleviating symptoms of the disease, not on altering its natural history. The present Working Group of the National Heart, Lung, and Blood Institute brought together clinical, translational, and basic scientists with the overarching goal of identifying novel strategies to prevent the phenotypic expression of disease. Herein, we identify research initiatives that we hope will lead to novel therapeutic approaches for patients with HCM. The epidemiology of HCM suggests that it is present in ≈1 in 500 adults.9 Because of the delay in phenotypic expression of the disease, HCM is not commonly recognized clinically in young children, but when it is, it is much more frequently recognized in males.10 This is likely due to greater penetrance in young males.11,12 HCM is underdiagnosed clinically in blacks and in women, yet …

Effects of the N-terminal Domains of Myosin Binding Protein-C in an in Vitro Motility Assay EVIDENCE FOR LONG-LIVED CROSS-BRIDGES

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 ∼110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.

Calcium Regulation of Tension Redevelopment Kinetics with 2-Deoxy-ATP or Low [ATP] in Rabbit Skeletal Muscle

The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.5 mM, respectively. The activation dependence of k(tr) and unloaded shortening velocity (Vu) was measured with each substrate. With 5.0 mM ATP, Vu depended linearly on tension (P), whereas k(tr) exhibited a nonlinear dependence on P, being relatively independent of P at submaximum levels and rising steeply at P > 0.6-0.7 of maximum tension (Po). With dATP, Vu was 25% greater than control at Po and was elevated at all P > 0.15Po, whereas Po was unchanged. Furthermore, the Ca(+2) sensitivity of both k(tr) and P increased, such that the dependence of k(tr) on P was not significantly different from control, despite an elevation of Vu and maximal k(tr). In contrast, lowering [ATP] caused a slight (8%) elevation of Po, no change in the Ca(+2) sensitivity of P, and a decrease in Vu at all P. Moreover, k(tr) was decreased relative to control at P > 0.75Po, but was elevated at P < 0.75Po. These data demonstrate that the cross-bridge cycling rate dominates k(tr) at maximum but not submaximum levels of Ca(2+) activation.

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca2+ relations

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady‐state Ca2+‐activated force was studied. Native troponin C (TnC) was extracted from single, de‐membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca2+ coordination at N‐terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca2+, both sTnC and xxsTnC bind with similar apparent affinity to sTnC‐extracted thin filaments. Endogenous sTnC‐extracted fibres reconstituted with 100 % xxsTnC did not develop Ca2+‐activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca2+‐activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca2+ binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10–12 actins. As the number of FUs was decreased, Ca2+ sensitivity of force (pCa50) decreased proportionally. The slope of the force‐pCa relation (the Hill coefficient, nH) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, nH was reduced to 1.7 ± 0.2, compared with 3.8 ± 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near‐neighbour FUs along the thin filament (via head‐to‐tail tropomyosin interactions).

Substrate stiffness increases twitch power of neonatal cardiomyocytes in correlation with changes in myofibril structure and intracellular calcium

During neonatal development, there is an increase in myocardial stiffness that coincides with an increase in the contractility of the heart. In vitro assays have shown that substrate stiffness plays a role in regulating the twitch forces produced by immature cardiomyocytes. However, its effect on twitch power is unclear due to difficulties in measuring the twitch velocity of cardiomyocytes. Here, we introduce what we consider a novel approach to quantify twitch power by combining the temporal resolution of optical line scanning with the subcellular force resolution of micropost arrays. Using this approach, twitch power was found to be greater for cells cultured on stiffer posts, despite having lower twitch velocities. The increased power was attributed in part to improved myofibril structure (increased sarcomere length and Z-band width) and intracellular calcium levels. Immunofluorescent staining of α-actin revealed that cardiomyocytes had greater sarcomere length and Z-band width when cultured on stiffer arrays. Moreover, the concentration of intracellular calcium at rest and its rise with each twitch contraction was greater for cells on the stiffer posts. Altogether, these findings indicate that cardiomyocytes respond to substrate stiffness with biomechanical and biochemical changes that lead to an increase in cardiac contractility.

2-Deoxy-ATP Enhances Contractility of Rat Cardiac Muscle

To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V(max) of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K(m) for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F(max)), rate of tension redevelopment (k(tr)), and unloaded shortening velocity (V(u)) in trabeculae from both untreated and PTU-treated rats (at maximal Ca(2+) activation), and F-actin sliding speed in an in vitro motility assay (V(f)). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F(max) is unchanged even though k(tr), V(u), and V(f) are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.