Engineered human cardiac tissue.

The human heart is the first organ to develop during embryogenesis and is arguably the most essential organ for life. However, after birth, the heart has very little capacity to repair malformations such as congenital heart defects or to regenerate after an injury such as myocardial infarction. Cardiac tissue engineering addresses the need for a therapeutic biologic implant to restore cardiac structure and muscle mass. This review highlights current research in cardiac tissue engineering that uses human cardiomyocytes derived from embryonic stem cells. Other human cell sources are discussed because future human therapies will benefit from novel techniques using human-induced pluripotent stem cells and cardiomyocytes derived from direct reprogramming of somatic cells. Furthermore, this review examines the main approaches to creating engineered cardiac tissue with synthetic scaffolds, natural scaffolds, or no exogenous scaffold (i.e., "scaffold free"). The choice of scaffold and cells ultimately depends on the goals of the therapy, so the review considers how congenital heart defects define the design parameters for cardiac tissue engineering needed for surgical repair in pediatric cardiac patients.

Calcium binding kinetics of troponin C strongly modulate cooperative activation and tension kinetics in cardiac muscle.

Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca(2+) binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca(2+)-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca(2+) dissociation rates (k(off)), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTn>WT cTn>L48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca(2+), L48Q cTnC did not affect maximum tension (T(max)), thin filament activation (k(ACT)) and tension development (k(TR)) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced T(max), k(ACT) and k(TR) by 40-65% with little change in relaxation. Interestingly, k(ACT) was less than k(TR) with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca(2+)-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced T(max), Ca(2+) sensitivity of tension (pCa(50)), and slope (n(H)) of tension-pCa, while L48Q cTn increased pCa(50) and reduced n(H). Increased cross-bridge cycling with 2-deoxy-ATP increased pCa(50) with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca(2+)-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.

Developing vasculature and stroma in engineered human myocardium.

We recently developed a scaffold-free patch of human myocardium with human embryonic stem cell-derived cardiomyocytes and showed that stromal and endothelial cells form vascular networks in vitro and improve cardiomyocyte engraftment. Here, we hypothesize that stromal cells regulate the angiogenic phenotype by modulating the extracellular matrix (ECM). Human marrow stromal cells (hMSCs) support the greatest degree of endothelial cell organization, at 1.3- to 2.4-fold higher than other stromal cells tested. Stromal cells produce abundant ECM components in patches, including fibrillar collagen, hyaluronan, and versican. We identified two clonal hMSC lines that supported endothelial networks poorly and robustly. Interestingly, the pro-angiogenic hMSCs express high levels of versican, a chondroitin sulfate proteglycan that modulates angiogenesis and wound healing, whereas poorly angiogenic hMSCs produce little versican. When transplanted onto uninjured athymic rat hearts, patches with proangiogenic hMSCs develop ~ 50-fold more human vessels and form anastomoses with the host circulation, resulting in chimeric vessels containing erythrocytes. Thus, stromal cells play a key role in supporting vascularization of engineered human myocardium. Different stromal cell types vary widely in their proangiogenic ability, likely due in part to differences in ECM synthesis. Comparison of these cells defines an in vitro predictive platform for studying vascular development.

Cardiogenesis from human embryonic stem cells.

Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-ß superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/ß-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/ß-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

Thin filament Ca2+ binding properties and regulatory unit interactions alter kinetics of tension development and relaxation in rabbit skeletal muscle.

The influence of Ca(2+) binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca(2+) dissociation rates (k(off)) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca(2+) at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca(2+), the rate of tension redevelopment (k(TR)) was not altered for fibres containing sTnC mutants with decreased k(off) or mixtures of sTnC:xxsTnC. We examined the influence of k(off) on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca(2+)]. In myofibrils with M80Q sTnC(F27W) (decreased k(off)), maximal tension, activation rate (k(ACT)), k(TR) and rates of relaxation were not altered. With I60Q sTnC(F27W) (increased k(off)), maximal tension, k(ACT) and k(TR) decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased k(off), respectively. For all sTnC reconstitution conditions, Ca(2+) dependence of k(TR) in fibres showed Ca(2+) sensitivity of k(TR) (pCa(50)) shifted parallel to tension and low-Ca(2+) k(TR) was elevated. Together the data suggest the Ca(2+)-dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by k(off) of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca(2+).

Influence of enhanced troponin C Ca2+-binding affinity on cooperative thin filament activation in rabbit skeletal muscle.

We studied how enhanced skeletal troponin C (sTnC) Ca2+-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca2+ dissociation, resulting in the following order of rates: wild-type (WT) sTnC-sTn>sTnC(F27W)-sTn>M80Q sTnC-sTn>M80Q sTnCF27W-sTn. Reconstitution of sTnC-extracted fibres increased Ca2+ sensitivity of steady-state force (pCa(50)) by 0.08 for M80Q sTnC, 0.15 for sTnCF27W and 0.32 for M80Q sTnCF27W with minimal loss of slope (nH, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca2+ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnCF27W: xxsTnC. It is interesting that pCa50 increased by approximately 0.1 for M80Q sTnC and approximately 0.3 for M80Q sTnCF27W when near-neighbour RU interactions were reduced; these values are similar in magnitude to those for fibres reconstituted with 100% mutant sTnC. After reconstitution with sTnC: xxsTnC mixtures, nH decreased to a similar value for all mutant sTnCs. Altered sTnC Ca2+-binding properties (M80Q sTnCF27W) did not affect strong crossbridge inhibition by 2,3-butanedione monoxime when near-neighbour thin filament RU interactions were reduced. Together these results suggest increased sTnC Ca2+ affinity strongly influences Ca2+ sensitivity of steady-state force without affecting near-neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca2+ to thin filament activation.

Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling.

We examined the influence of cross-bridge cycling kinetics on the length dependence of steady-state force and the rate of force redevelopment (k(tr)) during Ca(2+)-activation at sarcomere lengths (SL) of 2.0 and 2.3 microm in skinned rat cardiac trabeculae. Cross-bridge kinetics were altered by either replacing ATP with 2-deoxy-ATP (dATP) or by reducing [ATP]. At each SL dATP increased maximal force (F(max)) and Ca(2+)-sensitivity of force (pCa(50)) and reduced the cooperativity (n(H)) of force-pCa relations, whereas reducing [ATP] to 0.5 mM (low ATP) increased pCa(50) and n(H) without changing F(max). The difference in pCa(50) between SL 2.0 and 2.3 microm (Delta pCa(50)) was comparable between ATP and dATP, but reduced with low ATP. Maximal k(tr) was elevated by dATP and reduced by low ATP. Ca(2+)-sensitivity of k(tr) increased with both dATP and low ATP and was unaffected by altered SL under all conditions. Significantly, at equivalent levels of submaximal force k(tr) was faster at short SL or increased lattice spacing. These data demonstrate that the SL dependence of force depends on cross-bridge kinetics and that the increase of force upon SL extension occurs without increasing the rate of transitions between nonforce and force-generating cross-bridge states, suggesting SL or lattice spacing may modulate preforce cross-bridge transitions.