Fangping Yuan

Chronic AMD3100 antagonism of SDF-1α–CXCR4 exacerbates cardiac dysfunction and remodeling after myocardial infarction

The role of the SDF-1alpha-CXCR4 axis in response to myocardial infarction is unknown. We addressed it using the CXCR4 antagonist, AMD3100, to block SDF-1alpha interaction with CXCR4 after chronic coronary artery ligation. Chronic AMD3100 treatment decreased ejection fraction and fractional shortening in mice 20days after myocardial infarction compared with vehicle-treated mice (echocardiography). Morphometric analysis showed hearts of AMD3100-treated infarcted mice to have expanded scar, to be hypertrophic (confirmed by myocyte cross-section area) and dilated, with increased LV end systolic and end diastolic dimensions, and to have decreased scar collagen content; p-AKT levels were attenuated and this was accompanied by increased apoptosis. Despite increased injury, c-kit(pos) cardiac progenitor cells (CPCs) were increased in the risk region of AMD3100-treated infarcted mice; CPCs were CD34(neg)/CD45(neg) with the majority undergoing symmetric cell division. c-kit(pos)/MHC(pos) CPCs also increased in the risk region of the AMD3100-treated infarcted group. In this group, GSK-3beta signaling was attenuated compared to vehicle-treated, possibly accounting for increased proliferation and increased cardiac committed MHC(pos) CPCs. Increased proliferation following AMD3100 treatment was supported by increased levels of cyclin D1, a consequence of increased prolyl isomerase, Pin1, and decreased cyclin D1 phosphorylation. In summary, pharmacologic antagonism of CXCR4 demonstrates that SDF-1alpha-CXCR4 signaling plays an important role during and after myocardial infarction and that it exerts pleiotropic salubrious effects, protecting the myocardium from apoptotic cell death, facilitating scar formation, restricting CPC proliferation, and directing CPCs toward a cardiac fate.

Effects of Physiologic Mechanical Stimulation on Embryonic Chick Cardiomyocytes Using a Microfluidic Cardiac Cell Culture Model

Hemodynamic mechanical cues play a critical role in the early development and functional maturation of cardiomyocytes (CM). Therefore, tissue engineering approaches that incorporate immature CM into functional cardiac tissues capable of recovering or replacing damaged cardiac muscle require physiologically relevant environments to provide the appropriate mechanical cues. The goal of this work is to better understand the subcellular responses of immature cardiomyocytes using an in vitro cardiac cell culture model that realistically mimics in vivo mechanical conditions, including cyclical fluid flows, chamber pressures, and tissue strains that could be experienced by implanted cardiac tissues. Cardiomyocytes were cultured in a novel microfluidic cardiac cell culture model (CCCM) to achieve accurate replication of the mechanical cues experienced by ventricular CM. Day 10 chick embryonic ventricular CM (3.5 × 104 cell clusters per cell chamber) were cultured for 4 days in the CCCM under cyclic mechanical stimulation (10 mmHg, 8–15% stretch, 2 Hz frequency) and ventricular cells from the same embryo were cultured in a static condition for 4 days as controls. Additionally, ventricular cell suspensions and ventricular tissue from day 16 chick embryo were collected and analyzed for comparison with CCCM cultured CM. The gene expressions and protein synthesis of calcium handling proteins decreased significantly during the isolation process. Mechanical stimulation of the cultured CM using the CCCM resulted in an augmentation of gene expression and protein synthesis of calcium handling proteins compared to the 2D constructs cultured in the static conditions. Further, the CCCM conditioned 2D constructs have a higher beat rate and contractility response to isoproterenol. These results demonstrate that early mechanical stimulation of embryonic cardiac tissue is necessary for tissue proliferation and for protein synthesis of the calcium handling constituents required for tissue contractility. Thus, physiologic mechanical conditioning may be essential for generating functional cardiac patches for replacement of injured cardiac tissue.

The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages

Human induced pluripotent stem cells (hiPSCs) are a robust source for cardiac regenerative therapy due to their potential to support autologous and allogeneic transplant paradigms. The in vitro generation of three-dimensional myocardial tissue constructs using biomaterials as an implantable hiPSC-derived myocardium provides a path to realize sustainable myocardial regeneration. We generated engineered cardiac tissues (ECTs) from three cellular compositions of cardiomyocytes (CMs), endothelial cells (ECs), and vascular mural cells (MCs) differentiated from hiPSCs. We then determined the impact of cell composition on ECT structural and functional properties. In vitro force measurement showed that CM+EC+MC ECTs possessed preferential electromechanical properties versus ECTs without vascular cells indicating that incorporation of vascular cells augmented tissue maturation and function. The inclusion of MCs facilitated more mature CM sarcomeric structure, preferential alignment, and activated multiple tissue maturation pathways. The CM+EC+MC ECTs implanted onto infarcted, immune tolerant rat hearts engrafted, displayed both host and graft-derived vasculature, and ameliorated myocardial dysfunction. Thus, a composition of CMs and multiple vascular lineages derived from hiPSCs and incorporated into ECTs promotes functional maturation and demonstrates myocardial replacement and perfusion relevant for clinical translation.

Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue

The current study describes a scalable, porous large-format engineered cardiac tissue (LF-ECT) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies. We explored multiple 15 × 15 mm ECT geometries using molds with rectangular internal staggered posts (mesh, ME), without posts (plain sheet, PS), or long parallel posts (multiple linear bundles, ML) and a gel matrix containing hiPSC-derived cardiomyocytes, endothelial, and vascular mural cells matured in vitro for 14 days. ME-ECTs displayed the lowest dead cell ratio (p < 0.001) and matured into 0.5 mm diameter myofiber bundles with greater 3D cell alignment and higher active stress than PS-ECTs. Increased initial ECT cell number beyond 6 M per construct resulted in reduced cell survival and lower active stress. The 6M-ME-ECTs implanted onto 1 week post-infarct immune tolerant rat hearts engrafted, displayed evidence for host vascular coupling, and recovered myocardial structure and function with reduced scar area. We generated a larger (30 × 30 mm) ME-ECT to confirm scalability. Thus, large-format ECTs generated from hiPSC-derived cardiac cells may be feasible for large animal preclinical cardiac regeneration paradigms.

Neonatal Murine Engineered Cardiac Tissue Toxicology Model: Impact of Metallothionein Overexpression on Cadmium-Induced Injury

Engineered cardiac tissues (ECTs) serve as robust in vitro models to study human cardiac diseases including cardiac toxicity assays due to rapid structural and functional maturation and the ability to vary ECT composition. Metallothionein (MT) has been shown to be cardioprotective for environmental toxicants including heavy metals. To date, studies on the role of cardiomyocyte (CM)-specific MT expression and function have occurred in dissociated single cell assays or expensive in vivo small animal models. Therefore, we generated 3D ECTs using neonatal mouse ventricular cells from wild-type (WT) and the CM-specific overexpressing MT-transgenic (MT-TG) to determine the effect of MT overexpression on ECT maturation and function. Because Cadmium (Cd) is an environmentally prevalent heavy metal toxicant with direct negative impact on cardiac structure and function, we then determined the effect of MT overexpression to reduce Cd mediated CM toxicity within ECTs. We found: (1) structural and functional maturation was similar in WT and MT-TG ECTs; (2) Cd exposure negatively impacted ECT cell survival, maturation, and function; and (3) MT-ECTs showed reduced Cd toxicity as defined by reduced cleaved caspase 3, reduced Bax/Bcl2 ratio, reduced TdT-mediated dUTP nick-end labeling positive cells, reduced CM loss after Cd treatment, and delayed onset of cardiac dysfunction after Cd treatment. Thus, neonatal murine ECTs can serve as a robust in vitro model for heavy metal toxicity screening and as a platform to evaluate the role cardioprotective mechanisms, such as the MT-TG model, on environmentally relevant toxicants.

Engineered Cardiac Tissues Generated from Immature Cardiac and Stem Cell-Derived Cells: Multiple Approaches and Outcomes

The translation of in vitro engineered cardiac tissues (ECTs) from immature cardiac and stem cell-derived cells toward clinical therapies is benefiting from the following major advances: (1) rapid progress in the generation of immature cardiac cells from the cardiac and noncardiac cells of multiple species including normal and disease human cells, (2) incorporation of multiple cell lineages into 3D tissues, (3) multiple scalable 3D formulations including injectable gels and implantable tissues, and (4) insights into the regulation of cardiomyocyte proliferation and functional maturation. These advances are based on insights gained from investigating the regulation of cardiac morphogenesis and adaptation. Our lab continues to explore this approach, including changes in gene expression that occur in response to mechanical loading and tyrosine kinase inhibition, the incorporation of vascular fragments into ECTs, and the fabrication of porous implantable electrical sensors for in vitro conditioning and postimplantation testing. Significant challenges remain including optimizing ECT survival postimplantation and limited evidence of ECT functional coupling to the recipient myocardium. One clear focus of current research is the optimization and expansion of the cellular constituents, including CM, required for clinical-grade ECTs. Another major area of investigation will be large animal preclinical models that more accurately represent human CV failure and that can generate data in support of regulatory approval for phase I human clinical trials. The generation of reproducible human ECTs creates the opportunity to develop in vitro myocardial surrogate tissues for novel drug therapeutics and toxicity assays.