Dissecting healthy and diseased hearts

Poster no abstract available

Developing engineered cardiac tissue models from HL-1 cardiomyocytes and mouse embryonic fibroblasts

\u3cp\u3eEngineered cardiac tissue models become increasingly important for understanding normal and diseased cardiac physiology. The use of in-vitro engineered disease models can give more insight in the changing structurefunction properties during pathological condition; therefore, contributing to the development of new cardiac therapies. It is hypothesized that during cardiac disorders of impaired mechanotransduction, the ratios of cardiomyocytes, fibroblasts and their supporting endogenous extracellular matrix (ECM) change. Furthermore, these changes are comparable and predictable of the different stages of diseased cardiac tissue. The aim of this study is to investigate the effect of different cardiomyocyte/fibroblast ratios on tissue morphology and function. Co-cultures of the HL-1 cardiomyocyte cell line and mouse embryonic fibroblasts (MEFs) at different ratios were used in 2D feasibility studies. Cyclic mechanical straining was applied to mimic cardiac tissue deformation during contraction. Both HL-1 and MEFs survived in co-culture although clustering of HL-1 cells was observed. The cluster size of HL-1 was dependent on the amount of MEFs. Mechanical stimulation of cultures showed strain avoidance response of MEFs while co-culture with HL-1 prevented this response. The data obtained provides new insights in the usefulness of cardiac cell-line-derived HL-1 and MEFs in the development of cardiac tissue models.\u3c/p\u3

Mimicking cardiac fibrosis in a dish:fibroblast density rather than collagen density weakens cardiomyocyte function

\u3cp\u3eCardiac fibrosis is one of the most devastating effects of cardiac disease. Current in vitro models of cardiac fibrosis do not sufficiently mimic the complex in vivo environment of the cardiomyocyte. We determined the local composition and mechanical properties of the myocardium in established mouse models of genetic and acquired fibrosis and tested the effect of myocardial composition on cardiomyocyte contractility in vitro by systematically manipulating the number of fibroblasts and collagen concentration in a platform of engineered cardiac microtissues. The in vitro results showed that while increasing collagen content had little effect on microtissue contraction, increasing fibroblast density caused a significant reduction in contraction force. In addition, the beating frequency dropped significantly in tissues consisting of 50% cardiac fibroblasts or higher. Despite apparent dissimilarities between native and in vitro fibrosis, the latter allows for the independent analysis of local determinants of fibrosis, which is not possible in vivo.\u3c/p\u3

Colorful protein-based fluorescent probes for collagen imaging

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling