Project 11: Redox regulation of human heart muscle function

PI Göttingen: W.-H. Zimmermann; PI London: M. Mayr; PhD: I. Trautsch - finished Dr. rer. nat.

Scientific background and preliminary results

Our understanding of the functional relevance of redox altered signalling pathways in the human heart is incomplete. With the help of human pluripotent stem cells, genetic reporters (e.g., roGFP2), and tissue engineering paradigms we are able to simulate human heart muscle in a dish and visualize alterations in specific redox couples. The systematic association of specific redox couples and redox potentials (assessed on a cell specific level by cell specific expression of for example Grx1-roGFP2) with contractile performances (such as force of contraction, resting tension, rhythm and rate of contraction) and the cell type specific cellular and extracellular proteome (mass spec using cell type specific SILAC labels) will enable a systems biology approach to decipher the interdependencies of specific proteome alterations and heart muscle function.

We have recently developed highly efficient and reproducible protocols for cardiomyocytederivation from human pluripotent stem cells (Hudson et al., patent WO2015/040142) and protocols for the construction of engineered heart muscle (EHM) under defined serum-free conditions with structural and functional properties of postnatal myocardium (Tiburcy et al., patent WO2015/025030). Using TALEN and CRISPR/Cas gene editing tools we have been able to develop several human pluripotent stem cell lines with stable expression of reporters (GFP, ODDLuc) and gene deletions (e.g., SERPINH1). The availability of genetically encoded reporters, such as Grx1-roGFP2 allowed us to generate redox sensor cardiomyocytes by lentiviral transduction. These cardiomyocytes are able to report specific changes in EGSH.

EHM can be subjected to defined pharmacological (e.g., angiotensin II and isoprenaline) and physical (e.g., electrical stimulation) stimuli to either support tissue formation or induce a heart failure phenotype (Godier-Furnemont et al., Biomaterials, 60, 2015, 82-91). We anticipate that these stimuli induce distinct patterns of EGSH changes in a cell type specific context, leading to beneficial or detrimental function on the cellular and multicellular level. In agreement with this notion, we have observed distinct EGSH responses in cardiomyocytes and fibroblasts using the Grx1-roGFP2 tool.

In vivo, it is not only the intimate cross-talk between different cells types in the heart that would cause enhanced or disturbed function. Moreover, cross-talk between organs is considered of major importance. An example is the common finding of diastolic dysfunction in patients with liver cirrhosis. With our ability to engineer EHM and bioengineered liver tissue (BLT) in vitro we can simulate heart-liver cross-talk in vitro, while keeping the system simple. With this organ-on-a-chip approach we anticipate to be able to address the question whether damaged liver does indeed

interact via secreted or released factors with heart muscle and thus contribute the development of diastolic dysfunction. Visualization of EGSH in BLT and EHM alike with the available cytosolically or mitochondrially located Grx1-roGFP2 sensors will enable us to define the role of redox alterations in heart-liver cross-talk.

Hypotheses of the PhD project

We anticipate that the relevance of redox alteration for heart muscle performance can be further elucidated by the robust expression of roGFP2 sensors from the AAVS1 safe harbor site. Cell type specificity in EHM, used as human heart muscle surrogate, will be achieved by the application of highly specific directed differentiation protocols and SILAC labelling of the resulting cardiomyocyte and fibroblast populations used for EHM construction. The following hypotheses will be tested by PhD-students recruited within the framework of the IRTG 1816-project:

1. Human embryonic stem cells can be modified to express the Grx1-roGFP2 sensor stably from the AAVS1 locus.

2. Cardiomyocytes and fibroblasts from Grx1-roGFP2-ESC can be applied to construct EHM with the opportunity for imaging of the EGSH either in cardiomyocytes or fibroblasts.

3. Application of SILAC labelled cardiomyocytes with unlabeled fibroblasts and vice versa will allow for the definition of the cell type specific redox proteome.

4. Pharmacologically and by electrical stimulation induced heart failure phenotypes in EHM will elicit distinct patterns of EGSH alterations in cardiomyocytes and fibroblasts.

5. Liver-heart interactions can be simulated in an engineered organ-on-a-chip approach to gain insight into the mechanism underlying liver damage induced diastolic dysfunction.

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