Cardiovascular diseases are among the most frequent causes of death throughout the world. Cardiomyopathies, like the hypertrophic, arrhythmogenic, dilatative and restrictive cardiomyopathy, are heart muscle diseases, often genetically caused. Mostly, the affected genes encode for proteins of the sarcomere. For hypertrophic cardiomyopathy, mainly the gene encoding for myosin binding protein C is affected, but also genes encoding for the subunits of the heterotrimeric troponin complex. The most frequently affected gene in restrictive cardiomyopathy is encoding for troponin I. We investigate the pathomechanisms of such mutations in genes encoding for troponin subunits in vitro on protein level as well as in isolated cardiomyocytes.
Other factors possibly leading to heart diseases are e.g. stress, hypertension, atherosclerosis, inflammation etc. Recently we demonstrated an important role of the soluble adenylyl cyclase for the development of stress-induced cardiac hypertrophy. The underlying signaling pathways remain to be analyzed, though. As the soluble adenylyl cyclase is involved not only in cell growth but also in cell proliferation, we also investigate its role during tumor development in cooperation with the dermatology department of the St. Josef Hospital Bochum. Soluble adenylyl cyclase is a potential new target for treatment of pathologic growth and proliferation.
Cardiomyocytes, like most other cells, shed exosomes (communication units) containing specific cargo like miRNA and proteins, depending on cell conditions. The cargo can thus potentially be used as a diagnostic biomarker. In a current study we isolate microparticles from blood plasma from patients with different cardiovascular diseases, and characterize their cargo and their effects e.g. on survival and autophagy of isolated cardiomyocytes as well as endothelial cells (HUVEC) to detect potential biomarkers.
Methods:
- Isolation of recombinant proteins and proteins from tissue
- Mutagenesis
- Reconstitution of functional troponin complexes
- Analysis of protein interactions (cosedimentation, SPR, Microscale thermophoresis)
- Function analyses: Calcium binding (using fluorescent-labelled proteins), effects on calcium dependent activation of thin filaments
- isolation of cardiomyocytes from adult and neonatal rat hearts
- adenoviral and AAV mediated gene transfer for overexpression or knock down of proteins
- localization studies in cardiomyocytes
- contraction measurements in cardiomyocytes
- signal transduction (Western blot)
- cell survival, necrosis, apoptosis, autophagy assays
- in-cell protein interactions in H9C2 cells
- isolation of microparticles from blood plasma and cell culture supernatants
- FACS