Elke Hammer (Greifswald / DE), Sabine Ameling (Greifswald / DE), Stephanie Könemann (Greifswald / DE), Alexander Benkner (Greifswald / DE), Stephan Michalik (Greifswald / DE), Anja Wiechert (Greifswald / DE), Manuela Gesell Salazar (Greifswald / DE), Olga Tsoy (Hamburg / DE), Jan Baumbach (Hamburg / DE), Linda Weder (Kiel / DE), Derk Frank (Kiel / DE), Uwe Völker (Greifswald / DE)
Background: Heart failure (HF) of different origin is associated with alternative splicing of synaptopodin 2-like protein transcripts also named as cytoskeletal heart-enriched actin-associated protein (CHAP). In HF myocardial tissue display increased protein levels of the short isoform ChapB, while the amount of the canonical longer isoform ChapA remains unchanged. However, the cardiotropic function of the different isoforms is still largely unknown.
Methods: ChapA and ChapB were cloned and overexpressed (OE) in neonatal rat ventricular cardiomyocytes (NRVCM) by transfection. Microscopy was used to determine the protein localisation and the influence of the isoform levels on cell size. The impact of altered isoform levels on the protein pattern was analysed by LC-MS/MS, affected pathways were identified and the results were compared with protein and transcript data of cardiac tissue from in-house mouse HF models.
Results: WesternBlot and LC-MS/MS confirmed the successful OE of ChapA and ChapB. Changes in isoform levels were not associated with hypertrophic growth of the cells. Both isoforms were found to be localised in the cytoplasm as well as the nucleus. The cardiotropic influence of the ChapA OE was stronger than that of the ChapB OE. OE of the isoform ChapA, representing the major isoform in the healthy heart, shows a positive effect on cell viability and adhesion of cardiomyocytes. Higher levels of ChapB, indicative of pathological states, are associated with alterations in mitochondrial oxidases and ADP/ATP translocators, as well as changes in basal membrane proteins including Trim72, which is involved in membrane repair. Some of the findings were verified by correlating analyses of transcript and protein levels in murine HF models.
Conclusion:
Global protein profiling has been employed to provide molecular data on proteins that appear to be directly affected by Chap isoform levels in cardiomyocytes. Apparently, high ChapB levels in HF have an acute impact on membrane structure and mitochondrial ATP transport into the cytoplasm, thereby affecting the energy supply of the cells. Further investigation is required to ascertain biological significance which can be achieved by protein-protein-interaction studies and measurement of ATP levels in cells.