Klinik für Kinder- und Jugendmedizin - Pädiatrische Kardiologie

Project 4: Cell death and stress response in embryonic versus adult cardiomyocytes

Besides different regenerative approaches to replace diseased cells with new cardiomyocytes and restore cardiac function after injury, another important strategy is to prevent cell loss in the first place and increase the stress tolerance and survival of cardiomyocytes upon the impact of unfavorable conditions, a process referred to as cardioprotection. A prerequisite for the future development of new cardioprotective therapeutic concepts, however, is the identification of molecular mechanisms that influence cardiomyocyte stress response, death and survival. In this regard, it is generally believed that embryonic and fetal cardiomyocytes comprise a higher stress resistance than adult cardiomyocytes.

When investigating molecular and cellular mechanisms of embryonic heart regeneration in cHccs+/- female mice (see project 1), one of the most striking observations was the activation of a multitude of cell protective mechanisms specifically in embryonic HCCS deficient cardiomyocytes, including antioxidative defense, antiapoptotic and cell survival pathways and maintenance of protein homeostasis. To test whether this cardioprotective signalling in response to mitochondrial dysfunction is unique to the prenatal heart, we established an inducible, cardiomyocyte specific Hccs KO model in the adult mouse heart. Strikingly, several cell protective mechanisms identified in embryonic cardiomyocytes cannot be activated in the adult heart upon HCCS deficiency. These data support the hypothesis that loss of embryonic cardioprotection contributes to disease susceptibility of the postnatal heart. Ongoing projects aim to systematically investigate the differences in stress response, cell death and survival in prenatal versus postnatal cardiomyocytes in vitro. Therefore, primary cardiomyocytes from embryonic, neonatal and adult mouse hearts as well as stable cardiac cell lines are cultured in the presence of specific inhibitors of the mitochondrial respiratory chain and cell death and survival is monitored. The molecular stress response of the different cardiomyocyte populations is furthermore investigated by RNA expression profiling as well as Western Blot and immunofluorescence analyses. A special focus lies on genes and signalling pathways that are induced in embryonic but not adult cardiomyocytes, as these might represent attractive targets to improve survival of the latter. To test the functional relevance of some of the most promising candidates, siRNA knockdown in embryonic cardiomyocytes is performed to monitor the consequences for cell survival. In a reverse approach, overexpression of these genes in adult cardiomyocytes should improve their survival upon mitochondrial stress. Taken together, these studies should help to identify new therapeutic targets for cardioprotection in the adult heart.

Figure: Characterization of isolated adult mouse cardiomyocytes.
Primary cardiomyocytes were isolated from adult mouse hearts applying a retrograde organ perfusion protocol including enzymatic digestion of cardiac tissue. The left panel shows a phase contrast image of rod shaped cardiomyocytes in culture. The right panel shows an immunofluorescence image (confocal laser scanning microscopy, scale bar = 25 µm) of cardiomyocytes stained with an antibody against the sarcomere protein α-Actinin (in green). The latter clearly unmasks the cross striation pattern of mature sarcomere structures (nuclei are stained with DAPI and are depicted in blue).

Related Publications:
Drenckhahn JD, Schwarz QP, Gray S, Laskowski A, Kiriazis H, Ming Z, Harvey RP, Du XJ, Thorburn DR, Cox TC. Compensatory growth of healthy cardiac cells in the presence of diseased cells restores tissue homeostasis during heart development. Dev Cell 2008;15:521-533.

 
 
 
 

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