Gmaschitz, T. | bioRxiv Science

bioRxiv2024

A Synergistic Desmin-SPARC Axis Regulates Cardiac Stem Cell Differentiation and Promotes Cardiomyogenesis through Autocrine Regulation

BACKGROUNDThe mammalian heart contains cardiac stem cells throughout life, but it has not been possible to harness or stimulate these cells to repair damaged myocardium in vivo. Assuming physiological relevance of these cells, which have evolved and have been maintained throughout evolution, we are investigating their function using mouse cardiac stem cell lines as an in vitro model system. METHODSHere we use genetically modified embryonic stem cells and cardiac stem cells from the mouse as model systems to study the influence of desmin and Secreted Protein Acidic and Rich in Cysteine (SPARC) on cardiomyogenesis in embryoid bodies and cardiac bodies. We analyze their expression in self-renewing and differentiating stem cells by fluorescence microscopy, RT-qPCR, quantitative Western blotting and fluorescence activated cell sorting, and assess their influence on the expression of myocardial transcription factors. RESULTSIn embryoid bodies, desmin induces expression and secretion of SPARC, which promotes cardiomyogenesis. Cardiac stem cells secrete substantial amounts of SPARC, which also promotes cardiomyogenesis in a concentration-dependent, autocrine manner and promotes expression of myocardial transcription factors and desmin. Desmin and SPARC interact genetically and form a positive feedback loop and secreted SPARC negatively influences sparc mRNA expression. Finally, SPARC rescues cardiomyogenic desmin-haploinsufficiency in cardiac stem cells in a glycosylation-dependent manner, increases the phosphorylation of Smad2 and induces the expression of gata4, nkx2.5 and mef2C. CONCLUSIONSDemonstration that desmin-induced autocrine secretion of SPARC in cardiac stem cells promotes cardiomyogenesis raises the possibility that a physiological function of cardiac stem cells in the adult and aging heart may be the gland-like secretion of factors such as SPARC that modulate age-related and adverse environmental influences and thereby contribute to cardiac homeostasis throughout life.

developmental biology↗

bioRxiv2024

Confounding factors in targeted degradation of short-lived proteins

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using mathematical modelling of protein degradation, we demonstrate that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

cancer biology↗