Actin polymerization during myofibrillogenesis and its regulation by mechanosensing

Cardiovascular diseases are the most frequent cause of death worldwide. Therapeutic approaches need to take into account the complexity of the system from the gene to the level of the entire organ. Of particular interest is the cellular microenvironment, which signals to the cell via chemical and physical/mechanical pathways and is critical for biological processes, such as growth, differentiation or cell death. The issue, how matrix properties affect myocyte differentiation is particularly critical for therapies based on stem cells, or the treatment of congenital heart diseases. During myocyte differentiation, myofibrillogenesis results in the formation of arrays of basic contractile units, the sarcomeres. Depending on the model organism, various hypotheses have been proposed to explain their assembly. The models share common ideas, such as early actin polymerization from integrin adhesions, outside-in signaling sites with a well-documented importance for mechanosensing. However, variances persist about the existence of precursor structures or the incorporation of non-muscle myosin II. Other questions remain about if and how mechanical signals result in the activation of the early actin polymerization, or the further myofibril maturation, and which proteins assemble the actin. We propose to study these critical questions by combining the methods of cell biology, biophysics and nanotechnology in a three-tiered approach in which we observe the effects of A) passive resistance and varying rigidity; B) active force; C) no force; This will be achieved with elastomer pillar arrays to define the matrix elasticity and the sites of integrin attachments; magnetic tweezers and cell stretching devices to apply force; and myosin abrogation or lipid bilayer experiment to inhibit forces. We will then follow the localization of target proteins (i.e. actin assembly factors or sarcomeric markers) in heart and skeletal muscle cells over time with microscopy methods, including super resolution microscopy. Inhibition and knock down experiments will complement the studies. We expect this approach to give us novel and valuable information to understand the processes involved in myofibrillogenesis. This will possibly also shed light on the mechanisms of heart and muscle diseases and should result in novel therapeutic targets or strategies. (AHA Program: Postdoctoral Fellowship)