The sarcomere
In cardiac and skeletal muscle cells, myosin and actin are organized into thick and thin filament protein complexes to form sarcomeres, the most basic contractile unit. Their cyclic interaction is initiated by the release of calcium and powered by energy from captured from the hydrolysis of adenosine triphosphate (ATP). This results in the shortening of sarcomere, and thus shortening of cellular length.
Quantifying motion in-vitro
Myosin-thick and actin-thin filament complexes are isolated from model organisms and human hearts. Calcium dependent sliding of the filaments is recorded using fluorescence-based total internal reflectance (TIRF) microscopy. Myosin-binding protein C (MyBP-C) is a protein located within the C-zone of the thick filament, is a key regulator of sliding. Work from our lab has helped define the role of MyBP-C in the regulation of contractility and provide insight into why mutations in the gene for the protein lead to a debilitating disease, known as hypertrophic cardiomyopathy (HCM).
Protein localization
The organization of actin, myosin, MyBP-C and the calcium release sites (ryanodine receptors) are critical for efficient cardiac muscle contraction. Thin slices of frozen cardiac muscle can be cut and the subcellular localization of these proteins determined by electron microscopy (left) and stochastic optical reconstruction microscopy (STORM) super-resolution imaging (right). We are currently STORM to look at the localization of actin, myosin and MyBP-C in different states of cellular muscle contraction.
Protein Stoichiometry
Multiple natural variants exist for many of the sarcomeric proteins and their functions are altered by structural post-translational modifications such phosphorylation and oxidation. A combination of mass-spectrometry based proteomic techniques are used to quantify the abundance of these cellular proteins and their levels of modifications. In addition we have recently begun studies which rely on isotopic labeling of these proteins in-vivo to quantify their replacement and resynthesis in health and disease.
