The cellular basis of the Frank-Starling mechanism is sarcomere length modulation of myofilament Ca2+ sensitivity. The molecular mechanisms that underlie length sensitivity are unknown, but recent evidence has implicated the giant protein titin as possible sarcomeric strain sensor responsible for the phenomenon by an, as of yet unidentified, signal transduction pathway. Our studies over the past decades have employed various biophysical approaches to elucidate the underlying mechanisms of the problem at the level of: multicellular muscle bundles, single cardiac contractile cells, and sub-cellular single myofibrils. Our studies were performed both at steady state as well as during rapid activation/relaxation dynamics, while structure was assessed by utilizing fluorescent probe and x-ray diffraction analysis. Our results indicate that myofilament length dependent activation involves structural changes in both thick and thin filaments that are mediated by titin strain that may involve length dependent as well as phosphorylation dependent interaction of regulatory light chain, troponin-I, and myosin binding protein C interacting with actin and myosin to mediate the length sensing property of the cardiac sarcomere.