Size and speed of the working stroke of cardiac myosin in situ

The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force T_P to a value T (0.8–0.2 T_P). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s⁻¹ at high load to ∼8 nm⋅hs⁻¹ and 6,000 s⁻¹ at low load. Increases in sarcomere length (1.9–2.2 μm) and external [Ca²⁺]_o (1–2.5 mM), which produce an increase of T_P, do not affect the dependence on T, normalized for T_P, of the size and speed of the working stroke. Thus, length- and Ca²⁺-dependent increase of T_P and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.The performance of heart depends on the power developed by the myocardium, which in turn is strongly dependent on the end-diastolic volume modulating the systolic pressure development (Frank–Starling law of the heart). At the level of the sarcomere, the structural unit of striated muscle, the Frank–Starling law originates from the increase in the force of contraction with an increase in sarcomere length (length-dependent activation). Mutations of sarcomere proteins affect power output and are considered responsible for various forms of cardiomyopathy (1, 2). Over 250 mutations in cardiac myosin II have been reported as the cause of cardiomyopathies (1, 3, 4). Defining the mechanokinetic properties of the cardiac myosin in situ is therefore fundamental to understand the pathomechanisms of these cardiomyopathies and to provide previously unidentified therapeutic opportunities.In the sarcomere, the myosin motors are organized in two bipolar arrays extending from the thick filament and pulling the thin actin-containing filaments from the opposite sides of the sarcomere toward its center. In each array, the myosin motors are connected in parallel via their attachments to the thick filament and the resulting collective motor provides steady force and shortening by cyclic asynchronous ATP-driven actin–myosin interactions. Thus, the performance of the heart relies on the integration of the mechanokinetic properties of the myosin motor and the properties emerging from its array arrangement in the half-sarcomere (hs). Using sarcomere-level mechanics in intact cells from the skeletal muscle, it has been shown that the isotonic velocity transient following stepwise changes in force imposed on the otherwise isometric contraction contains information on both the working stroke of the myosin motor and the steady-state force–velocity (T–V) relation resulting from the cyclic actin–myosin interactions and accounting for the power output (5–9).Here, this approach is applied for the first time (to our knowledge) to a multicellular cardiac preparation like the intact trabecula dissected from the right ventricle of the rat heart. A striation follower (10) proved to be a reliable tool for measurement of sarcomere length changes with nanometer–microsecond resolution owing to optical averaging of the image of the sarcomeres that reduces the background noise originating from intracellular and intercellular components of the trabecula. Following the original idea by ter Keurs et al. (11), the sarcomere shortening recorded during the force development in a fixed-end twitch is used as a feedforward signal to maintain sarcomere length constant during the next twitch. By switching from length control to force control, a stepwise drop in force was imposed at the peak of force (T_P) to record the isotonic velocity transient. In this way, the amplitude and speed of the rapid phase of the transient (phase 2), which is the mechanical manifestation of the myosin working stroke, could be determined. Increases in sarcomere length (SL) from 1.9 to 2.2 μm and in the external Ca²⁺ concentration ([Ca²⁺]_o) from 1 to 2.5 mM, which produce an increase in T_P, do not affect the myosin working stroke. This indicates that length-dependent potentiation of cardiac contractility is fully accounted for by an increase in the number of attached myosin motors. These experiments demonstrate that our sarcomere-level mechanical methods have the full potential for the in situ investigation of cardiomyopathy-causing mutations in cardiac myosin.