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

ABSTRACT: 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 TP to a value T(0.8-0.2 TP). 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 ?m) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP 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.

SUBMITTER: Caremani M

PROVIDER: S-EPMC4822625 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Caremani Marco M Pinzauti Francesca F Reconditi Massimo M Piazzesi Gabriella G Stienen Ger J M GJ Lombardi Vincenzo V Linari Marco M

Proceedings of the National Academy of Sciences of the United States of America 20160316 13

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 stimul ...[more]

PMID: 26984499

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Project description:Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (3-fold) the maximum steady-state actin-activated ATPase activity ( k cat ) and decreases (6-fold) the actin concentration at which ATPase is one-half maximal ( K ATPase ). We also found a 3 to 4-fold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio 3-fold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt-bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt-bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.Significance statementHeart disease can be caused by inherited mutations in beta-cardiac myosin, the molecular motor that powers systolic contraction in the ventricles of the heart. However, it remains unclear how these mutations lead to contractile dysfunction and pathogenic remodeling of the heart. We investigated a unique dilated cardiomyopathy mutation (E525K) that dramatically stabilizes the autoinhibited state while enhancing intrinsic motor function. Thus, we examined how this mutation impacts transient kinetic steps of the ATPase cycle, motile properties, and structural changes associated with the power stroke and phosphate release. Our results provide a kinetic and structural basis for how beta-cardiac myosin mutations may disrupt molecular-level contractile function in complex ways, which may inform the development of targeted therapeutics.

Dataset Information

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

Publications

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

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