Direct Regulation of Striated Muscle Myosins by Nitric Oxide and Endogenous Nitrosothiols

, both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins..These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated

Measurement of time-averaged isometric force after exposure to NO.

<p>A: A streptavidin-coated bead is held in a laser trap and touched to the trailing (+) end of a moving, biotinylated, TRITC-phalloidin labeled actin filament. Displacement of the bead from trap center was followed using back focal plane interferometry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011209#pone.0011209-Allersma1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011209#pone.0011209-Guilford1" target="_blank">[35]</a>. B: Force versus number of potentially bound, force-generating heads (a reflection of filament length) for three conditions, control (•, black lines), 10 µM DEA NONOate (○, red lines) and 100 µM DEA NONOate (▾, green lines). Steeper slopes indicate proportionately higher forces per head. Solid lines indicate the regression fit, while the dashed lines show 95% confidence intervals. Each data point is an independent force measurement (N = 25, 20 and 10 for 0, 10, and 100 µM DEA NONOate respectively). C: Mixtures experiment for rat cardiac myosin. The line shows the fit of equation 1 from Harris et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011209#pone.0011209-Harris1" target="_blank">[11]</a> yielding a relative force production 2.1-fold higher after DEA NONOate-treated compared to control. N = 3.</p

Stereo-selective effects of SNO-L-cysteine on myosin.

<p>A: Dose-response showing that SNO-L-cysteine, the naturally occurring isomer, has pronounced effects on actin filament velocity, while SNO-D-cys does not. B: Effects of 5 µM L- and D-isomers of SNO-cys on actin filament velocities over skeletal (dark bars) and cardiac (light bars) myosin, and recovery by exposure to ultraviolet light – strong support of nitrosylation as the underlying modification. *Different from control (p<0.05). **Different from SNO-L-cys alone (p<0.05). N = 5.</p

Myosin nitrosylated <i>in vivo</i> and <i>in vitro</i>.

<p>A: Rat skeletal and cardiac myosin heavy (MHC) and light chains were nitrosylated by <i>in vitro</i> exposure to SNO-L-cysteine (L) over the control level (C). The ventricular/slow skeletal isoform of the essential light chain (ELC) could be nitrosylated, as could the regulatory light chain (RLC) and fast essential light chain of skeletal muscle myosin. The regulatory light chain of cardiac myosin was not significantly nitrosylated. B: Myosin heavy chain was endogenously (E) nitrosylated in rat skeletal and cardiac muscle, and also in human myocardium (MHC AMCA). A protein staining of the same gel is shown (MHC total) to illustrate how nitrosylation is normalized against protein mass to reveal low levels of nitrosylation between zero (A, ascorbate-treated) and maximum (L, SNO-L-cys-treated). See the text for details of the normalization procedure. Without normalization, variations in recovery between protein bands (as shown, typical) will mask single (or a few) nitrosylated cysteines in large proteins like myosin. N = 3 in all cases.</p