Actin-binding compounds that affect the kinetics of the interaction of cardiac myosin with actin

We measured the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the microsecond structural dynamics of actin and the steady-state activity of actin-activated myosin ATPase. In the present study, we have further characterized their mechanisms of action by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin, and is restored upon release of S1 by MgATP. We measured the effects of compounds under equilibrium and steady-state conditions, as affected by S1 and ATP, and also under transient conditions, in stopped-flow experiments following rapid addition of ATP to S1-bound PFA. We observe that these compounds affect the early steps of the myosin ATPase cycle to different extents (mild, moderate, and severe). The compounds decrease the equilibrium constant for the formation of the collision complex and the rate constant for subsequent isomerization to the ternary complex, indicating increased ATP affinity and trapping of ATP in the myosin active site. These compound effects on actin structure inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for possible treatment of hypercontractile forms of hypertrophic cardiomyopathy (HCM). This work helps to elucidate the mechanisms of action of these compounds, several of which are currently used therapeutically, and it sets the stage for future HTS campaigns on a larger scale, to discover new drugs for treatment of heart failure.

Immunohistochemistry for phospho-myosin light chain 2 in adult murine skin

Phosphorylation of the myosin regulatory light chain 2 is a post-translational modification commonly used to report for myosin ATPase activity and actomyosin contractility. While its use in cell culture has been broadly reported in several studies, data on immunostaining in tissues has been sparse and inconsistent. In this protocol we report a methodology to stain phospho-myosin light chain 2 (pMLC2 Ser19) and double phospho-myosin light chain 2 (ppMLC2Thr18/ Ser19) in adult murine epidermis.

The enduring relationship between myosin enzymatic activity and the speed of muscle contraction

Moss and Solaro recall Bárány’s landmark study that identified myosin ATPase as the fundamental driver of contraction speed.

Phenotypic Modulation of Skeletal Muscle Fibers in LPIN1-Deficient Lipodystrophic (fld) Mice

Lipin-1 ( Lpin1)–deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the β-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1-/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid–Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1-/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1-/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.

ZO-1 protein is required for hydrogen peroxide to increase MDCK cell paracellular permeability in an ERK 1/2-dependent manner

Hydrogen peroxide (H2O2) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H2O2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H2O2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H2O2 to increase paracellular permeability. H2O2 treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. H2O2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H2O2 increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to H2O2 was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. H2O2 treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that H2O2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H2O2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.