Forced expression of essential myosin light chain isoforms demonstrates their role in smooth muscle force production

The molecular determinants of the contractile properties of smooth muscle are poorly understood, and have been suggested to be controlled by splice variant expression of the myosin heavy chain near the 25/50-kDa junction (Kelley, C. A., Takahashi, M., Yu, J. H., and Adelstein, R. S. (1993) J. Biol. Chem. 268, 12848-12854) as well as by differences in the expression of an acidic (MLC(17a)) and a basic (MLC(17b)) isoform of the 17-kDa essential myosin light chain (Nabeshima, Y., Nonomura, Y., and Fujii-Kuriyama, Y. (1987) J. Biol. Chem. 262, 106508-10612). To investigate the molecular mechanism that regulates the mechanical properties of smooth muscle, we determined the effect of forced expression of MLC(17a) and MLC(17b) on the rate of force activation during agonist-stimulated contractions of single cultured chicken embryonic aortic and gizzard smooth muscle cells. Forced expression of MLC(17a) in aortic smooth muscle cells increased (p < 0.05) the rate of force activation, forced expression of MLC(17b) in gizzard smooth muscle cells decreased (p < 0.05) the rate of force activation, while forced expression of the endogenous MLC(17) isoform had no effect on the rate of force activation. These results demonstrate that MLC(17) is a molecular determinant of the contractile properties of smooth muscle. MLC(17) could affect the contractile properties of smooth muscle by either changing the stiffness of the myosin lever arm or modulating the rate of a load-dependent step and/or transition in the actomyosin ATPase cycle.     

The Emperor's New Clothes: PDE5 and the Heart

Phosphodiesterase-5 (PDE5) is highly expressed in the pulmonary vasculature, but its expression in the myocardium is controversial. Cyclic guanosine monophosphate (cGMP) activates protein kinase G (PKG), which has been hypothesized to blunt cardiac hypertrophy and negative remodeling in heart failure. Although PDE5 has been suggested to play a significant role in the breakdown of cGMP in cardiomyocytes and hence PKG regulation in the myocardium, the RELAX trial, which tested effect of PDE5 inhibition on exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF) failed to show a beneficial effect. These results highlight the controversy regarding the role and expression of PDE5 in the healthy and failing heart. This study used one- and two-dimensional electrophoresis and Western blotting to examine PDE5 expression in mouse (before and after trans-aortic constriction), dog (control and HFpEF) as well as human (healthy and failing) heart. We were unable to detect PDE5 in any cardiac tissue lysate, whereas PDE5 was present in the murine and bovine lung samples used as positive controls. These results indicate that if PDE5 is expressed in cardiac tissue, it is present in very low quantities, as PDE5 was not detected in either humans or any model of heart failure examined. Therefore in cardiac muscle, it is unlikely that PDE5 is involved the regulation of cGMP-PKG signaling, and hence PDE5 does not represent a suitable drug target for the treatment of cardiac hypertrophy. These results highlight the importance of rigorous investigation prior to clinical trial design.     

PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle

Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.     

PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle

In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.     

Agonist-induced force enhancement: the role of isoforms and phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase.

The magnitude of agonist-induced Ca(2+) sensitization of force is tissue-dependent, but an explanation for this diversity is unknown. Ca(2+) sensitization is thought to involve a G-protein-mediated inhibition of myosin light chain phosphatase activity by phosphorylation of the myosin-targeting subunit (MYPT). The MYPT has two isoforms that differ by a central insert, which lies near this phosphorylation site. Expression of MYPT isoforms is both developmentally regulated and tissue-specific. We hypothesized that the presence or absence of the central insert determines the magnitude of agonist-induced Ca(2+) sensitization. Throughout development, the chicken aorta exclusively expresses the splice-in MYPT isoform, and guanosine 5'-O-(thiotriphosphate) (GTPgammaS) produces a significant force enhancement. Early during development, the chicken gizzard expresses the splice-in MYPT isoform, and GTPgammaS produced a Ca(2+) sensitization. In the gizzard coincident with the shift in expression from the splice-in to splice-out MYPT isoform, GTPgammaS no longer produced force enhancement. In addition, adenosine 5'-O-(thiotriphosphate) (ATPgammaS) phosphorylated only adult gizzard tissue, the only tissue that did not demonstrate a Ca(2+) sensitization. These results suggest that the relative expression of splice-in/splice-out MYPT isoforms determines the magnitude of agonist-induced force enhancement and that MYPT phosphorylation is not required for Ca(2+) sensitization.     

Selected contribution: mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells.

Cultured airway smooth muscle cells subjected to cyclic deformational strain have increased cell content of myosin light chain kinase (MLCK) and myosin and increased formation of actin filaments. To determine how these changes may increase cell contractility, we measured isometric force production with changes in cytosolic calcium in individual permeabilized cells. The pCa for 50% maximal force production was 6.6+/-0.4 in the strain cells compared with 5.9+/-0.3 in control cells, signifying increased calcium sensitivity in strain cells. Maximal force production was also greater in strain cells (8.6+/-2.9 vs. 5.7+/-3.1 microN). The increased maximal force production in strain cells persisted after irreversible thiophosphorylation of myosin light chain, signifying that increased force could not be explained by differences in myosin light chain phosphorylation. Cells strained for brief periods sufficient to increase cytoskeletal organization but insufficient to increase contractile protein content also produced more force, suggesting that strain-induced cytoskeletal reorganization also increases force production.     

A dynamic approach reveals non-muscle myosin influences the overall smooth muscle cross-bridge cycling rate

Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B^+/− strain (8.73 ± 1.10 Hz vs 16.56 ± 1.26 Hz, P < 0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B^+/−) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle V_max during force maintenance.     

The frequency response of smooth muscle stiffness during Ca2+-activated contraction

To investigate the mechanism of smooth muscle contraction, the frequency response of the muscle stiffness of single beta-escin permeabilized smooth muscle cells in the relaxed state was studied. Also, the response was continuously monitored for 3 min from the beginning of the exchange of relaxing solution to activating solution, and then at 5-min intervals for up to 20 min. The frequency response (30 Hz bandwidth, 0.33 Hz (or 0.2 Hz) resolution) was calculated from the Fourier-transformed force and length sampled during a 3-s (or 5-s) constant-amplitude length perturbation of increasing-frequency (1-32 Hz) sine waves. In the relaxed state, a large negative phase angle was observed, which suggests the existence of attached energy generating cross-bridges. As the activation progressed, the muscle stiffness and phase angle steadily increased; these increases gradually extended to higher frequencies, and reached a steady state by 100 s after activation or approximately 40 s after stiffness began to increase. The results suggest that a fixed distribution of cross-bridge states was reached after 40 s of Ca2+ activation and the cross-bridge cycling rate did not change during the period of force maintenance.     

Muscle contraction generates discrete sound bursts.

Isolated frog sartorius muscles were stimulated to shorten under lightly loaded conditions. A piezoelectric transducer was placed alongside the muscle to record sounds generated during contraction. Shortening was accompanied by the generation of a series of discrete sound bursts. The bursts were found to be moderately repeatable among successive contractions; 44% repeated from contraction to contraction. The duration of each sound burst was on the order of 400 mus, and the temperature dependence of the interval between successive bursts had a Q10 of approximately 2. Sound intensity was variable: average acoustic power ranged from 0.05-0.4 mW/g, or approximately 1% of the heat generated during contraction. The generation of discrete bursts of sound during contraction, rather than continuous sound, implies that contractile behavior may be discontinuous.