University of Virginia Health System

Dr. Avril Somlyo

The molecular mechanisms of excitation-contraction coupling, contractile regulation and the basis of contraction in mammalian smooth muscle are the long-term interests of our laboratory. We are currently working on a new signal transduction pathway, which is activated when physiological transmitters or drugs bind to cell membrane receptors and lead to a marked increase in the sensitivity of the contractile proteins to calcium (Ca2+-sensitization). We have found that this G-protein-coupled process, which operates under physiological conditions, inhibits myosin light chain phosphatase, resulting in an increased population of phosphorylated (activated), cycling myosin motors. The phosphatase is a trimeric complex with a 110 kDa subunit which targets the activity to myosin. Current biochemical and molecular biological studies are directed to establishing the messengers between the membrane receptors/G-proteins and the myosin-associated phosphatase, as well as elucidating the mechanism of inhibition of the enzyme. The small G-protein RhoA and Rho-kinase participate in this signaling cascade. This same pathway modulates cell migration, and we are exploring its role in tumor cell metastasis. Biochemical and molecular biological techniques and physiological studies of permeabilized smooth muscle cells are used to identify the signaling molecules and their targets, as well as cross-talk among various other signaling pathways.

Transgenic and knockout mice are used to elucidate the role of telokin, a molecule found in small-but not large-blood vessels. Telokin is phosphorylated upon cyclic nucleotide-induced relaxation; we are testing the hypothesis that it functions by activating myosin phosphatase. Contraction in smooth muscle, mediated by interactions between actin filaments and crossbridges on adjacent myosin filaments, is triggered by the rise in cytoplasmic calcium and consequent phosphorylation of the 20 kDa light chains of myosin. The crossbridges are the motors that utilize ATP as an energy source for the generation of force and work. The kinetics of this motor and its modulation in muscle are characterized in our laboratory by using photolysis of caged nucleotides, caged Ca2+ and caged Bapta and monitoring force and stiffness transients. We use a new, fluorescently labeled, genetically engineered mutant of a bacterial phosphate-binding protein to follow the kinetic relationship between force development and phosphate release from the ATP binding pocket of the crossbridge following photolysis of caged ATP.

The rationale for studying crossbridge kinetics in permeabilized fibers, rather than in solutions of purified proteins, is that the biological ATPase cycle is modulated by the physical strain on the crossbridges. We have demonstrated that slow, tonic smooth muscles, unlike fast, phasic smooth muscles, have a high affinity for ADP and relatively low affinity for ATP, leading to prolongation of the strongly bound state in the crossbridge cycle and marked slowing of relaxation of force by MgADP. Current efforts are toward determining the myosin isoformic variations responsible for this high ADP affinity and its potential role in the maintenance of high force at low ATPase activity, a characteristic of the high economy of energy usage by smooth muscle. Our goal for these studies is to understand the fundamental properties of the crossbridge motors, their differences and their regulation in fast and in slowly contracting smooth muscles.

The long-term aim of our work is to provide insights into the abnormalities of disease states involving smooth muscle, such as hypertension. Another focus in our laboratory is the mapping of subcellular calcium distribution and handling in cardiac muscle during physiological stimuli and calcium overload. Electron probe X-ray microanalysis is used to determine and image, at high spatial resolution, elemental distributions within cardiac muscle organelles and membranes. We are particularly interested in the affinity of mitochondria for calcium during normal systole and diastole and in the role of calcium binding to intercalated disks during cardiac arrhythmias.