(20,21,23,33,64), neurite outgrowth (1, 8, 57), endothelial cell contraction (23,69,70), and resistance to mechanical perturbations (8,11,31). These myosin II-based cellular events have been considered to be regulated solely by an increase in cytosolic Ca 2ϩ according to the classic smooth muscle model of Ca 2ϩ -coupled contraction. However, within the last 10 years myosin II motor activity has been shown to be activated by Ca 2ϩ -independent pathways (39,41,63,68) in addition to Ca 2ϩ -dependent pathways (7,23,32,70). Ca 2ϩ -dependent cell contraction results from an influx of Ca 2ϩ from the extracellular space or the release of Ca 2ϩ from sequestered internal stores. Ca 2ϩ binds to calmodulin (CaM); the Ca 2ϩ /CaM complex in turn binds to and activates the serine/threonine protein kinase, myosin light chain kinase (MLCK). The active MLCK complex catalyzes myosin II regulatory light chain (RLC) phosphorylation at two sites, Ser-19 and Thr-18. Phosphorylation at these sites is required for myosin II filament formation (6), myosin II interaction with F-actin (23), and an increase in myosin II ATPase activity (7). These phosphorylation driven events are essential for initiation and maintenance of myosin II-based contraction. MLCK, the enzyme responsible for initiating Ca 2ϩ -dependent contraction, exists in several isoforms, resulting from splice variants with distinct intracellular localizations and tissue distributions (3,5,32,52). To date, all MLCK isoforms have been shown biochemically to be strictly dependent on Ca 2ϩ /CaM for activation and are known to catalyze phosphorylation of a single substrate, myosin II RLC.The discovery of the Rho family of proteins initiated study of possible Ca 2ϩ -independent regulation of myosin II motor activity and contraction. A range of protein kinases have now been shown to activate myosin II motor activity by catalyzing phosphorylation of myosin II RLC in vitro or in vivo, including Rho-kinase (2), p21-associated protein kinase (PAK) (9, 73), and integrin-linked kinase (ILK) (10). The most intensively studied of the Ca 2ϩ -independent activators of myosin II motor activity and contraction are those regulated by the Rho family of proteins. Rho is a member of the Ras superfamily of small GTPases, which functions as a molecular switch cycling between the active GTP-bound and inactive GDP-bound state. In the active GTP state, RhoA interacts with its effector molecules to initiate downstream responses. Of several possible Rho effector proteins (4, 17) that bind to GTP-bound Rho, the serine/threonine kinase, Rho-kinase, is the one that has been implicated in the regulation of myosin II and contraction.Rho-kinase exists in two isoforms, ROK␣/ROCK-II (43, 46) and ROK␤/ROCK-I/p160 ROCK (29, 42); both are ubiquitously expressed in various tissues and cells. Rho-kinase isoforms range in mass from 150 to 160 kDa and contain a kinase domain in their NH 2 -terminal domains, a central coil-coiled domain, and a pleckstrin homology domain in the COOH-terminal domains. The coil-coiled domai...

During the initial phase of cardiac looping, known as c-looping, the heart bends and twists into a c-shaped tube with the convex outer curvature normally directed toward the right side of the embryo. Despite intensive study for more than 80 years, the biophysical mechanisms that drive and regulate looping remain poorly understood, although some investigators have speculated that differential cytoskeletal contraction supplies the driving force for c-looping. The purpose of this investigation was to test this hypothesis. To inhibit contraction, embryonic chick hearts at stages 10-12 (10-16 somites, 33-48 h) were exposed to the myosin inhibitors 2,3-butanedione monoxime (BDM), ML-7, Y-27632, and blebbistatin. Experiments were conducted in both whole embryo culture and, to focus on bending alone, isolated heart culture. Measurements of heart stiffness and phosphorylation of the myosin regulatory light chains showed that BDM, Y-27632, and blebbistatin significantly reduced myocardial contractility, while ML-7 had a lesser effect. None of these drugs significantly affected looping during the studied stages. These results suggest that active contraction is not required for normal c-looping of the embryonic chick heart between stages 10 and 12.

The morphogenetic process of cardiac looping transforms the straight heart tube into a curved tube that resembles the shape of the future four-chambered heart. Although great progress has been made in identifying the molecular and genetic factors involved in looping, the physical mechanisms that drive this process have remained poorly understood. Recent work, however, has shed new light on this complicated problem. After briefly reviewing the current state of knowledge, we propose a relatively comprehensive hypothesis for the mechanics of the first phase of looping, termed c-looping, as the straight heart tube deforms into a c-shaped tube. According to this hypothesis, differential hypertrophic growth in the myocardium supplies the main forces that cause the heart tube to bend ventrally, while regional growth and cytoskeletal contraction in the omphalomesenteric veins (primitive atria) and compressive loads exerted by the splanchnopleuric membrane drive rightward torsion. A computational model based on realistic embryonic heart geometry is used to test the physical plausibility of this hypothesis. The behavior of the model is in reasonable agreement with available experimental data from control and perturbed embryos, offering support for our hypothesis. The results also suggest, however, that several other mechanisms contribute secondarily to normal looping, and we speculate that these mechanisms play backup roles when looping is perturbed. Finally, some outstanding questions are discussed for future study.

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Compounds can be screened for pharmaceutical activity either by detecting interactions with specified target molecules such as receptors or enzymes (molecular screening) or observing effects on the structure or physiological activities of cells or tissues (phenotypic screening). Screening at the molecular level has been greatly enhanced by fluorescence methods. Especially the combination of confocal detection with measurements of the amplitudes and time courses of fluorescence fluctuations have reduced sample volumes to < microliters and have increased throughputs to >100000 compounds per day. Screening at the molecular level, however, does not provide information about the effects of test compounds on cellular functions. Phenotypic screening, although much slower than molecular screening, does provide information about effects on cell or tissue structure or function and therefore can be used to eliminate at an early stage compounds that are toxic or do not produce the desired cellular response. Tissue constructs reconstituted using cells of specified types and defined extracellular matrix components provide test systems for detecting the effects of test compounds on cellular mechanical functions such as the development of contractile force and on cell and matrix structure and stiffness. For example, constructs based on vascular smooth muscle cells provide information about effects on cellular contractile force that can be used to identify agents that control blood pressure. Tissue constructs that mimic skeletal, smooth and heart muscles and connective tissues have been produced and can be used to study mechanical and structural responses to active compounds.