Phosphorylation of the regulatory light chains of myosin II (rMLC) by the Ca(2+)/calmodulin-dependent myosin light-chain kinase (MLCK) and dephosphorylation by a type 1 phosphatase (MLCP), which is targeted to myosin by a regulatory subunit (MYPT1), are the predominant mechanisms of regulation of smooth muscle tone. The activities of both enzymes are modulated by several protein kinases. MLCK is inhibited by the Ca(2+)/calmodulin-dependent protein kinase II, whereas the activity of MLCP is increased by cGMP and perhaps also cAMP-dependent protein kinases. In either case, this results in a decrease in the Ca(2+) sensitivity of rMLC phosphorylation and force production. The activity of MLCP is inhibited by Rho-associated kinase, one of the effectors of the monomeric GTPase Rho, and protein kinase C, leading to an increase in Ca(2+) sensitivity. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades.

Background-Cardiac troponins in blood are the most preferred markers of myocardial damage. The fact that they are normally not found in the circulation provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. After myocardial injury, the troponins enter the circulation, where they can be used for diagnosis of acute coronary syndromes. Thus, the cardiac troponins are paramount for disease classification and risk stratification. However, little is known about the long-term effects of the released troponins on cardiac function. Methods and Results-In this study we prepared recombinant murine cardiac troponin I (mc-TnI) and murine cardiac troponin T and used them to immunize mice. We report that A/J mice immunized with mc-TnI developed severe inflammation of the myocardium with increased expression of inflammatory chemokines RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1␣, MIP-1␤, MIP-2, T-cell activation gene 3, and eotaxin and chemokine receptors CCR1, CCR2, and CCR5. The inflammation was followed by cardiomegaly, fibrosis, reduced fractional shortening, and 30% mortality over 270 days. In contrast, mice immunized with murine cardiac troponin T or with the control buffer showed little or no inflammation and no death. Furthermore, we demonstrate that mice preimmunized with mc-TnI before left anterior descending coronary artery ligation showed greater infarct size, more fibrosis, higher inflammation score, and reduced fractional shortening. Conclusions-Overall, our results show for the first time that provocation of an autoimmune response to mc-TnI induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.

Next to changes in cytosolic [Ca(2+)], members of the Rho subfamily of small GTPases, in particular Rho and its effector Rho kinase, also known as ROK or ROCK, emerged as key regulators of smooth muscle function in health and disease. In this review, we will focus on the regulation of the contractile machinery by Rho/ROK signaling and its interaction with PKC and cyclic nucleotide signaling. We will briefly discuss the emerging evidence that remodeling of cortical actin is necessary for contraction.

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).

To study the dynamics of individual half-sarcomeres in striated muscle contraction, myofibrils prepared from rabbit psoas muscle and left ventricles of guinea pig were immunostained with two conjugated antibody complexes consisting of a primary antibody against either alpha-actinin or myomesin and a secondary fluorescently labeled Fab-fragment. We simultaneously measured force kinetics and determined the positions of the Z-line and M-band signals by fluorescence video microscopy and sophisticated computer vision (tracking) algorithms. Upon calcium activation, sarcomeres and half-sarcomeres shortened nonuniformly. Shortening occurred first rapidly and exponentially during the force rise and then slowly during the force plateau. In psoas myofibrils, time-resolved displacements of the A-band in sarcomeres were observed, i.e., the two halves of individual sarcomeres behaved nonuniformly. Nonuniformity in length changes between the two halves of sarcomeres was comparable to that between two adjacent half-sarcomeres of neighboring sarcomeres. Sequential lengthening of half-sarcomeres was observed in cardiac myofibrils during the rapid phase of force relaxation. The independent dynamics of the halves in a sarcomere reveals the half-sarcomere as the functional unit rather than the structural unit, the sarcomere. The technique will facilitate the study of filament sliding within individual half-sarcomeres and the mechanics of intersegmental chemomechanical coupling in multisegmental striated muscles.

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca²⁺ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca²⁺-induced Ca²⁺-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

We demonstrate for the first time that the prevalence of cTnI-Abs in patients with AMI has an impact on the improvement of the LVEF over a study period of 6-9 months.

We examined length changes of individual half-sarcomeres during and after stretch in actively contracting, single rabbit psoas myofibrils containing 10-30 sarcomeres. The myofibrils were fluorescently immunostained so that both Z-lines and M-bands of sarcomeres could be monitored by video microscopy simultaneously with the force measurement. Half-sarcomere lengths were determined by processing of video images and tracking the fluorescent Z-line and M-band signals. Upon Ca 2+ activation, during the rise in force, active half-sarcomeres predominantly shorten but to different extents so that an active myofibril consists of half-sarcomeres of different lengths and thus asymmetric sarcomeres, i.e. shifted A-bands, indicating different amounts of filament overlap in the two halves. When force reached a plateau, the myofibril was stretched by 15-20% resting length (L 0 ) at a velocity of ∼0.2 L 0 s -1 . The myofibril force response to a ramp stretch is similar to that reported from muscle fibres. Despite the ∼2.5-fold increase in force due to the stretch, the variability in half-sarcomere length remained almost constant during the stretch and A-band shifts did not progress further, independent of whether half-sarcomeres shortened or lengthened during the initial Ca 2+ activation. Moreover, albeit half-sarcomeres lengthened to different extents during a stretch, rapid elongation of individual sarcomeres beyond filament overlap ('popping') was not observed. Thus, in contrast to predictions of the 'popping sarcomere' hypothesis, a stretch rather stabilizes the uniformity of half-sarcomere lengths and sarcomere symmetry. In general, the half-sarcomere length changes (dynamics) before and after stretch were slow and the dynamics after stretch were not readily predictable on the basis of the steady-state force-sarcomere length relation.