Excitation-contraction Pathway Research Articles

Affinity Purification-Mass Spectrometry and Single Fiber Physiology/Proteomics Reveals Mechanistic Insights of C18ORF25.

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25. Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

The Role of Parvalbumin, Sarcoplasmatic Reticulum Calcium Pump Rate, Rates of Cross-Bridge Dynamics, and Ryanodine Receptor Calcium Current on Peripheral Muscle Fatigue: A Simulation Study.

A biophysical model of the excitation-contraction pathway, which has previously been validated for slow-twitch and fast-twitch skeletal muscles, is employed to investigate key biophysical processes leading to peripheral muscle fatigue. Special emphasis hereby is on investigating how the model's original parameter sets can be interpolated such that realistic behaviour with respect to contraction time and fatigue progression can be obtained for a continuous distribution of the model's parameters across the muscle units, as found for the functional properties of muscles. The parameters are divided into 5 groups describing (i) the sarcoplasmatic reticulum calcium pump rate, (ii) the cross-bridge dynamics rates, (iii) the ryanodine receptor calcium current, (iv) the rates of binding of magnesium and calcium ions to parvalbumin and corresponding dissociations, and (v) the remaining processes. The simulations reveal that the first two parameter groups are sensitive to contraction time but not fatigue, the third parameter group affects both considered properties, and the fourth parameter group is only sensitive to fatigue progression. Hence, within the scope of the underlying model, further experimental studies should investigate parvalbumin dynamics and the ryanodine receptor calcium current to enhance the understanding of peripheral muscle fatigue.

Modeling the Chemoelectromechanical Behavior of Skeletal Muscle Using the Parallel Open-Source Software Library OpenCMISS

An extensible, flexible, multiscale, and multiphysics model for nonisometric skeletal muscle behavior is presented. The skeletal muscle chemoelectromechanical model is based on a bottom-up approach modeling the entire excitation-contraction pathway by strongly coupling a detailed biophysical model of a half-sarcomere to the propagation of action potentials along skeletal muscle fibers and linking cellular parameters to a transversely isotropic continuum-mechanical constitutive equation describing the overall mechanical behavior of skeletal muscle tissue. Since the multiscale model exhibits separable time scales, a special emphasis is placed on employing computationally efficient staggered solution schemes. Further, the implementation builds on the open-source software library OpenCMISS and uses state-of-the-art parallelization techniques taking advantage of the unique anatomical fiber architecture of skeletal muscles. OpenCMISS utilizes standardized data structures for geometrical aspects (FieldML) and cellular models (CellML). Both standards are designed to allow for a maximum flexibility, reproducibility, and extensibility. The results demonstrate the model's capability of simulating different aspects of nonisometric muscle contraction and efficiently simulating the chemoelectromechanical behavior in complex skeletal muscles such as the tibialis anterior muscle.

A mathematical model of fatigue in skeletal muscle force contraction

The ability for muscle to repeatedly generate force is limited by fatigue. The cellular mechanisms behind muscle fatigue are complex and potentially include breakdown at many points along the excitation-contraction pathway. In this paper we construct a mathematical model of the skeletal muscle excitation-contraction pathway based on the cellular biochemical events that link excitation to contraction. The model includes descriptions of membrane voltage, calcium cycling and crossbridge dynamics and was parameterised and validated using the response characteristics of mouse skeletal muscle to a range of electrical stimuli. This model was used to uncover the complexities of skeletal muscle fatigue. We also parameterised our model to describe force kinetics in fast and slow twitch fibre types, which have a number of biochemical and biophysical differences. How these differences interact to generate different force/fatigue responses in fast- and slow- twitch fibres is not well understood and we used our modelling approach to bring new insights to this relationship.

41. High Efficiency, Catheter-Based, Gene Transfer to the Large Animal Heart

Congestive heart failure (HF) is a leading cause of hospitalization, disability and death. Despite significant advances in pharmacotherapy, HF remains a progressive disorder with an unacceptably high mortality rate. While the molecular and cellular basis of the impairment of myocardial contractility has been determined in detail, current beneficial therapies for heart failure only act indirectly, by interfering with neurohormonal control of the heart. Accordingly, considerable attention has recently been directed at the identification of appropriate targets for gene therapy. In particular, studies directed at the excitation-contraction pathway of the heart have identified genes (particularly phospholamban and SERCA) that can be manipulated to improve contractility. As such, translation into clinical practice now requires the development of safe, minimally invasive methods that provide homogeneous delivery to the heart.

Regulation of the contractile element of airway smooth muscle

Smooth muscle of the mammalian airways controls airway diameter and resistance to airflow. Smooth muscle tone is in turn controlled by a variety of external signals that are transduced to useful work by contractile proteins. The protein components of the contractile element of airway smooth muscle are similar to those found in other smooth muscles and include actin, myosin, tropomyosin, caldesmon, and calponin. There has been significant recent progress in studies of contractile system regulation of airway smooth muscle. Regulation of myosin light chain kinase, identification of the sites phosphorylated on the regulatory myosin light chains, and the effect of myosin phosphorylation on stress development and crossbridge cycling rates have all been studied in some detail. We infer from these studies that besides myosin phosphorylation there is an important role for a thin filament Ca(2+)-dependent regulatory mechanism. The potentially important thin filament proteins caldesmon and calponin are present in tracheal smooth muscle and may be phosphorylated during contraction. The use of intracellular Ca2+ indicators to estimate changes in intracellular Ca2+ ([Ca2+]i) and the development of several skinned fiber preparations have broadened the scope of physiological studies with airway smooth muscle and have suggested that the contractile element sensitivity to Ca2+ is not fixed but might be modulated by undefined messengers or excitation-contraction pathways. This adds an additional challenge to the continuing effort to define the messengers and regulatory proteins that couple activation of membrane receptors to the contractile element in airway smooth muscle.

Further evidence for the existence of alpha 2-mediated adrenergic vasoconstriction in human vessels.

To confirm the presence of alpha 2-mediated vasoconstriction in human vasculature, the effect of selective alpha 1- and alpha 2-agonists (methoxamine and B-HT 933) and antagonists (indoramin and Yohimbine) was studied in fourteen patients with mild, uncomplicated, essential hypertension. Drugs were infused, into the brachial artery at systemically ineffective rates, and concomitant changes in forearm blood flow were measured by strain gauge venous plethysmography. During control conditions, cumulative infusions either of methoxamine or B-HT 933 caused dose-related vasoconstriction, while both indoramin and yohimbine doubled forearm blood flow. Subsequently, the alpha 1-adrenoceptor mediated vasoconstriction produced by methoxamine was shown to be completely blocked by indoramin pretreatment, and to be left unchanged by yohimbine. The alpha 2-vascular stimulation by B-HT 933 was antagonized by previous yohimbine but not by indoramin pretreatment, thus fulfilling the pharmacological requirements for identification of distinct alpha-adrenoceptor mediated excitation-contraction pathways. The data provide further evidence of the existence of alpha 2-mediated vasoconstriction in human forearm vessels.

Excitation-Contraction Coupling in Hypertrophied Myocardium

In hypertensive cardiac hypertrophy, inotropic responsiveness of alpha and beta adrenergic stimuli is reduced. We have previously shown that hearts from two-kidney, one-clip renal hypertensive rats (RHR) have increased beta-adrenergic receptor density and a defect in the guanine nucleotide regulatory protein, leading to decreased adenylate cyclase activity. In spontaneously hypertensive rats (SHR), beta-receptor density was decreased with no change in adenylate cyclase. In these present experiments, we have shown that the alpha 1-adrenergic receptor changes are in the opposite direction, decreasing in RHR and increasing in SHR. All these changes are reversible within 4 weeks following removal of the clipped kidney in RHR, at which time blood pressure and heart weight have also returned towards normal. Further studies on the excitation-contraction pathway have indicated that c-AMP-stimulated protein kinase is decreased in SHR with no changes seen in RHR. Subcutaneous infusion of epinephrine leads to some increase of cardiac mass associated with decreased beta-adrenergic receptors element and decreased adenylate cyclase activity. However, following angiotensin II infusion, even though hypertrophy is more pronounced, no changes in receptors or cyclase are detected. We conclude that different models of hypertensive cardiac hypertrophy associated with different biochemical defects in the adrenergic excitation response pathway, and that if some of these changes become irreversible, further cardiac deterioration and even heart failure may ensue.