Active Tension Development Research Articles

Neural regulation of slow waves and phasic contractions in the distal stomach: a mathematical model

Objective. Neural regulation of gastric motility occurs partly through the regulation of gastric bioelectrical slow waves (SWs) and phasic contractions. The interaction of the tissues and organs involved in this regulatory process is complex. We sought to infer the relative importance of cellular mechanisms in inhibitory neural regulation of the stomach by enteric neurons and the interaction of inhibitory and excitatory electrical field stimulation. Approach. A novel mathematical model of gastric motility regulation by enteric neurons was developed and scenarios were simulated to determine the mechanisms through which enteric neural influence is exerted. This model was coupled to revised and extended electrophysiological models of gastric SWs and smooth muscle cells (SMCs). Main results. The mathematical model predicted that regulation of contractile apparatus sensitivity to intracellular calcium in the SMC was the major inhibition mechanism of active tension development, and that the effect on SW amplitude depended on the inhibition of non-specific cation currents more than the inhibition of calcium-activated chloride current (kiNSCC = 0.77 vs kiAno1 = 0.33). The model predicted that the interaction between inhibitory and excitatory neural regulation, when applied with simultaneous and equal intensity, resulted in an inhibition of contraction amplitude almost equivalent to that of inhibitory stimulation (79% vs 77% decrease), while the effect on frequency was overall excitatory, though less than excitatory stimulation alone (66% vs 47% increase). Significance. The mathematical model predicts the effects of inhibitory and excitatory enteric neural stimulation on gastric motility function, as well as the effects when inhibitory and excitatory enteric neural stimulation interact. Incorporation of the model into organ-level simulations will provide insights regarding pathological mechanisms that underpin gastric functional disorders, and allow for in silico testing of the effects of clinical neuromodulation protocols for the treatment of these disorders.

A new active contraction model for the myocardium using a modified hill model

This study develops a new hybrid active contraction model for myocardial dynamics abstracted from sarcomere by combining the phenomenologically active-stress based Hill model and the micro-structurally motivated active strain approach. This new model consists of a passive branch and a parallel active branch that consists of a serial passive element for active tension transmission and a contractile unit for active tension development. This rheology represents an additive decomposition of the total stress into a passive and active response. The active stress is formulated following the active strain approach based on the sliding filament theory by multiplicatively decomposing the stretch of the contractile element into a fictitious and an active part. The length-dependence and force-velocity are further incorporated in the active strain. We estimate the passive stiffness of the serial passive element using literature data, which is 250 kPa, then the active stress is computed from the serial passive element in the active branch because of its force transmission structure. This one-dimensional contraction model is further generalized to three dimensions for modelling myocardial dynamics. Our results demonstrate that the proposed active contraction model has a high descriptive capability for various experiments, including both isometric and isotonic contraction compared to existing active strain approaches. We also show that it can simulate physiologically accurate cardiac dynamics in humans. The excellent agreement with experimental data and a local sensitivity study highlight the importance of length-dependence and force-velocity in the active strain approach. Our results further show that there exists a tight interaction between the length-dependence and force-velocity relationships. This new hybrid model serves as a step forward in personalized cardiac modelling using an active-strain based contraction model and has the potential to understand the multi-scale coupling in active contraction according to the sliding filament theory.

Cardiac electro-mechanical activity in a deforming human cardiac tissue: modeling, existence-uniqueness, finite element computation and application to multiple ischemic disease.

In this study, the cardiac electro-mechanical model in a deforming domain is taken with the addition of mechanical feedback and stretch-activated channel current coupled with the ten Tusscher human ventricular cell level model that results in a coupled PDE-ODE system. The existence and uniqueness of such a coupled system in a deforming domain is proved. At first, the existence of a solution is proved in the deformed domain. The local existence of the solution is proved using the regularization and the Faedo-Galerkin technique. Then, the global existence is proved using the energy estimates in appropriate Banach spaces, Gronwall lemma, and the compactness procedure. The existence of the solution in an undeformed domain is proved using the lower semi-continuity of the norms. Uniqueness is proved using Young's inequality, Gronwall lemma, and the Cauchy-Schwartz inequality. For the application purpose, this model is applied to understand the electro-mechanical activity in ischemic cardiac tissue. It also takes care of the development of active tension, conductive, convective, and ionic feedback. The Second Piola-Kirchoff stress tensor arising in Lagrangian mapping between reference and moving frames is taken as a combination of active, passive, and volumetric components. We investigated the effect of varying strength of hyperkalemia and hypoxia, in the ischemic subregions of human cardiac tissue with local multiple ischemic subregions, on the electro-mechanical activity of healthy and ischemic zones. This system is solved numerically using the [Formula: see text] finite element method in space and the implicit-explicit Euler method in time. Discontinuities arising with the modeled multiple ischemic regions are treated to the desired order of accuracy by a simple regularization technique using the interpolating polynomials. We examined the cardiac electro-mechanical activity for several cases in multiple hyperkalemic and hypoxic human cardiac tissue. We concluded that local multiple ischemic subregions severely affect the cardiac electro-mechanical activity more, in terms of action potential (v) and mechanical parameters, intracellular calcium ion concentration [Formula: see text], active tension ([Formula: see text]), stretch ([Formula: see text]) and stretch rate ([Formula: see text]), of a healthy cell in its vicinity, compared to a single Hyperkalemic or Hypoxic subregion. The four moderate hypoxically generated ischemic subregions affect the waveform of the stretch along the fiber and the stretch rate more than a single severe ischemic subregion.

Estimating cardiac active tension from wall motion-An inverse problem of cardiac biomechanics.

The contraction of the human heart is a complex process as a consequence of the interaction of internal and external forces. In current clinical routine, the resulting deformation can be imaged during an entire heart beat. However, the active tension development cannot be measured in vivo but may provide valuable diagnostic information. In this work, we present a novel numerical method for solving an inverse problem of cardiac biomechanics-estimating the dynamic active tension field, provided the motion of the myocardial wall is known. This ill-posed non-linear problem is solved using second order Tikhonov regularization in space and time. We conducted a sensitivity analysis by varying the fiber orientation in the range of measurement accuracy. To achieve RMSE <20% of the maximal tension, the fiber orientation needs to be provided with an accuracy of 10°. Also, variation was added to the deformation data in the range of segmentation accuracy. Here, imposing temporal regularization led to an eightfold decrease in the error down to 12%. Furthermore, non-contracting regions representing myocardial infarct scars were introduced in the left ventricle and could be identified accurately in the inverse solution (sensitivity >0.95). The results obtained with non-matching input data are promising and indicate directions for further improvement of the method. In future, this method will be extended to estimate the active tension field based on motion data from clinical images, which could provide important insights in terms of a new diagnostic tool for the identification and treatment of diseased heart tissue.

Increased Contractile Function of Human Saphenous Vein Grafts Harvested by "No-Touch" Technique.

Saphenous vein grafts are the most common conduits used for coronary artery bypass grafting (CABG); however, no more than 60% of vein grafts remain open after 10 years and graft failure is associated with poor clinical outcome. The “no-touch” harvesting technique—where a sheet of perivascular tissue is retained around the vein—improves graft patency to over 80% after 16 years of follow-up, but the mechanism for the improved patency rate is unclear. In this study, we investigated acute functional differences between vein grafts harvested conventionally and by “no-touch” technique and explored the importance of perivascular tissue for reducing surgical trauma, minimizing excessive distension, and releasing vasoactive paracrine factors. Segments of human saphenous veins were obtained from CABG surgery and their functional properties investigated by isometric and isobaric myography. We found a broad diameter-tension relationship for human saphenous veins, with peak capacity for active tension development at diameters corresponding to transmural pressures around 60 mmHg. Across the investigated transmural pressure range between 10 and 120 mmHg, maximal tension development was higher for “no-touch” compared to conventionally harvested saphenous veins. Contractile responses to serotonin, noradrenaline, and depolarization induced with elevated extracellular [K+] were significantly larger for saphenous veins harvested by “no-touch” compared to conventional technique. Conventional vein grafts are routinely pressurized manually in order to test for leaks; however, avoiding this distension procedure did not change the acute contractile function of the conventionally excised saphenous veins. In contrast, even though surgical removal of perivascular tissue during conventional harvesting was associated with a substantial decrease in force development, removal of perivascular tissue by careful dissection under a stereomicroscope only marginally affected contractile responses of veins harvested by “no-touch” technique. In conclusion, we show that saphenous veins harvested by “no-touch” technique have greater contractile capacity than veins harvested by conventional technique. The different capacity for smooth muscle contraction is not due to vasoactive substances released by the perivascular tissue. Instead, we propose that the larger tension development of saphenous veins harvested by “no-touch” technique reflects reduced surgical damage, which may have long-term consequences that contribute to the superior graft patency.

The Skeletal Muscle Molecular Clock Regulates Titin Isoform Expression

The circadian clock transcription factors, BMAL1 and CLOCK, are fundamental transcriptional regulators of cell time keeping and critical cell specific genes important for homeostasis. To determine the specific role of the molecular clock in adult skeletal muscle, our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse. Notably, skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. To begin to discern the molecular mechanisms that link the changes in the molecular clock with changes in muscle function, we tested whether iMSBmal1-/- muscle would also exhibit changes in sarcomeric protein expression. We determined that the tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant increase (38% in iMSBmal1-/- mice vs. 19% in iMSBmal1+/+ mice) in expression of a longer isoform of titin. It is established that titin, the giant filamentous protein that maintains sarcomeric structure, underlies passive tension, and contributes to active tension development. To determine if this increased heterogeneity of titin isoforms within the TA had an effect on sarcomere length, we performed immunohistochemistry. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length was increased following Bmal1 knockout in skeletal muscle. Further studies with our model are currently ongoing and will lead to increased knowledge of the importance of titin isoform maintenance in a variety of pathological conditions.

Role of leptin in coronary vascular dysfunction and proliferation

The purpose of this investigation was to test the hypothesis that leptin augments vasoconstriction and impairs dilation via a Rho kinase‐dependent pathway and promotes proliferation of coronary smooth muscle. Coronary arteries from domestic swine were cleaned of surrounding tissue and incubated with/without leptin (30 ng/mL) for 30 min. Leptin administration significantly augmented contractile responses to KCl (10 – 60 mM), with active tension development increased by ~1.3 g at doses &gt;40mM, and reduced maximal vasodilation to adenosine (30 μM) ~18% compared to untreated controls. To examine the extent to which longer‐term exposure to leptin influences coronary vascular reactivity, arteries were placed in organ culture for 3 days. Following incubation in serum‐free media, leptin treatment significantly augmented overall active tension development to KCl (10 – 60 mM), with an average increase of ~2.0 g at doses &gt;40 mM, and reduced maximal vasodilation to adenosine (30 μM) ~50% compared to untreated controls. The acute and chronic effects of leptin administration on maximal KCl (60 mM) contractions were essentially abolished in the presence of the Rho kinase inhibitor, fasudil (1 μM). Short‐term leptin exposure significantly increased the level of Rho kinase activity, while no difference in Rho kinase (Rock‐2) protein abundance was detected. In contrast, long‐term leptin administration in cultured arteries significantly increased the abundance of Rock‐2 protein, while no difference in Rho kinase activity level was detected. Global proteomic assessment of arteries cultured (3 days) in the presence or absence of leptin (30 ng/mL) revealed significant alterations in the coronary artery proteome and Ingenuity Pathway Analysis identified associations between leptin treatment and numerous cellular processes, namely cellular growth and proliferation (35 proteins). To assess the effects of leptin on vascular smooth muscle proliferation, additional studies were conducted in which coronary arteries were cultured in serum‐containing media with/without leptin (30 ng/mL) for 2 or 8 days. 5‐Bromo‐2′‐deoxyuridine (BrdU) staining revealed a significantly higher percentage of BrdU‐positive nuclei in leptin treated arteries (~10%) compared to untreated controls at both time points. Taken together, data from this investigation indicate that leptin alters coronary smooth muscle reactivity and potentiates contraction via a Rho kinase‐dependent pathway. Additionally, these findings support the potential for leptin to modulate a key network of pathways that promote vascular smooth muscle proliferation.Support or Funding InformationIndiana University Health, Indiana University School of Medicine Strategic Research Initiative (CECARE); HL117620 (J.D. Tune‐Mather); TL1 TR001107 and UL TR001108 (A. Shekhar).

Coronary Vascular Effects of Leptin and Calpastatin in Lean vs. Obese Hearts

Recent evidence suggests a role for leptin and calpastatin in the pathogenesis of obesity‐induced coronary vascular dysfunction. However, how these factors affect coronary smooth muscle reactivity in lean vs. obese hearts is not well defined. Coronary arteries from lean and obese Ossabaw swine were isolated, cleaned of all surrounding adipose tissue, and incubated with or without leptin (3 – 30 ng/mL) or calpastatin (10 μM) for 30 min. Arteries were then pre‐constricted with the thromboxane mimetic U46619 (1μM). In untreated arteries, vasodilation to adenosine (0.01 – 30 μM) was decreased ~25% in obese compared to lean arteries. Administration of leptin dose‐dependently reduced vasodilation to adenosine, with the 30 ng/mL concentration attenuating dilation ~25% in both lean and obese arteries. In contrast, the presence of calpastatin impaired responses to adenosine (10 μM) in lean (~26%), but not obese arteries. In additional studies, coronary arteries from lean swine were cultured at 37oC in the presence/absence of leptin (30 ng/mL) or calpastatin (10 μM) for 3 days and subsequently subjected to functional isometric tension studies. Relative to untreated cultured arteries, incubation with leptin augmented active tension development to KCl‐induced depolarization (30 mM) and diminished vasodilation in response to adenosine (10 μM). Incubation with calpastatin modestly reduced vasodilation to adenosine but did not influence active tension development to KCl. These findings indicate that leptin and calpastatin differentially influence vascular function and suggest that these factors could contribute to the augmented contractile, impaired vasodilator phenotype of the obese coronary circulation.

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr(1065) on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr(1065) in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr(1065) (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr(1065) phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr(1065) phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.

Human skeletal muscle biochemical diversity

The molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain, MHC) and mechanosensitive signaling (titin) have been well studied in animals but less is known about their roles in humans. The purpose of this study was to perform a comprehensive analysis of titin, collagen and MHC isoform distributions in a large number of human muscles, to search for common themes and trends in the muscular organization of the human body. In this study, 599 biopsies were obtained from six human cadaveric donors (mean age 83 years). Three assays were performed on each biopsy - titin molecular mass determination, hydroxyproline content (a surrogate for collagen content) and MHC isoform distribution. Titin molecular mass was increased in more distal muscles of the upper and lower limbs. This trend was also observed for collagen. Percentage MHC-1 data followed a pattern similar to collagen in muscles of the upper extremity but this trend was reversed in the lower extremity. Titin molecular mass was the best predictor of anatomical region and muscle functional group. On average, human muscles had more slow myosin than other mammals. Also, larger titins were generally associated with faster muscles. These trends suggest that distal muscles should have higher passive tension than proximal ones, and that titin size variability may potentially act to 'tune' the protein's mechanotransduction capability.

Biochemical diversity of human skeletal muscle

INTRODUCTIONThe molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain [MHC]), and mechanosensitive signaling (titin) are well studied in animals but less is known about their roles in humans.METHODS599 biopsies were obtained from 6 human cadaveric donors. Three assays were performed on each biopsy ‐ titin molecular weight determination, hydroxyproline content (a surrogate for collagen content), and MHC isoform distribution.RESULTSDiscriminant function analysis (DFA) showed that titin MW was the strongest predictive factor of anatomic region and muscle functional group. On average, human muscles had more slow myosin than lower mammals. Average %MHC‐1 of muscles in this study was 65% (compare to 6% for mouse, 8% for rat, and 19% for rabbit). Overall, larger titins were associated with faster muscles.DISCUSSIONThe finding that titin was the strongest discriminating factor for DFA with anatomic region and muscle functional group as grouping variables is unexpected; titin is currently primarily considered to be a signal transduction cascade activator or determinant of passive tension. The titin‐MHC‐1 relationship we observed is opposite to that previously observed in rabbits, where large titins were associated with slower muscles.

Membrane acid‐base transporters modulate artery structure

Acid‐base transport and pH control modulate cell proliferation and migration with possible consequences for artery remodeling. Here, we used knockout mice for the Na+/H+‐exchanger NHE1 and Na+,HCO3−‐cotransporter NBCn1 to study their role in determining arterial structure. Measurements were performed using pH‐sensitive fluorophores, myography and stereological analyses. NHE1 mediated the Na+/H+‐exchange in mesenteric arteries, and knockout of NHE1 was associated with reduced media thickness and an equivalent inhibition of maximum active tension development. The reduced media thickness was due to a smaller smooth muscle cell volume and cross‐sectional area with no change in the number of smooth muscle cells per unit artery length. NBCn1 mediated the Na+,HCO3−‐cotransport in carotid arteries. Following partial carotid artery ligation, the inward remodeling (reduction in carotid artery diameter and increase in media area) in wild type mice was more than 50% attenuated in NBCn1 knockout mice. In conclusion, we show that the acid‐base transport proteins NBCn1 and NHE1 play an important role for determining artery structure. Supported by Danish Council for Independent Research and Danish Heart Foundation.

Biochemical Diversity of Human Skeletal Muscle

INTRODUCTIONThe molecular components largely responsible for muscle attributes such as passive tension development (titin and collagen), active tension development (myosin heavy chain [MHC]), and mechanosensitive signaling (titin) are well studied in animals. We performed a comprehensive analysis of these components in human muscle to search for common themes and trends in the muscular organization of the human body.METHODS599 biopsies from 6 donors were obtained early postmortem. Three assays were performed on each biopsy - titin molecular weight determination (n=586), hydroxyproline content (a surrogate for collagen content) (n=599), and MHC isoform distribution (n=599).Muscles were analyzed individually and in functional groups. Analysis of variance (ANOVA) was used to compare individual muscles and discriminant function analysis (DFA) was used to determine which dependent variables were the best predictors of muscle group.RESULTSDFA demonstrated that titin MW was the strongest predictive factor of anatomic region and muscle functional group.On average, human muscles were very “slow” (i.e., had more slow- myosin than muscles of lower mammals). Average % MHC-1 of muscles in this study was 65% (compare to 6% for mouse, 8% for rat, and 19% for rabbit). Overall, larger titins were associated with faster muscles.DISCUSSION:The finding that titin was the strongest discriminating factor for DFA with anatomic region and muscle functional group as grouping variables is unexpected; titin is currently primarily considered to be a signal transduction cascade activator or determinant of passive tension. The idea that titin MW is a good predictor of muscle anatomic localization is previously unexplored and warrants further study.The titin-% MHC-1 relationship we observed is opposite to that previously reported in rabbits, where large titins were associated with slower muscles. This may represent a different design paradigm in human vs rabbit muscular organization.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Contraction of Tracheal Smooth Muscle Tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.

Is the Cross-Bridge Stiffness Proportional to Tension during Muscle Fiber Activation?

The cross-bridge stiffness can be used to estimate the number of S1 that are bound to actin during contraction, which is a critical parameter for elucidating the fundamental mechanism of the myosin motor. At present, the development of active tension and the increase in muscle stiffness due to S1 binding to actin are thought to be linearly related to the number of cross-bridges formed upon activation. The nonlinearity of total stiffness with respect to active force is thought to arise from the contribution of actin and myosin filament stiffness to total sarcomere elasticity. In this work, we reexamined the relation of total stiffness to tension during activation and during exposure to N-benzyl-p-toluene sulphonamide, an inhibitor of cross-bridge formation. In addition to filament and cross-bridge elasticity, our findings are best accounted for by the inclusion of an extra elasticity in parallel with the cross-bridges, which is formed upon activation but is insensitive to the subsequent level of cross-bridge formation. By analyzing the rupture tension of the muscle (an independent measure of cross-bridge formation) at different levels of activation, we found that this additional elasticity could be explained as the stiffness of a population of no-force-generating cross-bridges. These findings call into question the assumption that active force development can be taken as directly proportional to the cross-bridge number.

β-Catenin regulates airway smooth muscle contraction

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

The effect of acute ethanol exposure on the chronotropic and inotropic function of the rat right atrium.

Consumption of ethanol (CH2CH3OH), both acutely and chronically, is known to affect cardiac function and may alter the autonomic control of the heart. This study investigated the effects of two modes of acute exposure to ethanol on the chronotropy and inotropy of the rat right atrium with emphasis on alterations in the adrenergic responses. Atria from rats infused with an anesthetizing level of ethanol for 21 h showed a tendency for a greater increase of the unstimulated beating rate with isoproterenol (ISO), while both unstimulated inotropy and the inotropic response to ISO were significantly decreased compared with the control. Right atria in the presence of ethanol in-vitro demonstrated decreased basal active tension development and decreased inotropic responses to ISO. No alteration of the chronotropic response to ISO was evident with any concentration of ethanol. These results demonstrate both an immediate as well as a persistent effect of ethanol on right atrial chronotropy and inotropy. Alterations in the G-stimulatory subunit of the adenylate cyclase system and alterations in myofilament binding of Ca2+ are consistent with these observed ethanol effects.

Length-Dependent Active Tension Development In Single Intact Cardiomyocytes, Isolated From Different Regions Of Guinea Pig Heart

Introduction: Information on the force-length relation of intact myocytes from different regions of the heart is scarce. We therefore studied myocytes, isolated from apical and basal areas of guinea pig left and right ventricles (cell numbers: LVA 22, LVB 29, RVA 11, RVB 12). Methods: Force-length relations were measured by attaching carbon fibers to myocytes, allowing application of diastolic stretch while measuring passive and active force.1 Cells were kept at 36±1°C and paced at 2 Hz. Recorded forces were normalized to cell cross-sectional area and used to construct end-diastolic, end-systolic and active tension-length relations (EDTL, ESTL and ATL=ESTL-EDTL; respectively). In addition, the ratio of the slopes of ESTL and EDTL was used as a cross-section independent factor to characterise the Frank-Starling Gain (FSG) in individual cells. Results: For all tissue regions ESTL, EDTL and, hence, ATL, are linear over the range of end-diastolic sarcomere lengths studied (1.88-2.15 μm). Plotting the slope values of ATL vs. EDTL for all cells shows a positive correlation (slope 1.29, R2=0.24, 74 cells). In addition, FSG is larger than one for all cells studied: RVB 3.91±0.54), RVA (2.84±0.30), LVB (2.77±0.17), and LVA (3.20±0.24). Conclusions: Using the carbon fiber technique, it is possible to probe length-dependence of passive and active tension at the single cell level, without the interference of extracellular structures. The Frank-Starling Gain varies between the different regions of the heart and the positive correlation between ATL and EDTL confirms that in intact cells passive force bearing structures (such as titin) are likely to play a role in modulating length dependent activation.[1] Iribe et al, Force-length relations in isolated intact cardiomyocytes subjected to dynamic changes in mechanical load. AJP 2007/292:1487-1497.

Mysteries of Renal Autoregulation

Autoregulation is an important renal regulatory mechanism that provides an important protective role in glomerular hemodynamics.1 The phenomenon of autoregulatory behavior has been recognized for many decades, but a clear understanding of the underlying mechanisms involved in mediating resistance adjustments remains to be established. As illustrated in Figure 1, pressure-mediated autoregulatory behavior operates by recognizing a change in renal perfusion pressure, or renal vascular transmural pressure, and initiating induction of autoregulatory resistance changes intended to stabilize renal blood flow and/or glomerular filtration rate. The net result is that renal blood flow and glomerular filtration rate remain relatively stable over a wide range of renal perfusion pressures. Autoregulatory behavior is exquisitely sensitive in healthy normal kidneys but significantly less efficient in pathological settings, such as diabetes mellitus and some forms of hypertension. Complete loss of autoregulatory control results in renal blood flow that behaves more passively, thus increasing variability and rendering flow more directly determined by renal perfusion pressure.1,2 Figure 1. Outline of the pressure-induced signaling scheme postulated for autoregulatory adjustments in the afferent arteriolar diameter. Whole kidney autoregulation reflects regulatory input from the myogenic and tubuloglomerular feedback mechanisms at the very least.3–7 Recently, contributions of a third and possibly a fourth mechanism have been suggested.6,7 Consequently, with continued study, the mechanisms underlying renal autoregulatory resistance adjustments are slowly becoming clearer. In addition to the P1 receptor hypothesis put forth to explain signal transduction for tubuloglomerular feedback-dependent resistance changes, we have explored the hypothesis that pressure-mediated autoregulatory responses are mediated by extracellular ATP and activation of P2X1 receptors.3,8,9 Conclusive identification of the signaling molecules that mediate autoregulation remains a point of debate, but it appears clear that the release of a purine-based moiety is required for autoregulatory adjustments in afferent arteriolar resistance.3–5,8–10 This …

Dynamic association between α‐actinin and β‐integrin regulates contraction of canine tracheal smooth muscle

The adhesion junctions of smooth muscle cells may be dynamically regulated during smooth muscle contraction, and this dynamic regulation may be important for the development of active tension. In the present study, the role of alpha-actinin during smooth muscle contraction was evaluated in tracheal smooth muscle tissues and freshly dissociated cells. Stimulation with acetylcholine (ACh) increased the localization of alpha-actinin at the membrane of freshly dissociated smooth muscle cells, and increased the amount of beta1 integrin that coprecipitated with alpha-actinin from muscle tissue homogenates. GFP-alpha-actinin fusion proteins were expressed in muscle tissues and visualized in live freshly dissociated cells. GFP-alpha-actinin translocated to the membrane within seconds of stimulation of the cells with ACh. Expression of the integrin-binding rod domain of alpha-actinin in smooth muscle tissues depressed active contraction in response to ACh. Expression of the alpha-actinin rod domain also inhibited the translocation of endogenous alpha-actinin to the membrane, and inhibited the association of endogenous alpha-actinin with beta1-integrin in alpha-actinin immunoprecipitates from tissue extracts. However, the expression of alpha-actinin rod domain peptides did not inhibit increases in myosin light chain phosphorylation or actin polymerization in response to stimulation with ACh. Results suggest that contractile stimulation of smooth muscle causes the rapid recruitment of alpha-actinin to beta-integrin complexes at the membrane, and that the recruitment of alpha-actinin to integrin complexes is necessary for active tension development in smooth muscle.