Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

I(CaL) inhibition prevents arrhythmogenic Ca(2+) waves caused by abnormal Ca(2+) sensitivity of RyR or SR Ca(2+) accumulation.

AIMS: Arrhythmogenic Ca(2+) waves result from uncontrolled Ca(2+) release from the sarcoplasmic reticulum (SR) that occurs with increased Ca(2+) sensitivity of the ryanodine receptor (RyR) or excessive Ca(2+) accumulation during ß-adrenergic stimulation. We hypothesized that inhibition of the L-type Ca(2+) current (I(CaL)) could prevent such Ca(2+) waves in both situations. METHODS AND RESULTS: Ca(2+) waves were induced in mouse left ventricular cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca(2+) sensitivity. I(CaL) inhibition by verapamil (0.5 µM) reduced Ca(2+) wave probability in cardiomyocytes during electrostimulation, and during a 10 s rest period after ceasing stimulation. A separate type of Ca(2+) release events occurred during the decay phase of the Ca(2+) transient and was not prevented by verapamil. Verapamil decreased Ca(2+) spark frequency, but not in permeabilized cells, indicating that this was not due to direct effects on RyR. The antiarrhythmic effect of verapamil was due to reduced SR Ca(2+) content following I(CaL) inhibition. Computational modelling supported that the level of I(CaL) inhibition obtained experimentally was sufficient to reduce the SR Ca(2+) content. Ca(2+) wave prevention through reduced SR Ca(2+) content was also effective in heterozygous ankyrin B knockout mice with excessive SR Ca(2+) accumulation during ß-adrenergic stimulation. CONCLUSION: I(CaL) inhibition prevents diastolic Ca(2+) waves caused by increased Ca(2+) sensitivity of RyR or excessive SR Ca(2+) accumulation during ß-adrenergic stimulation. In contrast, unstimulated early Ca(2+) release during the decay of the Ca(2+) transient is not prevented, and merits further study to understand the full antiarrhythmic potential of I(CaL) inhibition.

An open sarcolemmal adenosine triphosphate-sensitive potassium channel is necessary for detrimental myocyte swelling secondary to stress.

BACKGROUND: Stress (exposure to hyperkalemic cardioplegia, metabolic inhibition, or osmotic) results in significant myocyte swelling and reduced contractility. In contrast to wild-type mice, these detrimental consequences are not observed in mice lacking the Kir6.2 subunit of the sarcolemmal ATP-sensitive potassium (sK(ATP)) channel after exposure to hyperkalemic cardioplegia. The hypothesis for this study was that an open sK(ATP) channel (Kir6.2 and SUR2A subunits) is necessary for detrimental myocyte swelling to occur in response to stress. METHODS AND RESULTS: To investigate the role of the sK(ATP) channel in stress-induced myocyte swelling, high-dose pharmacological sK(ATP) channel blockade and genetic deletion (knockout of Kir6.2 subunit) were used. Myocytes were exposed sequentially to Tyrode control (20 minutes), test (stress) solution (20 minutes), and Tyrode control (20 minutes). To evaluate pharmacological channel blockade, myocytes were exposed to hyperkalemic cardioplegia (stress) with and without a K(ATP) channel blocker. To evaluate the effects of genetic deletion, wild-type and sK(ATP) knockout [Kir6.2(-/-)] myocytes were exposed to metabolic inhibition (stress). Myocyte volume was recorded using image-grabbing software. Detrimental myocyte swelling was prevented by high-dose sK(ATP) channel blockade (glibenclamide or HMR 1098) but not mitochondrial K(ATP) channel blockade (5-hydroxydecanoate) during exposure to hyperkalemic cardioplegia. Genetic deletion of the sK(ATP) channel prevented significant myocyte swelling in response to metabolic inhibition. CONCLUSIONS: K(ATP) channel openers prevent detrimental myocyte swelling and reduce contractility in response to stress through an unknown mechanism. Paradoxically, the present data support a role for sK(ATP) channel activation in myocyte volume derangement in response to stress.

ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-α and PGC-1.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate genes involved in energy metabolism and inflammation. For biological activity, PPARs require cognate lipid ligands, heterodimerization with retinoic X receptors, and coactivation by PPAR-γ coactivator-1α or PPAR-γ coactivator-1ß (PGC-1α or PGC-1ß, encoded by Ppargc1a and Ppargc1b, respectively). Here we show that lipolysis of cellular triglycerides by adipose triglyceride lipase (patatin-like phospholipase domain containing protein 2, encoded by Pnpla2; hereafter referred to as Atgl) generates essential mediator(s) involved in the generation of lipid ligands for PPAR activation. Atgl deficiency in mice decreases mRNA levels of PPAR-α and PPAR-δ target genes. In the heart, this leads to decreased PGC-1α and PGC-1ß expression and severely disrupted mitochondrial substrate oxidation and respiration; this is followed by excessive lipid accumulation, cardiac insufficiency and lethal cardiomyopathy. Reconstituting normal PPAR target gene expression by pharmacological treatment of Atgl-deficient mice with PPAR-α agonists completely reverses the mitochondrial defects, restores normal heart function and prevents premature death. These findings reveal a potential treatment for the excessive cardiac lipid accumulation and often-lethal cardiomyopathy in people with neutral lipid storage disease, a disease marked by reduced or absent ATGL activity.

Distrofias musculares de cinturas autosómicas recesivas / Autosomal recessive limb-girdle muscular dystrophy

Resumen. Las distrofias musculares son un grupo heterogéneo de enfermedades hereditarias, caracterizadas por debilidad y pérdida muscular de origen no neurogénico. Son causadas por mutaciones de uno o más genes involucrados en la formación de las células musculares. El descubrimiento de las diversas proteínas presentes en el músculo comenzó con el descubrimiento de la distrofina, 130 años después de la descripción clínica de la distrofia muscular. Actualmente, debido al mejor conocimiento de la biología del músculo normal y del enfermo, se ha logrado realizar una clasificación molecular de los diferentes tipos de distrofias musculares, de acuerdo con la proteína que se encuentre afectada. Esto ha sido particularmente importante para las distrofias musculares de cinturas, las cuales presentan características clínicas que pueden llevar a confundirlas con la distrofia muscular de Duchenne. Por otro lado, en años recientes se ha favorecido el desarrollo de terapias que en un futuro cercano podrían dar una solución para la restauración de la función de la fibra muscular (AU)

Summary. Muscular dystrophies are a heterogeneous group of hereditary diseases characterized by loss of muscle and weakness of non neurogenic origin. They are caused by mutations in one or more genes involved in the formation of muscle cells. The discovery of several proteins in the muscle began with the discovery of dystrophin, 130 years after the clinical description of muscular dystrophy. Currently, due to a better understanding of the biology of normal and diseased muscle, has achieved a classification at the molecular level of different types of muscular dystrophies, according to the protein that is affected. This has been particularly important for limb girdle muscular dystrophies, which present clinical features that can lead to confusion with Duchenne muscular dystrophy. Moreover, in recent years has encouraged the development of therapies in the near future could provide a solution for restoring the function of the muscle fiber (AU)

The role of mammalian cardiac t-tubules in excitation-contraction coupling: experimental and computational approaches.

The sarcolemmal membrane of mammalian cardiac ventricular myocytes is characterized by the presence of invaginations called transverse tubules (t-tubules). Transverse tubules occur at the Z-line as transverse elements with longitudinal extensions. While the existence of t-tubules has been known for some time, recent experimental studies have suggested that their structure and function are more complex than previously believed. There are, however, aspects of t-tubule function that are not currently amenable to experimental investigation, but can be investigated using computational and mathematical approaches. Such studies have helped elucidate further the possible role of t-tubules in cell function. This review summarizes recent experimental and complementary computational studies which highlight the important role of t-tubules in cardiac excitation-contraction coupling.

Hypersensitivity of myofilament response to Ca2+ in association with maladaptation of estrogen-deficient heart under diabetes complication.

The amelioration of cardioprotective effect of estrogen in diabetes suggests potential interactive action of estrogen and insulin on myofilament activation. We compared Ca2+-dependent Mg2+-ATPase activity of isolated myofibrillar preparations from hearts of sham and 10-wk ovariectomized rats with or without simultaneous 8 wk-induction of diabetes and from diabetic-ovariectomized rats with estrogen and/or insulin supplementation. Similar magnitude of suppressed maximum myofibrillar ATPase activity was demonstrated in ovariectomized, diabetic, and diabetic-ovariectomized rat hearts. Such suppressed activity and the relative suppression in alpha-myosin heavy chain level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca2+ hypersensitivity detected only in the ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rats, which was restored upon estrogen supplementation. Binding kinetics of beta1-adrenergic receptors and immunoblots of beta1-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of the myofilament response to Ca2+. The amount of beta1-adrenoceptors significantly increased concomitant with Ca2+ hypersensitivity of the myofilament, without differences in the receptor binding affinity among the groups. In contrast, changes in HSP72 paralleled that of maximum myofibrillar ATPase activity. These results indicate that hypersensitivity of cardiac myofilament to Ca2+ is specifically induced in ovariectomized rats even under diabetes complication and that alterations in the expression of beta1-adrenoceptors may, in part, play a mechanistic role underlying the cardioprotective effects of estrogen that act together with Ca2+ hypersensitivity of the myofilament in determining the gender difference in cardiac activation.

Incorporated sarcolemmal fish oil fatty acids shorten pig ventricular action potentials.

BACKGROUND: Omega-3 polyunsaturated fatty acids (omega3-PUFAs) from fish oil reduce the risk of sudden death presumably by preventing life-threatening arrhythmias. Acutely administered omega3-PUFAs modulate the activity of several cardiac ion channels, but the chronic effects of a diet enriched with fish oil leading to omega3-PUFA-incorporation into the sarcolemma on membrane currents are unknown. METHODS: Pigs received a diet either rich in omega3-PUFAs or in omega9-fatty acids for 8 weeks. Ventricular myocytes (VMs) were isolated and used for patch-clamp studies. RESULTS: omega3-VMs contained higher amounts of omega3-PUFAs and had a shorter action potential (AP) with a more negative plateau than control VM. In omega3 VMs, L-type Ca(2+) current (I(Ca,L)) and Na(+)-Ca(2+) exchange current (I(NCX)) were reduced by approximately 20% and 60%, respectively, and inward rectifier K(+) current (I(K1)) and slow delayed rectifier K(+) current (I(Ks)) were increased by approximately 50% and 70%, respectively, compared to control. Densities of rapid delayed rectifier K(+) current, Ca(2+)-activated Cl(-) current, and Na(+) current (I(Na)) were unchanged, although voltage-dependence of I(Na) inactivation was more negative in omega3 VMs. CONCLUSIONS: A fish oil diet increases omega3-PUFA content in the ventricular sarcolemma, decreases I(Ca,L) and I(NCX), and increases I(K1) and I(Ks), resulting in AP shortening. Incorporation of omega3-PUFAs in the sarcolemma may have consequences for arrhythmias independent of circulating omega3-PUFAs.

A novel scheme of dystrophin disruption for the progression of advanced heart failure.

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.

Partitioning of polyunsaturated fatty acids, which prevent cardiac arrhythmias, into phospholipid cell membranes.

It has been demonstrated in animal studies that polyunsaturated fatty acids (PUFA) prevent ischemia-induced malignant ventricular arrhythmias, a major cause of sudden cardiac death in humans. To learn how these PUFA, at low micromolar concentrations, exert their antiarrhythmic activity, we studied their effects in vitro on the contractions of isolated cardiac myocytes and the conductances of their sarcolemmal ion channels. These fatty acids directly stabilize electrically every cardiac myocyte by modulating the conductances of specific ion channels in their sarcolemma. In this study, we determined the molar ratio of PUFA to the moles of phospholipid (PL) in cell membranes to learn if the ratio is so low as to preclude the possibility that the primary site of action of PUFA is on the packing of the membrane PL. [(3)H]-arachidonic acid (AA) was used to measure the incorporation of PUFA, and the inorganic phosphorous of the PL was determined as a measure of the moles of PL in the cell membrane. Our results indicate that the mole percent of AA to moles of phospolipid is very low (< or =1.0) at the concentrations that affect myocyte contraction and the conductance of voltage-dependent Na(+) and L-type Ca(2)+ channels in rat cardiomyocytes and in alpha-subunits of human myocardial Na(+) channels. In conclusion, it seems highly unlikely that these fatty acids are affecting the packing of PL within cell membranes as their way of modulating changes in cell membrane ion currents and in preventing arrhythmias in our contractility studies. -- Pound, E. M., J. X. Kang, and A. Leaf. Partitioning of polyunsaturated fatty acids, which prevent cardiac arrhythmias, into phospholipid cell membranes. J. Lipid Res. 2001. 42: 346--351.

n-3 fatty acids in the prevention of cardiac arrhythmias.

In animals and probably in humans n-3 polyunsaturated fatty acids (PUFA) are antiarrhythmic. A report follows on the recent studies of the antiarrhythmic actions of PUFA. The PUFA stabilize the electrical activity of isolated cardiac myocytes by inhibiting sarcolemmal ion channels, so that a stronger electrical stimulus is required to elicit an action potential and the relative refractory period is markedly prolonged. This appears at present to be the probable major antiarrhythmic mechanism of PUFA.

Computer simulation of field distribution and excitation of denervated muscle fibers caused by surface electrodes.

In the course of this study, 2 submodels have been developed and combined, the 2-D finite element modeling of the electrical potential distribution in the human thigh and a Hodgkin and Huxley (HH) type model to calculate fiber excitation and action potential propagation. To determine the excitation of the target muscle fiber with the help of the activating function, the fiber's orientation within the muscle has to be known. The electric field along the fiber has to be calculated as a function of the applied electric current and the potential at the electrodes, respectively. The excitement of the muscle fibers varies across a wide range depending on the active and passive membrane parameters and the intracellular and extracellular mediums. Persisting denervation leads to a decay of muscle cells, and a partial substitution by fibroblasts occurs. The electrical activation of these tissues is more difficult, and biphasic stimulation pulses up to 200 ms in duration and 60-100 V in amplitude are needed to cause a contraction of the denervated muscle. An example shows the field distribution and the simulated activity in one representative muscle fiber of a well trained m. rectus femoris.

Sarcolemmal chloride and potassium channels from normal and myotonic mouse muscle studied in lipid supplemented vesicles.

By patch-clamp analysis of lipid supplemented vesicles prepared from the sarcolemma of mouse wildtype skeletal muscle, we could identify two known types of potassium channels, the inward rectifier and Ca2+ activated BK channels, and five types of chloride channels designated CIC-a, CIC-b, CIC-c, CIC-d, and CIC-e. CIC-b corresponds to a known chloride channel, whereas CICa, -c, -d and -e have not been described previously. The diversity may be due to a heterooligomeric composition of different subunits. None of these chloride channels nor the potassium channels were found in the sarcoplasmic reticulum fraction. In vesicles from myotonic mice, Clc-d and -e were not found.

Coregulation of adrenoceptors and the lipid environment in heart muscle during repeated adrenergic stimulation.

The purpose of this study was to examine changes in binding properties of alpha 1-adrenoceptors, beta-adrenoceptors and L-Ca channels in rat heart muscle in relation to changes in the lipid environment within the membrane, i.e. fatty acyl chain composition of membrane phospholipids, occurring during repeated adrenergic stimulation. The effect of daily administration of epinephrine for seven days upon the maximum number of binding sites of adrenoceptors and upon the fatty acyl chain composition of phosphatidylcholine and phosphatidylethanolamine was examined in 10 months old rats. Decrease in Bmax of adrenoceptors during repeated adrenergic stimulation coincided with remodeling of the membrane phospholipids, with replacement of 18:2n-6 by 20:4n-6 in phosphatidylcholine and by 22:6n-3 in phosphatidylethanolamine, respectively. The effect of repeated adrenergic stimulation was also examined in rats fed different dietary fats and oils, i.e. butter, corn oil or cod liver oil, in hearts with markedly different levels of 18:2n-6, 20:4n-6 and 22:6n-3. The binding properties of the adrenoceptors and L-Ca channels did not relate to the fatty acyl chain composition of bulk phospholipids, but the epinephrine induced decrease in Bmax of the receptors was always accompanied by replacement of 18:2n-6 by 20:4n-6 in phosphatidylcholine and by 22:6n-3 in phosphatidylethanolamine, regardless of the initial level of these fatty acyl chains in the phospholipids. It is concluded that adaption to repeated adrenergic stimulation may include coregulation of the lipid environment within the membrane and the binding properties of adrenoceptors, and possibly other functionally coupled proteins such as L-Ca channels, residing in the membrane.

Ion channel activity in lobster skeletal muscle membrane.

Ion channel activity in the sarcolemmal membrane of muscle fibers is critical for regulating the excitability, and therefore the contractility, of muscle. To begin the characterization of the biophysical properties of the sarcolemmal membrane of lobster exoskeletal muscle fibers, recordings were made from excised patches of membrane from enzymatically induced muscle fiber blebs. Blebs formed as evaginations of the muscle sarcolemmal membrane and were sufficiently free of extracellular debris to allow the formation of gigaohm seals. Under simple experimental conditions using bi-ionic symmetrical recording solutions and maintained holding potentials, a variety of single channel types with conductances in the range 32-380 pS were detected. Two of these ion channel species are described in detail, both are cation channels selective for potassium. They can be distinguished from each other on the basis of their single-channel conductance and gating properties. The results suggest that current flows through a large number of ion channels that open spontaneously in bleb membranes in the absence of exogenous metabolites or hormones.

Indanyloxyacetic acid-sensitive chloride channels from outer membranes of skeletal muscle.

In mature mammalian muscle, the chloride conductance of the membrane is an important factor in the regulation of excitability. Up to now, no ligand was available for the biochemical characterization of muscle chloride channels. In order to localize and characterize these channels, we have used indanyloxyacetic acid (IAA)-94, a ligand previously used for epithelial Cl- channels (Landry, D. W., Reitman, M., Cragoe, E. J., Jr., and Al-Awqati, Q. (1987) J. Gen. Physiol. 90, 779-798; Landry, D. W., Akabas, M. H., Redhead, C., Edelman, A., Cragoe, E. J., Jr., and Al-Awqati, Q. (1989) Science 244, 1469-1472). IAA induced myotonic responses when microinjected into mature mouse muscle fibers, indicating a blockade of Cl- channels from the cytoplasmic side. Membrane vesicles were prepared from rabbit skeletal muscle and separated by sucrose gradient centrifugation. Fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmatic reticulum (SR), and triads and mitochondria (TR/M). The fraction enriched for SL was characterized by high specific binding capacity for [3H]saxitoxin (Na+ channel), whereas TT-rich fractions bound [3H]PN 200-110 (dihydropyridine receptor) with high specific activity. Upon patch-clamping of lipid supplemented vesicles, IAA-sensitive Cl- channels were found in the SL fraction but not in the SR. Highest specific activities in electrical diffusion potential sensitive 36Cl transport and [3H]IAA-94 binding were found in the SL. SL vesicles were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate and subjected to IAA-Sepharose affinity chromatography. Specifically bound protein was eluted with 100 microM IAA-94 and either analyzed by SDS-gel electrophoresis or reconstituted into phospholipid vesicles. The eluate contained four polypeptides (specifically bound, mapp 110-120 and 60 kDa; unspecifically bound mapp 67 and 50 kDa) and was highly enriched for IAA-sensitive chloride channels as shown by patch-clamping after reconstitution. The IAA-sensitive 100/280-picosiemens chloride channels of the sarcolemma are likely to be responsible for its major chloride conductance and thereby for the stabilization of resting potential.

The effect of Agauria salicifolia leaf extract on the sodium current of tetrodotoxin-treated frog skeletal muscle fibres.

An ethanolic extract of Agauria salicifolia leaf (AS) was tested on voltage-clamped single muscle fibres from frogs, using the double sucrose-gap technique, in the presence of tetrodotoxin (TTX) to partially block the transient inward sodium current. Below 5 x 10(-10) g/ml TTX, 10(-6) g/l AS reversed the blocking effect of TTX on INa and increased the current amplitude. For TTX concentrations ranging from 5 x 10(-10) to 5 x 10(-8) g/ml, which partially blocked INa and lengthened its time to peak, 10(-6) g/l AS reinforced the blocking effect on the current. The inhibitory effect of AS was then studied in the presence of 10(-9) g/ml TTX which was assumed to block all the sarcolemmal sodium channels and not the tubular ones. It appeared to be strongly voltage-dependent since it was fully relieved when the cell was hyperpolarized by 20 mV and was maximum at holding potential (h.p.) -70 mV (100% blockage of INa instead of 50% at h.p. -90 mV). Contrary to observations in depolarised fibres (in which the inhibitory effect of AS decreased as concentration rose) the INa blockage increased with concentration in TTX-treated fibres. It is concluded that AS extract exerts an antagonistic effect on tubular sodium channels by acting on a single site which is different from the high affinity inhibitory site demasked in normal fibres by depolarization of the resting membrane potential.

The effects of Agauria salicifolia leaf extract on calcium current and excitation-contraction coupling of isolated frog muscle cells.

The effects of the ethanolic extract of Agauria salicifolia (AS) previously tested on sodium currents of normal and TTX-treated voltage-clamped skeletal and cardiac muscle cells was investigated on mechanisms involved in the excitation-contraction coupling of the same biological preparations. AS (10(-6) g/l) prolonged the action potential duration whereas the associated contraction was depressed. This is not due to the blocking action of AS on the tubular sodium current since similar results were obtained in the presence of high tetrodotoxin (TTX) concentrations. This is not due to some blocking action of AS on the L-type calcium current since this type of calcium current was enhanced by the extract. In contrast, the cardiac T-type calcium current was decreased by 10(-6) g/l AS. AS exerted a dose-dependent (tested for concentrations ranging from 5 x 10(-10) to 5 x 10(-5) g/l) inhibition of the two components of the contractile response elicited by durable depolarizations, with a prominent effect on the tonic phase. This effect was partially relieved by increasing the external divalent cation (Ca2+ or Cd2+) concentration. At the same concentration it shifted the inactivation/potential relationship for tension by 20 mV towards negative potentials. It is concluded that the inhibitory action of AS on excitation-contraction coupling is partly or completely due to the enhancement of the voltage- and Ca(2+)-dependent inactivation processes of the voltage-sensor.

Inward spread of activation in vertebrate muscle fibres.

1. A method for detecting the activation of individual myofibrils or groups of myofibrils within an isolated muscle fibre is described. It consists in making all the myofibrils wavy by setting the fibre in gelatine and compressing it longitudinally; active shortening of myofibrils can then be recognized by the straightening out of the waves.2. The time course of this straightening during a twitch was found by high-speed ciné micrography.3. There is a delay of activation between the superficial and central myofibrils, from which the velocity of inward spread of activation can be found.4. This velocity has a Q(10) of 2, and is about 7 cm/sec at 20 degrees C. The mechanism of the inward spread of activation is discussed.5. On relaxation the waves reappear, showing that there is a spontaneous elongation of the myofibrils.