Cat Heart Muscle in Vitro : VII. The temperature dependence of steady state K exchange in presence and absence of NaCl

In quiescent cat papillary muscles J _K, the rate of exchange of cellular K with K⁴² in the steady state, has been measured in the presence and absence of NaCl over a wide range of temperatures. J _K was found to be independent of the presence of external NaCl under the steady state conditions investigated. The Arrhenius plot for K exchange was linear over a range of temperatures from 2.5 to 37.5°C in the absence of NaCl, and from 17.5 to 37.5°C in the presence of NaCl. The corresponding apparent activation energies were, respectively, 10,200 and 8,800 calories/mole. J _K in the absence of NaCl was not affected by 10^-5 M ouabain. These results are consistent with a passive distribution for the K of heart muscle cells. The observations suggest that a carrier-mediated forced exchange of K for Na does not occur during the steady state in mammalian heart muscle.     

The Rate of Oxygen Uptake of Quiescent Cardiac Muscle

The rate of oxygen uptake of quiescent papillary muscle of the cat heart has been determined in a flow respirometer with the use of the oxygen electrode. The apparent rate of oxygen uptake as a function of the diameter of the muscle was also determined. It was found that papillary muscles from cat hearts use oxygen at a rate of 2.84 (microliters/mg. wet weight)/hour at a temperature of 35°C. Such muscles can be adequately supplied by diffusion when their surface is uniformly exposed to an atmosphere containing 95 per cent oxygen only if their diameter is 0.64 mm. or less. Papillary muscles from kitten hearts use oxygen at a rate of 4.05 (microliters/mg. wet weight)/hour at a temperature of 35°C. Such muscles can be adequately supplied by diffusion when their surface is uniformly exposed to an atmosphere containing 95 per cent oxygen only if their diameter is 0.53 mm. or less. If the muscles are small enough to be adequately supplied with oxygen by diffusion, the rate of oxygen uptake does not increase when the muscle is stretched.     

Cat Heart Muscle in Vitro : VIII. Active transport of sodium in papillary muscles

The cells of cat right ventricular papillary muscles were depleted of K and caused to accumulate Na and water by preincubation at 2–3°C. The time courses of changes in cellular ion content and volume and of the resting membrane potential (V_m) were then followed after abrupt rewarming to 27–28°C. At physiological external K concentration ([K]_o = 5.32 mM) recovery of cellular ion and water contents was complete within 30 minutes, the maximal observable rates of K uptake and Na extrusion (Δmmol cell ion/(kg dry weight) (min.)) being 3.4 and 3.6, respectively. The recovery rate was markedly slowed at [K]_o = 1.0 mM. Rewarming caused V_m measured in cells at the muscle surface to recover within from <1 to 9 minutes, but only slight restoration of cellular ion contents (measured in whole muscles) had occurred after 10 minutes. Studies of recovery in NaCl-free sucrose Ringer''s solution made it possible to separate the ouabain-insensitive outward diffusion of Na as a salt from a simultaneous ouabain-sensitive Na extrusion which is associated with a net cellular K uptake. A hypothesis consistent with these observations is that rewarming may activate a ouabain-sensitive "electrogenic" mechanism, most probably the net active transport of Na out of the cell, from which net K uptake may then follow passively.     

Electrically induced muscle cramps induce hypertrophy of calf muscles in healthy adults

Objectives:

Skeletal muscles usually cramp at short lengths, where the tension that can be exerted by muscle fibers is low. Since high tension is an important anabolic stimulus, it is questionable if cramps can induce hypertrophy and strength gains. In the present study we investigated if electrically induced cramps (EIMCs) can elicit these adaptations.

Methods:

15 healthy male adults were randomly assigned to an intervention (IG; n=10) and a control group (CG; n=5). The cramp protocol (CP) applied twice a week to one leg of the IG, consisted of 3x6 EIMCs, of 5 s each. Calf muscles of the opposite leg were stimulated equally, but were hindered from cramping by fixating the ankle at 0° plantar flexion (nCP).

Results:

After six weeks, the cross sectional area of the triceps surae was similarly increased in both the CP (+9.0±3.4%) and the nCP (+6.8±3.7%). By contrast, force of maximal voluntary contractions, measured at 0° and 30° plantar flexion, increased significantly only in nCP (0°: +8.5±8.8%; 30°: 11.7±13.7%).

Conclusion:

The present data indicate that muscle cramps can induce hypertrophy in calf muscles, though lacking high tension as an important anabolic stimulus.     

Myocardial and Skeletal Muscle Concentrations of Digoxin in Patients on Long-term Therapy

The digoxin content was measured in samples of left ventricular papillary muscle, skeletal muscle, and plasma obtained during mitral valve replacement from eight patients on maintenance treatment with the drug. The content in papillary muscle ranged from 15·5 to 132 ng/g (mean 77·7) and in skeletal muscle from 7·5 to 23 ng/g (mean 11·3). The ratio of myocardial digoxin concentration to plasma concentration varied between patients from 39:1 to 155:1. No simple relationship exists between plasma levels of digoxin and its concentration in the heart muscle, but total myocardial concentration may not accurately reflect therapeutic activity.     

Temperature Dependence of Vasopressin Action on the Toad Bladder

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 20° to 38°C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 4° to 20°C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°–9°C) and 4.1 kcal/mole (9°–20°C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20°C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 µosmol of NaCl was exchanged for 1 µl of H₂O across the denatured membrane.     

ATP Consumption by Sarcoplasmic Reticulum Ca2+ Pumps Accounts for 40-50% of Resting Metabolic Rate in Mouse Fast and Slow Twitch Skeletal Muscle

The main purpose of this study was to directly quantify the relative contribution of Ca²⁺ cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO₂, µL/g wet weight/s) measured polarographically at 30^°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca²⁺ cycling to resting metabolic rate, the concentration of MgCl₂ in the bath was increased to 10 mM to block Ca²⁺ release through the ryanodine receptor, thus eliminating a major source of Ca²⁺ leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca²⁺-ATPases (SERCAs). The relative (%) reduction in muscle VO₂ in response to 10 mM MgCl₂ was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca²⁺ cycling in resting isolated muscles. The results of the MgCl₂ experiments indicate that ATP consumption by SERCAs is responsible for 40–50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30^°C, or 12–15% of whole body resting VO₂. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.     

Muscle Activity and Inactivity Periods during Normal Daily Life

Recent findings suggest that not only the lack of physical activity, but also prolonged times of sedentary behaviour where major locomotor muscles are inactive, significantly increase the risk of chronic diseases. The purpose of this study was to provide details of quadriceps and hamstring muscle inactivity and activity during normal daily life of ordinary people. Eighty-four volunteers (44 females, 40 males, 44.1±17.3 years, 172.3±6.1 cm, 70.1±10.2 kg) were measured during normal daily life using shorts measuring muscle electromyographic (EMG) activity (recording time 11.3±2.0 hours). EMG was normalized to isometric MVC (EMG_MVC) during knee flexion and extension, and inactivity threshold of each muscle group was defined as 90% of EMG activity during standing (2.5±1.7% of EMG_MVC). During normal daily life the average EMG amplitude was 4.0±2.6% and average activity burst amplitude was 5.8±3.4% of EMG_MVC (mean duration of 1.4±1.4 s) which is below the EMG level required for walking (5 km/h corresponding to EMG level of about 10% of EMG_MVC). Using the proposed individual inactivity threshold, thigh muscles were inactive 67.5±11.9% of the total recording time and the longest inactivity periods lasted for 13.9±7.3 min (2.5–38.3 min). Women had more activity bursts and spent more time at intensities above 40% EMG_MVC than men (p<0.05). In conclusion, during normal daily life the locomotor muscles are inactive about 7.5 hours, and only a small fraction of muscle''s maximal voluntary activation capacity is used averaging only 4% of the maximal recruitment of the thigh muscles. Some daily non-exercise activities such as stair climbing produce much higher muscle activity levels than brisk walking, and replacing sitting by standing can considerably increase cumulative daily muscle activity.     

Nerve stimulation decreases malonyl-CoA in skeletal muscle.

This study was designed to determine the effect of in situ electrical stimulation of the sciatic nerve on malonyl-CoA, an inhibitor of carnitine palmitoyl transferase, in the gastrocnemius/plantaris muscle group of rats. The left sciatic nerve was stimulated at a frequency of 5 Hz with 100-ms trains of impulses (50 Hz) for 1, 3, or 5 min. At the end of stimulation, the left and right (nonstimulated) gastrocnemius/plantaris muscle groups were clamp-frozen and later analyzed for malonyl-CoA and other metabolites. No change was observed in the noncontracting contralateral muscles in malonyl-CoA, ATP, creatine phosphate (CP), or citrate. In the stimulated muscles, malonyl-CoA decreased from 1.7 +/- 0.1 to 1.0 +/- 0.1 nmol/g (P less than 0.05), and CP decreased from 15.8 +/- 0.9 to 12.2 +/- 1.0 mumol/g (P less than 0.05) after 3 min of stimulation. After 5 min of stimulation, malonyl-CoA was 1.0 +/- 0.1 nmol/g and CP was 10.3 +/- 1.3 mumol/g. When muscles were stimulated for 5 min with single impulses (5 Hz), malonyl-CoA was decreased from 1.8 +/- 0.3 to 1.0 +/- 0.1 nmol/g, with no change in CP, ATP, or adenosine 3',5'-cyclic monophosphate. Thus a decline in malonyl-CoA can be induced by muscle contraction independently of humoral influence.     

Energetics of Isometric and Isotonic Twitches in Toad Sartorius

Contractile energetics have been studied in twitches of toad sartorius muscle at 6-7°C. Isometric and isotonic energy production has been measured and plotted against a wide range of developed tensions and tension-time integrals. These parameters were varied by altering the isotonic load or by changing the preset isometric length. The isometric tension-independent heat was 1.12 ±0.18 (SD) mcal/g. The isometric heat coefficient Pl₀/H was 12.0 ±1.4 in muscles having twitch to tetanus ratios ranging from 0.4 to 0.6. Isometric enthalpy increased monotonically with tension or tension-time integral but the correlation between isometric heat and these parameters was poor. Isotonic enthalpy consumption was always higher than isometric enthalpy for any given tension or tension-time integral; however, isotonic heat production was consistently less than isometric heat production. The isotonic heat for the highest load (3 g) was not significantly different from the isometric tension-independent heat. Thus isotonic heat production first decreased and then increased with increasing tension or tension-time integral. In the discussion it is shown that the results conflict with all current interpretations of muscle energetics.     

Cat Heart Muscle in Vitro : I. Cell volumes and intracellular concentrations in papillary muscle

Methods have been developed for the simultaneous determination of total water, inulin space, and K and Na content in muscles of 0.5 to 10 mg. wet weight. These methods have been used to define steady state conditions with respect to intracellular K concentration in papillary muscles from cat hearts perfused and contracting isometrically at 27–28°C. and at 37–38°C. Cell volumes and intracellular ionic concentrations have been followed as a function of the external K concentration and compared with values predicted on the basis of electroneutrality and osmotic equilibrium.     

Increasing skeletal muscle carnitine content in older individuals increases whole‐body fat oxidation during moderate‐intensity exercise

Intramyocellular lipid (IMCL) utilization is impaired in older individuals, and IMCL accumulation is associated with insulin resistance. We hypothesized that increasing muscle total carnitine content in older men would increase fat oxidation and IMCL utilization during exercise, and improve insulin sensitivity. Fourteen healthy older men (69 ± 1 year, BMI 26.5 ± 0.8 kg/m²) performed 1 h of cycling at 50% VO₂max and, on a separate occasion, underwent a 60 mU/m²/min euglycaemic hyperinsulinaemic clamp before and after 25 weeks of daily ingestion of a 220 ml insulinogenic beverage (44.4 g carbohydrate, 13.8 g protein) containing 4.5 g placebo (n = 7) or L‐carnitine L‐tartrate (n = 7). During supplementation, participants performed twice‐weekly cycling for 1 h at 50% VO₂max. Placebo ingestion had no effect on muscle carnitine content or total fat oxidation during exercise at 50% VO₂max. L‐carnitine supplementation resulted in a 20% increase in muscle total carnitine content (20.1 ± 1.2 to 23.9 ± 1.7 mmol/kg/dm; p < 0.01) and a 20% increase in total fat oxidation (181.1 ± 15.0 to 220.4 ± 19.6 J/kg lbm/min; p < 0.01), predominantly due to increased IMCL utilization. These changes were associated with increased expression of genes involved in fat metabolism (ACAT1, DGKD & PLIN2; p < 0.05). There was no change in resting insulin‐stimulated whole‐body or skeletal muscle glucose disposal after supplementation. This is the first study to demonstrate that a carnitine‐mediated increase in fat oxidation is achievable in older individuals. This warrants further investigation given reduced lipid turnover is associated with poor metabolic health in older adults.     

Effects of Neuromuscular Electrical Stimulation on the Masticatory Muscles and Physiologic Sleep Variables in Adults with Cerebral Palsy: A Novel Therapeutic Approach

Cerebral palsy (CP) is a term employed to define a group of non-progressive neuromotor disorders caused by damage to the immature or developing brain, with consequent limitations regarding movement and posture. CP may impair orapharygeal muscle tone, leading to a compromised chewing function and to sleep disorders (such as obstructive sleep apnea). Thirteen adults with CP underwent bilateral masseter and temporalis neuromuscular electrical stimulation (NMES) therapy. The effects on the masticatory muscles and sleep variables were evaluated using electromyography (EMG) and polysomnography (PSG), respectively, prior and after 2 months of NMES. EMG consisted of 3 tests in different positions: rest, mouth opening and maximum clenching effort (MCE). EMG values in the rest position were 100% higher than values recorded prior to therapy for all muscles analyzed (p < 0.05); mean mouth opening increased from 38.0 ± 8.0 to 44.0 ± 10.0 cm (p = 0.03). A significant difference in MCE was found only for the right masseter. PSG revealed an improved in the AHI from 7.2±7.0/h to 2.3±1.5/h (p < 0.05); total sleep time improved from 185 min to 250 min (p = 0.04) and minimun SaO2 improved from 83.6 ± 3.0 to 86.4 ± 4.0 (p = 0.04). NMES performed over a two-month period led to improvements in the electrical activity of the masticatory muscles at rest, mouth opening, isometric contraction and sleep variables, including the elimination of obstructive sleep apnea events in patients with CP.

Trial Registration

ReBEC RBR994XFS http://www.ensaiosclinicos.gov.br     

CONTRACTILITY AND ULTRASTRUCTURE IN GLYCEROL-EXTRACTED MUSCLE FIBERS : I. The Relationship of Contractility to Sarcomere Length

This study was undertaken to determine whether glycerol-extracted rabbit psoas muscle fibers can develop tension and shorten after being stretched to such a length that the primary and secondary filaments no longer overlap. A method was devised to measure the initial sarcomere length and the ATP-induced isotonic shortening in prestretched isolated fibers subjected to a small preload (0.02 to 0.15 P₀). At all degrees of stretch, the fiber was able to shorten (60 to 75 per cent): to a sarcomere length of 0.7 µ when the initial length was 3.7 µ or less, and to an increasing length of 0.9 to 1.8 µ with increasing initial sarcomere length (3.8 to 4.4 µ). At sarcomere lengths of 3.8 to 4.5 µ, overlap of filaments was lost, as verified by electron microscopy. The variation in sarcomere length within individual fibers has been assessed by both light and electron microscopic measurements. In fibers up to 10 mm in length the stretch was evenly distributed along the fiber, and with sarcomere spacings greater than 4 µ there was only a slight chance of finding sarcomeres with filament overlap. These observations are in apparent contradiction to the assumption that an overlap of A and I filaments is necessary for tension generation and shortening.     

Intracellular Calcium Binding and Release in Frog Heart

The capacities and affinities of intracellular calcium-binding sites have been studied in frog ventricles, in which the concentration of Ca⁺⁺ in the sarcoplasm can be controlled as a result of treatment with EDTA. The total calcium content of calcium-depleted and nondepleted muscles at rest and muscles generating considerable tension was 0.8, 1.4, and 5.4 µmol/g of muscle, respectively. Net movement of calcium into or out of the cells occurred without change in tension when the sarcoplasmic concentration of Ca⁺⁺ was either of two values, less than 10^-7 M or approximately 5 x 10^-7 M. These data can be explained by the presence of two groups of intracellular calcium sinks which compete with the contractile proteins, one with a capacity of about 0.6 µmol/g and an affinity constant greater than 10⁷ M^-1 and a second with a capacity of 4.0 µmol/g and an affinity constant of about 2 x 10⁶ M^-1. The higher affinity calcium is released by anoxia, oligomycin, or abrupt changes in sarcoplasmic Ca⁺⁺. Muscles soaked in Sr-Ringer's contain electron densities in the sarcoplasmic reticulum and to a lesser extent in the mitochondria.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

Metabolism of propionate by sheep liver. Oxidation of propionate by homogenates

1. The rate and stability to aging of the metabolism of propionate by sheep-liver slices and sucrose homogenates were examined. Aging for up to 20min. at 37° in the absence of added substrate had little effect with slices, whole homogenates or homogenates without the nuclear fraction. 2. Metabolism of propionate by sucrose homogenates was confined to the mitochondrial fraction, but the mitochondrial supernatant (microsomes plus cell sap) stimulated propionate removal. 3. The rate of propionate metabolism by liver slices was higher in a high potassium phosphate–bicarbonate medium [0·88(±s.e.m. 0·16)μmole/mg. of N/hr.] than in Krebs–Ringer bicarbonate medium [0·44(±s.e.m. 0·13)μmole/mg. of N/hr.]. 4. Metabolism of propionate by sucrose homogenates freed from nuclei was dependent on the presence of oxygen, carbon dioxide and ATP. Propionate removal was stimulated 250% by Mg²⁺ ions and 670% by cytochrome c. 5. In the complete medium 2·39(±s.e.m. 0·15)μmoles of propionate were consumed/mg. of N/hr. 6. The ratio of oxygen consumption to propionate utilization was sufficient to account for the complete oxidation of half the propionate consumed. 7. The only products detected under these conditions were succinate, fumarate and malate. Propionate had no effect on the production of lactate from endogenous sources and did not itself give rise to lactate. 8. Methylmalonate did not accumulate when propionate was metabolized and was not oxidized. It was detected as an intermediate in the conversion of propionyl-CoA into succinate. The rate of this reaction sequence was adequate to account for the rate of propionate metabolism by sucrose homogenates or slices, provided that the rate of formation of propionyl-CoA was not limiting. 9. The methylmalonate pathway was predominantly a mitochondrial function. 10. The metabolism of propionate appeared to be dependent on active oxidative phosphorylation.     

Cat Heart Muscle in Vitro : III. The extracellular space

The "osmotic gradient" method, an intracellular microelectrode technique for determining whether an uncharged, water-soluble molecule enters cells or remains extracellular, is described. Using this method, a series of carbohydrates of graded molecular size were examined. In cat papillary muscles mannitol, molecular radius 4.0 Å, remained extracellular while arabinose, molecular radius 3.5 Å entered the cells. Measurement of the simultaneous uptake of H³-mannitol and C¹⁴-inulin showed that mannitol equilibrates with 40 per cent of total water in 1 hour, after which the mannitol space does not further increase. By contrast, inulin, molecular radius ~15 Å, equilibrates with 24 per cent of total water in 1 hour; thereafter the inulin space continues to increase very slowly. The intracellular K concentrations are significantly higher and the intracellular Na and Cl concentrations significantly lower when mannitol rather than inulin is used to measure the extracellular space. The intracellular Cl concentration determined with Cl³⁶ or Br⁸² is significantly higher than that calculated from the membrane potential assuming a passive Cl distribution. In addition, it is shown that choline enters and is probably metabolized by the cells of papillary muscle.     

Transient Phases of the Isometric Tetanus in Frog's Striated Muscle

In an isometric tetanus in frog's sartorius muscle tension approaches the plateau exponentially with rate constant α. α a depends on sarcomere length, s, and temperature, T, according to the Arrhenius equation See PDF for Equation for temperatures between 1 and 20°C and for sarcomere lengths 2.0–2.8 µm. The energy of activation, E, does not vary significantly with s; E = 13.9 ± 2.4 kcal/mole. A(s) decreases monotonically with s; A(2.1 µm) is about three times greater than A(2.8 µm). Late in relaxation active tension approaches zero exponentially with rate constant r. r decreases exponentially with increasing duration of tetanus, D, from r₀ in a twitch to r_∞ for large D. The rate constant for decrease of r with D increases with s and with T. r₀ and r_∞ obey the Arrhenius equation and decrease with increasing s.     

Sodium Exchange in Dog Ventricular Muscle : Relation to frequency of contraction and its possible role in the control of myocardial contractility

Sodium exchange was studied in the arterially perfused papillary muscle of the dog. Three kinetically defined phases accounted for all the myocardial sodium: phase 0 (vascular)-λ_o (exchange constant) = 3.6 min^-1 phase 1 (interstitial)-λ₁ = 0.62 min^-1; phase 2 (intracellular)-λ₂ < 0.020 min^-1 in quiescent muscles. The phase 2 exchange rate was proportional to frequency of contraction and increased by approximately 0.004 min^-1 for each 1 beat/min increment in rate in muscles demonstrating stable function. A sudden increase in frequency of contraction was followed by a marked increase in phase 2 sodium exchange if muscle function did not deteriorate. This increased exchange required 14 min to achieve a steady state. During this time active tension increased (positive staircase) and then declined to become stable as the sodium exchange stabilized. In muscles in which increased frequency of contraction produced a progressive decrease in active tension and contracture, sodium exchange failed to increase. The characteristics of sodium exchange are compared to those previously defined for calcium and potassium in the perfused dog papillary muscle. It is proposed that alteration in sodium exchange is a primary determinant of calcium and potassium movements and thereby plays a significant role in the control of myocardial contractility.