Fang Lou

Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology.

For the majority of Duchenne muscular dystrophy (DMD) mutations, antisense oligonucleotide (AON)-mediated exon skipping has the potential to restore a functional protein. Here we show that weekly intravenous injections of morpholino phosphorodiamidate (morpholino) AONs induce expression of functional levels of dystrophin in body-wide skeletal muscles of the dystrophic mdx mouse, with resulting improvement in muscle function. Although the level of dystrophin expression achieved varies considerably between muscles, antisense therapy may provide a realistic hope for the treatment of a majority of individuals with DMD.

Depression of tetanic force induced by loaded shortening of frog muscle fibres.

1. Single fibres isolated from the anterior tibialis muscle of Rana temporaria were allowed to shorten against a high load during a 2.5‐4.0 s fused tetanus (1‐3 degrees C) and the maximum force produced at the short length was compared with that recorded during a fixed‐end tetanus at the same overall fibre length. Changes in length of marked, consecutive segments (ca 0.5 mm in length) along the fibre were measured throughout the tetanus using a photoelectric recording system. 2. Loaded shortening (load ca 3/4 of maximum tetanic force) starting from approximately 2.55 microns sarcomere length and ending near slack fibre length depressed the tetanic force by 13 +/‐ 2% (mean +/‐ S.E.M., n = 10) and caused a marked redistribution of sarcomere length along the fibre. Unloaded shortening over the same range caused no force deficit and did not lead to increased dispersion of sarcomere length. 3. Loaded shortening below slack length produced less force depression and less non‐uniformity of sarcomere length than did a corresponding intervention above slack length. 4. The force deficit after loaded shortening, both above and below slack fibre length, was positively correlated (P < 0.005) to the coefficient of variation of the sarcomere length along the fibre. 5. The decrease in active force after loaded shortening, and its relation to increased dispersion of sarcomere length along the fibre, could be simulated closely by a computer model in which the muscle fibre was assumed to consist of eleven discrete segments acting in series with a passive elastic element. 6. Experiments were performed in which the length of an individual segment of the intact muscle fibre was strictly controlled throughout a tetanus. Loaded shortening of such a ‘length‐clamped’ segment caused no force depression during the subsequent isometric phase either above or below slack fibre length. 7. The results suggest strongly that force depression after loaded shortening of a single muscle fibre is attributable to non‐uniform sarcomere behaviour along the fibre. The experimental evidence supports the view that: (i) the myosin cross‐bridges act as independent force generators; and (ii) their steady‐state performance during a tetanus is unaffected by the preceding contractile activity.

Calcium‐ and myosin‐dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin‐containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double‐cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca2+‐binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca2+ dependence as active force production, with a Hill coefficient (nH) close to 3, consistent with a single co‐operative transition controlled by Ca2+ binding. Complete inhibition of active force by 25 μm blebbistatin had very little effect on the Ca2+‐dependent structural changes and in particular did not significantly reduce the value of nH. Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca2+ also changed the orientation of the C and E helices, and addition of Ca2+ in rigor produced further changes characterized by increased Ca2+ affinity but with nH close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co‐operative Ca2+ regulation of cardiac contractility must therefore be intrinsic to the thin filaments.

Changes in force and stiffness induced by fatigue and intracellular acidification in frog muscle fibres.

1. Changes in force and stiffness were recorded simultaneously during 1 s isometric (fixed ends) tetani of single fibres isolated from the anterior tibialis muscle of Rana temporaria (temperature 1‐3 degrees C; sarcomere length, 2.10 micron). Stiffness was measured as the change in force that occurred in response to a 4 kHz sinusoidal length oscillation of the fibre. Some experiments were performed in which stiffness was determined from a fast (0.2 ms) length step that was applied to a ‘tendon‐free’ segment of the muscle fibre during the tetanus plateau. 2. A moderate degree of fatigue was produced by decreasing the time between tetani from 300 s (control) to 15 s. By this treatment the maximum tetanic force (Ftet) was reversibly reduced to 70‐75% of the control value. Maximum tetanic stiffness (Stet) was related to Ftet according to the following regression (both variables expressed as percentage of their control values): Stet = 0.369 Ftet + 62.91 (correlation coefficient, 0.95; P less than 0.001). A 25% decrease in isometric force during fatigue was thus associated with merely 9% reduction of fibre stiffness. 3. Whereas the rate of rise of force during tetanus was markedly reduced by fatiguing stimulation, the rate of rise of stiffness was only slightly affected. 4. Intracellular acidification (produced by raised extracellular CO2 concentration) largely reproduced the contractile changes observed during fatigue. However, for a given decrease in tetanic force there was a smaller reduction in fibre stiffness during acidosis than during fatigue. 5. Caffeine (0.5 mM) added to the fibre after development of fatigue and intracellular acidosis greatly potentiated the isometric twitch but did not affect maximum tetanic force. This finding provides evidence that the contractile system was fully activated during the tetanus plateau both in the fatigued state and during acidosis. 6. The results suggest that the decrease in contractile strength after frequent tetanization (intervals between tetani, 15 s) is attributable to altered kinetics of cross‐bridge function leading to reduced number of active cross‐bridges and, most significantly, to reduced force output of the individual bridge. The possible role of increased intracellular H+ concentration in the development of muscle fatigue is discussed.

Variation in myoplasmic Ca2+ concentration during contraction and relaxation studied by the indicator fluo-3 in frog muscle fibres

1. The fluorescent dye fluo‐3, in its permeant acetoxymethyl form, was used to monitor calcium transients during twitch and tetanus of single fibres isolated from the anterior tibialis muscle of Rana temporaria (2‐5 degrees C). 2. Fluo‐3 was loaded into the muscle fibre by diffusion. Under the experimental conditions used, approximately 45% of maximal fluorescence was reached during a 1 s fused isometric tetanus. Fluo‐3 had no detectable effect on the mechanical response of the fibre. 3. The free calcium concentration in the myoplasm, [Ca2+]i, and its variation with time, was calculated from the fluorescence signal by accounting for the on‐ and off‐rate constants for the binding of calcium to the dye. The time course of the calcium transient during twitch and tetanus determined in this way agreed well with previous measurements based on fast‐reacting calcium‐sensitive dyes. 4. [Ca2+]i declined steeply during the initial phase of force relaxation in both twitch and tetanus, but exhibited a secondary rise that closely coincided with the pseudoexponential fall of tension after the shoulder in the tetanus myogram. The rate of decay of [Ca2+]i during relaxation and the rate of decline of force both became progressively reduced by repetitive stimulation. 5. Stretch and shortening ramps performed during the plateau of an isometric tetanus had no detectable effect upon the calcium transient during the movement. By contrast, shortening and stretch imposed during the linear phase of relaxation both led to an increase of [Ca2+]i and to a steepening of the relaxation phase. 6. The results strongly suggest that the non‐uniform length changes that are known to occur along a muscle fibre during relaxation enhance the release of calcium from the contractile system. The calcium mobilized in this way probably accounts for the transitory increase of [Ca2+]i that is observed during the latter part of force relaxation.

Myofibrillar fatigue versus failure of activation during repetitive stimulation of frog muscle fibres

1. Single fibres isolated from the anterior tibialis muscle of Rana temporaria (temperature, 2‐5 degrees C; sarcomere length, 2.10 microns) were fatigued using two separate protocols that led to different degrees of depression of tetanic force. Under control conditions the fibre was stimulated to produce a 1 s fused isometric tetanus at 300 s intervals. A moderate degree of fatigue (tetanic force reduced to 70‐80% of the control value) was produced by decreasing the intervals between tetani to 15 s (‘fatiguing protocol 1’). A more pronounced depression of tetanic force (to 40‐50% of the control value) was produced by evoking a single twitch at 1‐2 s intervals (‘fatiguing protocol 2’). 2. Fatiguing protocol 1 reduced the contracture response to submaximal and supramaximal concentrations of caffeine (3‐15 mM) in proportion to the decrease in tetanic force. These results support the view that fatiguing stimulation according to protocol 1 leads to a true ‘myofibrillar fatigue’ with no failure of activation of the muscle fibre. 3. Fatiguing protocol 2 reduced the amplitudes of isometric twitch and tetanus to below 10 and 50% of the control values, respectively. By contrast, the maximal contracture response to caffeine (15 mM) was depressed by merely 2‐3% of its prefatigue value. 4. Force and instantaneous fibre stiffness were recorded simultaneously during twitch and tetanus as fatigue was induced by protocol 2. During the initial part of fatigue (tetanic force reduced by 25% of control) stiffness was reduced by merely 9% in accordance with previous measurements during fatigue induced by protocol 1. However, with further depression of twitch and tetanus by protocol 2 there was a marked reduction of fibre stiffness. These results, together with the findings reported under point 3, strongly suggest that at an advanced state of fatigue induced by protocol 2 the decrease in active force is largely due to failure of activation of the contractile system. 5. Muscle fibres were quickly frozen for electron microscopical examination after shortening below slack length (to approximately 1.6 microns sarcomere spacing) during tetanic stimulation. In non‐fatigued fibres, and in fibres fatigued according to protocol 1, the myofibrils exhibited a straight appearance throughout the preparation suggesting that the entire volume of the fibre was properly activated. In fibres fatigued by protocol 2, on the other hand, only the most peripheral layers of myofibrils remained straight after shortening, whereas the centre of the fibre showed marked waviness indicating failure of the inward spread of activation in this case.

The relationship between the intracellular Ca2+ transient and the isometric twitch force in frog muscle fibres

The calcium‐sensitive fluorescent indicator fluo‐3 was used to monitor the intracellular free calcium concentration ([Ca2+]i) during isometric twitches in twenty‐nine single muscle fibres from the anterior tibialis muscle of Rana temporaria (sarcomere length, 2.2 microns; 2‐4 degrees C). The transient change in [Ca2+]i in response to a single stimulus was very brief. The time to peak and the duration of the Ca2+ signal, measured at 50% of the peak amplitude, were 8.3 +/‐ 0.2 and 22.1 +/‐ 1.4 ms (mean +/‐ S.E.M., n = 29), respectively. The mean peak amplitude of the Ca2+ transient was 3.2 +/‐ 0.1 microM, ranging from 2.46 to 3.92 microM among the different fibres. The isometric force started to rise 2.5 ms before [Ca2+]i reached its maximum value. When peak twitch force was attained, [Ca2+]i had already declined to approximately 10% of its maximum value. The peak force produced during a twitch was closely related to the decay phase of the Ca2+ transient, a slower decay of [Ca2+]i being associated with a greater amplitude of the twitch. The amplitude and duration of the Ca2+ transient varied in a systematic way relative to one another in different fibres, in that a greater amplitude was associated with a more rapid decay of the Ca2+ transient. NO3‐ and Zn2+ added to the external medium greatly enhanced the peak twitch force without markedly affecting the amplitude of the Ca2+ transient. However, both agents delayed the decay of [Ca2+]i. It is concluded that the decay phase of the Ca2+ transient is a more important determinant of the mechanical response during an isometric twitch than is the peak amplitude of the transient.

Contraction with shortening during stimulation or during relaxation: how do the energetic costs compare?

White muscle fibres from dogfish were used to compare the energetic costs of shortening by fully active muscle and by relaxing muscle. The muscle preparation was tetanized for 0.6s and shortened either during stimulation or during relaxation. The distance shortened was 1mm (about 15% L0, the muscle length optimum for force) and the velocity was 3.5 or 7.0mms−1 (about 15 or 30% V0, the maximum velocity of shortening). Isometric tetani at L0 were also investigated. Mechanical work and heat production were measured, and work + heat was taken as a measure of energetic cost. Both work and the energetic cost were higher with shortening during stimulation than with shortening during relaxation. The results suggest that shortening during relaxation, which is known to occur during locomotion in vivo, may be an energy-saving strategy.

Sustained performance by red and white muscle fibres from the dogfish Scyliorhinus canicula

SUMMARY The mechanical performance of red and white muscle fibres from dogfish was compared during a long series of contractions with sinusoidal movement or under isometric conditions at 12°C (normal in vivo temperature). Power output was measured during sinusoidal movement at 0.75 Hz and peak-to-peak amplitude about 12% L0. Tetanus duty cycle was 33% (0.44 s) at phase −8% (first stimulus at 0.107 s before shortening started). Initially, the red fibres produced only about one third as much power as the white fibres, 6.57±0.63 W kg−1 wet mass (mean ± s.e.m.) and 18.3±2.3, respectively. Red fibres were better at sustaining power output; it declined rapidly to about 60% of its initial value and then remained relatively steady for up to 450 cycles of movement. Force during shortening declined, but force during stretch did not increase: force always relaxed to a low value before stretch started. By contrast, net power output by white fibres declined rapidly to zero within about 50 cycles. Two changes contributed: decline in force during shortening and an increase in force during stretch because relaxation became progressively less complete during the series of contractions. In isometric series (0.44 s stimulation every 1.33 s, cycle frequency 0.75 Hz), red and white fibres sustained peak isometric force similarly; in the 50th cycle force was 59±3% and 56±4% of initial values. The time required for force to relax to 10% of its maximum value decreased during the series for red fibres and increased for white fibres.

Shortening during Stimulation vs. during Relaxation

White muscle fibres from dogfish were used to investigate the energetic cost of shortening by fully active muscle and by relaxing muscle. The muscle preparation was tetanized for 0.6 s and shortened by 1 mm (about 15% L0) at 7 mm/s (about 30% Vo) either during stimulation or during relaxation. Isometric tetani at L0 were also investigated. Mechanical work was calculated from force and length change. Work + heat was taken as a measure of energetic cost. Both work and energetic cost were higher for shortening during stimulation than during relaxation. We also evaluated separately the work and heat associated with the contractile component and with the series elastic component. Work stored in the series elasticity could be completely recovered as external work when the shortening occurred during relaxation.