Stefan Galler

Stretch activation, unloaded shortening velocity, and myosin heavy chain isoforms of rat skeletal muscle fibres.

1. Contractile properties were investigated on single skinned‐fibre preparations from rat leg muscles. Following the mechanical measurements, the myosin heavy chain (HC) composition of the same fibre was analysed by gradient gel electrophoresis. 2. Fibres were typed according to their myosin HC isoform composition (HCI, type I; HCIIA, type IIA; HCIID, type IID; HCIIB, type IIB). Many fibres showed the co‐existence of two myosin HC isoforms (hybrid fibres). 3. A strong correlation was found between fibre type and time characteristics of stretch‐induced delayed force increase (stretch activation) of fully Ca(2+)‐activated fibres. 4. The maximal unloaded shortening velocity (Vmax), as measured with the slack test, was lowest in type I fibres. Within the type II group, a continuum of Vmax values was found, with large overlaps of the different fibre types. 5. The results suggest that the kinetics of stretch activation is determined by the myosin HCs whereas unloaded fibre shortening seems to be determined by other myofibrillar proteins in addition to the myosin HCs. Assuming that stretch activation represents certain steps of the cross‐bridge turnover under isometric conditions and Vmax reflects cross‐bridge detachment under unloaded conditions it can be deduced that different myofibrillar proteins are responsible for different steps within the cross‐bridge turnover.

TWO FUNCTIONALLY DISTINCT MYOSIN HEAVY CHAIN ISOFORMS IN SLOW SKELETAL MUSCLE FIBRES

The head part of the myosin heavy chain (MHC) represents the essential component of the molecular force‐generating system of muscle [1–3] . To date, three fast but only one slow MHC isoforms have been identified in adult mammalian limb muscles [4, 5] . We show here two functionally different slow MHC isoforms, MHCIβ and MHCIa, coexisting in a considerable fraction of slow fibres of rabbit plantaris muscle. The two isoforms exhibit distinct electrophoretic mobilities and different kinetic properties. Thus, as it is known for the fast muscle, also the slow muscle seems to use different MHC isoforms in order to fulfil different functional demands.

Effects of nitric oxide on force-generating proteins of skeletal muscle.

Abstract Nitric oxide (NO) has recently been identified as a physiologically important intracellular messenger modulating the contractile activity of skeletal muscle [Kobzik L, Reid MB, Bredt DS, Stamler JS (1994) Nature 372: 546–548]. However, the mechanism of action of NO is not yet known. We used skinned (demembranated) muscle fibres to investigate the mechanism of NO function in muscle contraction. Maximally Ca2+-activated single fibres of rat skeletal muscle were exposed to physiologically relevant NO concentrations by adding NO donor molecules into the bath solution. Donor application caused a decline both in the contractile properties and in the myofibrillar adenosine triphosphatase (ATPase) activity. These results reveal a novel molecular mechanism of NO action: a direct inhibition of the force-generating proteins in skeletal muscle.

Force responses following stepwise length changes of rat skeletal muscle fibre types.

1. Force responses following stepwise length changes of Ca(2+)‐activated skinned leg muscle fibres (6 degrees C) of the rat were correlated with their myosin heavy chain (HC) isoforms (myosin HC I, fibre type I; myosin HC IIA, type IIA; myosin HC IID (HC IIX), type IID (type IIX); myosin HC IIB, type IIB) in order to study the mechanical properties of these molecules. 2. Marked differences in the time behaviour of force transients following quick releases of fibre length existed between various muscle fibres, and a conspicuous correlation with their myosin HC complement was noticed (order of velocity: IIB > IID > IIA > > I). No differences were found in the relationship between the applied length step and the resulting force (T1, T2 curves). 3. Our results suggest that the heads of various myosin heavy chain isoforms exhibit different kinetic properties. The differences concern the kinetics of the myosin head movements and the duration of cyclic interactions between myosin heads and thin filaments. The extent of force‐generating movements and the mean elongation of attached heads in the isometric state seem to be independent of the isoform.

Unloaded shortening of skinned mammalian skeletal muscle fibres: effects of the experimental approach and passive force

SummaryIn rabbit, rat and human skinned skeletal muscle fibres the length-time relationship of isotonic releases was determined after maximal Ca2+ activation. Slack test experiments provided information about unloaded conditions. Force clamp experiments of different load were extrapolated for zero load and compared with the slack test data. The course length-time relationship for unloaded conditions was similar using both approaches. However, slack test data showed a triphasic shape which could be fitted by three straight lines (phase I, II, III), whereas the data of force clamp experiments exhibited a steady curved shape. Consequently, the instantaneous slopes differed in the two relationships, but the distance which was shortened during the time interval of phase II was similar in both approaches. The ratio between these unloaded shortening velocities resulting from force clamp and slack test experiments was 1.01 ± 0.05 (sd) (n=25). The effects of passive force on the velocity of fibre shortening was investigated in skinned rabbit muscle fibres using slack test experiments. A significant increase in the unloaded shortening velocity was observed when the sarcomere length of the fibres was increased to values which exhibited considerable amounts of passive force. The high reproducibility of the isotonic releases required in this study was achieved by improving some methodological details. Using these improved techniques an identity between the relative fibre and sarcomere shortening was observed during the isotonic releases.

STRETCH ACTIVATION AND MYOSIN HEAVY CHAIN ISOFORMS OF RAT, RABBIT AND HUMAN SKELETAL MUSCLE FIBRES

The underlying mechanism of stretch-induced delayed force increase (stretch activation) of activated muscles is unknown. To assess the molecular correlate of this phenomenon, we measured stretch activation of single, Ca2+-activated skinned muscle fibres from rat, rabbit and the human and analysed their myosin heavy chain complement by SDS gradient gel electrophoresis. Stretch activation kinetics was found to be closely correlated with the myosin heavy chain isoform complement (I, IIa, IId/x and IIb). In hybrid fibres containing two myosin heavy chain isoforms (especially IId and IIb), the kinetics of stretch activation depended on the percentage distribution of the two isoforms. Muscle fibres of the same type but originating from different mammalian species exhibited similar kinetics of stretch activation. Considering the differing unloaded shortening velocities of these fibres, the time-limiting factors for stretch activation and unloaded shortening velocity appear not to be the same. The stretch activation kinetics of the fibre types IIB, IID and IIA more likely seemed to follow a Normal Gaussian distribution than that of type I fibres. Several type I fibres had extraordinarily slow kinetics. This observation corroborates biochemical data indicating the possible existence of more than one slow myosin heavy chain isoform

Kinetic properties of myosin heavy chain isoforms in single fibers from human skeletal muscle.

The head portion of the myosin heavy chain is essential in force generation. As previously shown, Ca2+‐activated muscle fibers from rat and rabbit display a strong correlation between their myosin heavy chain isoform composition and the kinetics of stretch activation, corresponding to an order of velocity: myosin heavy chain Ib>myosin heavy chain IId(x)>myosin heavy chain IIa≫myosin heavy chain I. Here, we show a similar correlation for human muscle fibers (myosin heavy chain IIb>myosin heavy chain IIa≫myosin heavy chain I), suggesting isoform‐specific differences between the kinetics of force‐generating power strokes. The kinetics of myosin heavy chain I are similar in human and rodents. This holds also true for myosin heavy chain IIa, but human myosin heavy chain IIb is slower than rodent myosin heavy chain IIb. It is similar to rodent myosin heavy chain IId(x).

Stretch activation and isoforms of myosin heavy chain and troponin-T of rat skeletal muscle fibres

Recent studies on single mammalian skeletal muscle fibres revealed a correlation between the kinetics of stretch-induced delayed force increase (stretch activation) and the isoforms of the myosin heavy chain. This observation suggests a causal relation between stretch activation and myosin heavy chain. However, the assumption is weakened by the fact that isoforms of other myofibrillar proteins tend to be coexpressed with myosin heavy chain isoforms. The relation between the isoforms of the tropomyosin-binding troponin subunit and myosin heavy chain is unknown. For a variety of reasons, tropomyosin-binding troponin subunit is a possible candidate for being involved in stretch activation. Therefore, we measured stretch activation of single, maximally Ca2+-activated skinned rat skeletal muscle fibres and characterized them by their myosin heavy chain composition, as well as by the isoform species of tropomyosin-binding troponin subunit. Four myosin heavy chain isoforms (I, IIa, IId or IIx and IIb) and six tropomyosin-binding troponin subunit isoforms (TnT1s, TnT2s, TnT1f, TnT2f, TnT3f, TnT4f) were distinguis hed. The following preferential coexpression patterns of the myosin heavy chain and tropomyosin-binding troponin subunit isoforms were observed: MHCI-TnT1s, MHCIIa-TnT3f, MHCIId-TnT1f, and MHCIIb-TnT4f. Stretch activation kinetics was found to be correlated with the myosin heavy chain isoform complement also in fibres not displaying one of the preferential MHC-TnTf isoform coexpression patterns. This corroborates the assumption of a causal relation between myosin heavy chain and stretch activation

The ionic mechanism of intracellular pH regulation in crayfish muscle fibres.

The ionic mechanism of intracellular pH (pHi) regulation was investigated in isolated muscle fibres of the carpopodite adductor in the crayfish Astacus fluviatilis by electrophysiological means with pH, Na+ and Cl‐ ‐sensitive liquid ion exchanger micro‐electrodes. In eighty‐six cells a mean pHi of 7.14 +/‐ 0.12 (S.D.) at a membrane potential of‐‐79.7 +/‐ 3.4 mV was found under control conditions which is about one pH unit more alkaline than predicted from passive distribution and indicates the presence of an acid‐extrusion mechanism. In order to study pHi recovery the cells were acid loaded by exposure either to NH4 Cl or CO2. The effects of HCO3‐ and DIDS (an inhibitor of the anion exchange) on pHi recovery as well as the HCO3‐ ‐dependent decrease of intracellular Cl‐ during pHi recovery indicate that in pHi regulation a mechanism of acid extrusion is involved which exchanges extracellular HCO3‐ for intracellular Cl‐. In CO2/HCO3‐ ‐free solution or in salines with DIDS, pHi recovery was retarded to the same degree, but the effects were not additive. Because of this the remaining pHi recovery must originate from an HCO3‐ ‐independent acid‐extrusion mechanism. In Na+ ‐free solution any pHi recovery was blocked; if pHi recovery occurred it was accompanied by an increase of intracellular Na+ activity (aiNa). From these results it was concluded that all acid extrusion mechanisms which contributed to pHi recovery are coupled to an influx of Na+. A Na+/H+/HCO3‐/Cl‐, and a separate Na+/H+, exchange are proposed as a model of pHi regulation in the crayfish muscle fibre. Similar kinds of acid extrusion mechanisms are found in the neurone of the crayfish (Moody, 1981), with the difference that in the muscle fibre pHi regulation is achieved mainly by the former process. The rate of pHi recovery is considerably lower in the muscle fibre than in the neurone or in the sensory cell (Moser, 1985) of crayfish.

Molecular basis of the catch state in molluscan smooth muscles: a catchy challenge.

The catch state (or ‘catch’) of molluscan smooth muscles is a passive holding state that occurs after cessation of stimulation. During catch, force and, in particular, resistance to stretch are maintained for long time periods with low (or no) energy consumption at basal intracellular free [Ca2+]. The catch state is initiated by Ca2+-stimulated dephosphorylation of the titin-like protein twitchin and is inhibited by cAMP-dependent phosphorylation of twitchin. In addition, catch is pH sensitive, but the reason for this is unknown. According to a traditional model, catch is due to slower cross-bridge cycles where myosin heads remain longer attached to the actin filaments after force generation, possibly caused by a hindered release of ADP from the myosin heads. However, this model was disproved by recent findings which showed that (i) inhibitors of myosin function, such as vanadate, do not affect catch force; (ii) factors which terminate the catch state do not accelerate myosin head detachment kinetics and (iii) a catch-like high resistance to stretch is still inducible when force development is prevented. Thus, catch probably involves passive linkage structures interconnecting the myofilaments (catch linkages). For example twitchin could (i) tie myosin heads to the thin filaments, (ii) mechanically lock them in a stretch resistant state or (iii) interconnect thick and thin filaments directly. However, it is questionable if these mechanisms are sufficient since twitchin seems to be about 15-times less abundant than myosin. Therefore, in addition, interconnections between thick filaments could exist, which could involve e.g. paramyosin or twitchin. Catch could even involve changes in the compliance of thick filaments. The function of myorod, found specifically in catch muscles in equal abundance with myosin, is not known. The suggestion is made here that catch linkages are present already during active contraction either as ratchet-like elements resisting stretch and not opposing shortening or in some kind of ‘standby’ mode ready to transform suddenly into the working mode by stretches or after Ca2+ removal following cessation of stimulation.