Karlheinz Hilber

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

IMPROVEMENT OF THE MEASUREMENTS ON SKINNED MUSCLE FIBRES BY FIXATION OF THE FIBRE ENDS WITH GLUTARALDEHYDE

Experiments with activated skinned muscle fibre segments are limited by the structural and mechanical instability of the preparations. The present study shows that fixation of the muscle fibre ends with glutaraldehyde significantly improves the reliability of such experiments. We tested the effects of a specific glutaraldehyde fixation technique on the structural stability and the mechanical properties of skinned rat and rabbit skeletal muscle fibres in an approach where the fibre segments are attached to the apparatus by gluing. Preparations with fixed and unfixed ends were compared. During the first few minutes of maximal activation, fibres with fixed and unfixed ends exhibited similar mechanical properties to one another, suggesting that our fixation procedure selectively impregnates the fibre ends without contaminating the remaining active fibre part. During prolonged maximal activations (3–60min), preparations with fixed ends exhibited a better stability, both in the sarcomere length signal (detected by laser diffraction) and in the unloaded shortening velocity. Thus, our technique of muscle fibre end fixation caused a substantial improvement in the mechanical measurements on skinned muscle preparations.

Mechanical properties and myosin heavy chain isoform composition of skinned skeletal muscle fibres from a human biopsy sample

Abstract Experiments were conducted to investigate the mechanics of contraction of chemically skinned muscle fibre segments of a biopsied sample of single human quadriceps muscle. Subsequently, the isoforms of the myosin heavy chain (MHC) were analysed by sodium dodecyl sulphate (SDS) gel electrophoresis.Of the 41 fibres, 26 contained MHCI (type I), 11 of the fibres contained MHCIIa (type IIA), and 4 of the fibres contained both MHCI and MHCIIa (of which MHCIIa was always slightly predominant (type IIC)). Distinct differences between fibre types were found in terms of the kinetics of force responses following stepwise length changes (order of velocity: IIA > IIC > I). The differences in maximal shortening velocity and in the kinetics of Ca2+-dependent activation were of the same order, but much less pronounced. Type I fibres had significantly greater fibre diameters than type IIA fibres. No significant differences were found among different fibre types in terms of isometric tension, resting sarcomere length or the length change needed to discharge the elasticity of maximally Ca2+-activated fibres (yo value). The distribution of shortening velocity and kinetics of stretch activation values suggest that two muscle fibre subtypes may exist in human type I fibres.

Shortening properties of two biochemically defined muscle fibre types of the Norway lobster Nephrops norvegicus L.

Mechanical properties of myofibrillar bundles from single chemically skinned fibres from the superficial abdominal flexor muscle of the Norway lobster Nephrops norvegicus were measured, and the protein content of these fibres was analysed by SDS-PAGE. Two slow fibre phenotypes (S1, S2) were distinguished on the basis of their myofibrillar protein assemblages. Data from 9 S1 and 8 S2 fibres obtained at similar sarcomere length demonstrate significant differences between the fibre types in maximal tension (N cm-2, S1: 10.5 ± 3.9; S2: 3.1 ± 0.8), in the delay of the peak of stretch activation (ms, S1: 122 ± 18; S2: 412 ± 202), in fibre stiffness (N cm-2 per nm half sarcomere, S1: 0.36 ± 0.19; S2: 0.09 ± 0.03) and in maximal shortening velocity (fibre length s-1, S1: 0.53 ± 0.10; S2: 0.27 ± 0.06). Furthermore, the maximal power output of the type S1 fibres was about five times larger than that of S2 fibres. The power output was maximal at lower loads in S1 fibres (relative load = 0.37 ± 0.04) than in S2 fibres (relative load = 0.44 ± 0.05). This study represents a comprehensive investigation of two slow muscle fibre types which are thought to be specialized for slow movements (S1 fibres) and for the postural control of the abdomen (S2 fibres).