Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens

Autor: Denise B. Cuizon, Gordon J. Lutz, Richard L. Lieber, Allen F. Ryan
Jazyk: angličtina
Rok vydání: 1998
Předmět:
Popis: Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS-PAGE and only one adult hindlimb MHC isoform has been cloned. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Ranapipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS-PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co-migrated with type 1 MHC. The combined data from immunohistochemistry and SDS-PAGE suggests that Rana fibre types are composed of four different MHCs. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno-identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation. Studies of frog skeletal muscle have provided the mechanical and energetic information that forms much of the basis for current theories of muscle contraction (Huxley, 1974). The ability to perform functional studies on living isolated single frog muscle fibres distinguishes the frog from other species and has allowed for high resolution mechanical and physiological studies. One reason to study muscle at the single fibre level is that it is a stable and relatively homogeneous mechanical system, more easily defined and controlled at the level of the sarcomere than whole muscle. Since fibres of a given muscle are heterogeneous with regard to speed of contraction and metabolism, single fibre studies permit more precise correlations to be made between structural, functional and biochemical properties. Studies of mammalian and avian single skinned fibres have established a detailed correlation between myofibrillar protein isoform composition and a fibre's mechanical properties and calcium sensitivity (Moss, Giulian & Greaser, 1985; Sweeney, Kushmerick, Mabuchi, Gergely & Sreter, 1986; Bottinelli, Betto, Schiaffino & Reggiani, 1994). Studies of skinned single fibres have also provided critical advances in our understanding of the molecular mechanisms of force generation and cross-bridge state transitions (Hibberd, Dantzig, Trentham & Goldman, 1985; Irving et al. 1995). However, there are serious limitations to the study of mammalian and avian single fibres. With the exception of mouse flexor brevis muscle (Westerblad & Allen, 1993), single fibre studies of mammalian and avian muscle are restricted to skinned preparations, in which the fibre membrane is removed or chemically disrupted. Skinning alters the fibre's mechanical properties, uncouples membrane-associated signalling, alters the structural integrity of some cytoskeletal elements, and permits the loss of soluble intracellular components. In the frog, the use of single living fibres permits study of muscle function in an intact cellular environment. Many contractile characteristics of muscle are defined by the myosin isoforms expressed within the fibres. The myosin molecule exists in skeletal muscle as a hexamer of two myosin heavy chains (MHCs) and four myosin light chains (MLCs), all of which combine to provide a muscle fibre with specific force- and velocity-dependent properties, mechanical power production capabilities, and energetic cost of force production (Curtin & Davies, 1973; Sweeney et al. 1986; Bottinelli et al. 1994). Thus, MHC composition is an important factor in the overall design of a muscle for producing a specific mechanical output. For example, recent studies of locomotion in frogs suggest that MHC composition may be matched to fibre mechanical gearing and joint moments such that, during locomotion, fibres operate at a velocity where near optimal power is generated (Lutz & Rome, 1994, 1996a, b). The close association between MHC isoform expression and muscle function is also demonstrated by studies of muscle plasticity. For instance, MHC expression changes during development (Miller & Stockdale, 1986) and exhibits plasticity even in fully differentiated adult muscle in response to conditions such as the state of innervation (Engel, Brooke & Nelson, 1966), injury by eccentric contraction (Lieber, Schmitz, Mishra & Friden, 1994), immobilization (Booth & Kelso, 1973), and various disease states (Karpati & Engel, 1968). It is believed that this adaptation represents the muscle's attempt to match its structural composition to the new functional demands (Pette, 1990). Despite the advantages of the frog single fibre preparation for studies of muscle function, relatively little is known about the MHC isoform composition of amphibian skeletal muscle. Since the pioneering work of Smith & Ovalle (1973) as many as three different twitch fibre types (types 1, 2 and 3) and two slow or tonic fibre types (types 4 and 5) have been recognized in amphibian skeletal muscle. This classification scheme was based on a combination of metabolic, morphological, ultrastructural and physiological properties. The different muscle fibre types possess a wide range of mechanical properties. For instance, in single Xenopus laevis hindlimb muscle fibres, maximum shortening velocity is 10-fold higher in the fastest twitch fibres than in the slowest tonic fibres (Lannergren, 1992). The different mechanical properties have been attributed to different myosin isoforms, based on a combination of isomyosin banding patterns on native-PAGE gels and MLC content on SDS-PAGE gels (Lannergren & Hoh, 1984; Lannergren, 1987). However, MHC isoform content cannot be unambiguously determined with this methodology. A detailed and systematic study of fibre types in ranid frog and Xenopus limb muscle using myosin isoform-based criteria has been performed (Rowlerson & Spurway, 1988). In Rana, the genus most commonly used for functional studies, three twitch fibre types (types 1, 2 and 3) and a tonic type were defined based on myosin-ATPase reactivity and differential reactivity to a panel of MHC monoclonal antibodies. In Xenopus, three twitch fibre types, a tonic type and an additional slow fibre type (Lannergren, 1979) were distinguished using the same criteria (Rowlerson & Spurway, 1988). These data provided strong evidence that the different fibre types in Rana and Xenopus limb muscle contained different MHC isoforms. Despite the indirect evidence that up to five MHC isoforms are present in frog muscle, only two different MHC isoforms have been detected by SDS-PAGE (Lannergren, 1987). In addition, only one adult frog hindlimb MHC has been cloned and its identity was not correlated with a particular fibre type (Radice & Malacinski, 1989). Therefore, the purpose of the present study was to characterize the MHC composition of the various fibre types in ranid hindlimb muscle at the mRNA and protein level. As the physiology of amphibian fibre types has been previously characterized, a link is established between differential MHC gene expression and the mechanical function of Rana fibre types. A brief version of this work was presented in abstract form (Lutz, Ryan & Lieber 1997).
Databáze: OpenAIRE
Externí odkaz: