Evolution of skeletal type e-c coupling: a novel means of controlling calcium delivery

Abstract

The functional separation between skeletal and cardiac muscles, which occurs at the threshold between vertebrates and invertebrates, involves the evolution of separate contractile and control proteins for the two types of striated muscles, as well as separate mechanisms of contractile activation. The functional link between electrical excitation of the surface membrane and activation of the contractile material (known as excitation-contraction [e-c] coupling) requires the interaction between a voltage sensor in the surface membrane, the dihydropyridine receptor (DHPR), and a calcium release channel in the sarcoplasmic reticulum, the ryanodine receptor (RyR). Skeletal and cardiac muscles have different isoforms of the two proteins and present two structurally and functionally distinct modes of interaction. We use structural clues to trace the evolution of the dichotomy from a single, generic type of e-c coupling to a diversified system involving a novel mechanism for skeletal muscle activation. Our results show that a significant structural transition marks the protochordate to the Craniate evolutionary step, with the appearance of skeletal muscle-specific RyR and DHPR isoforms.

Figure 1.

Phylogenetic tree showing details of the transition between low chordates and Craniata. At the right of the phylogenetic tree, the dispositions of the DHPR particles found in this study are indicated for the animal models used.

Figure 2.

Peripheral couplings in Amphioxus muscle. (A and B) Cross-sections of myotomes in Amphioxus show series of lamellae, each containing only a single myofibril. (A) At the fiber periphery several jSR sacs associate with the plasmalemma, forming peripheral couplings (arrows) opposite the Z line, I band, and the edges of the A band. (B) Feet (RyRs; arrows) are present in the junctional gap. (C and D) Freeze-fracture replicas of the cytoplasmic leaflet from the plasmalemma in Amphioxus lamellae. Dome-shaped plasmalemma domains are arranged in three circumferential rows (arrows). Additional domes are occasionally present (asterisks). (A) The rows are centered on the Z lines (Z) but cover a broad band of the surface. The shape and positioning of these domains are consistent with those of peripheral couplings (compare with A). (D, inset) The plasmalemmal domes are decorated by clusters of particles consistently larger than those decorating the rest of cytoplasmic leaflet. Bar, 100 nm.

Figure 3.

Longitudinal section of myotome muscle in hagfish. At the A-I band junctions, jSR associates with T tubules to form triads (A) with numerous feet (C, arrows). In this muscle, peripheral couplings between SR and plasmalemma are small, have few feet (B, arrows), and are not frequent. The cytoplasmic leaflet of plasmalemma in hagfish myotomes shows a flat membrane rich in particles and in caveolae; its neck openings appear as small circles (D). Small patches of membrane contain a population of large particles (D, arrows) that are clustered into tetrads (inset).

Figure 4.

Triads and peripheral couplings in a longitudinal section of myotome muscle in lamprey. (A) Triads located at the Z line (Z). In all cases feet occupy the junctional gap (arrowheads). (C and D) In freeze-fracture, the cytoplasmic leaflet of the plasmalemma shows groups of large particles located at the Z lines (Z). The groups are frequent, large, and often elongated in the circumferential direction. (B) This corresponds to the location of large peripheral couplings seen in thin sections.

Figure 5.

The unusual jSR in myotome muscles of garfish. (A) Longitudinal section shows a peculiar fingerlike shape of the jSR (asterisks) facing the T tubules (T) at the Z line (Z). (B) When cut in cross-section the fingers appear as small vesicles facing the T tubules. (C) Similarly elongated jSR fingers are associated with the plasmalemma, appearing as closely spaced jSR vesicles in cross-section. Regularly spaced feet are present in the junctional gap (arrowheads). (D and E) In freeze fracture of the surface membrane, groups of large particles are located in numerous parallel domains at the Z lines (arrows). The domains are elongated in the longitudinal direction and correspond in position and shape to the areas of junctions between the fingerlike jSR and the plasmalemma (compare with A). (E) The membrane domains occupied by large particles are slightly raised, whereas the membrane in between is slightly depressed.

Figure 6.

Particle clusters illustrate DHPR disposition. Details of large particle clusters in Amphioxus (A–D), hagfish (E–H), lamprey (I–L), and garfish (M–P). In Amphioxus, the particles are clustered, but show no special arrangement within the clusters. In hagfish, lamprey, and garfish, the particles tend to be grouped into small orthogonal arrangements of four (tetrads, within squares). The tetrads are not always complete, but they clearly form part of a larger array. Two arrangements are visible. In hagfish (E–H) and lamprey (I–L), the tetrads abut side by side; in garfish (M–P), on the other hand, the tetrads are in proximity to their corners. The latter arrangement is typical of most vertebrates.

Figure 7.

The arrangements of DHPR tetrads described in the text are superimposed on a possible arrangement of RyRs that may account for the tetrad disposition. Each pink circle indicates one DHPR molecule, and RyRs are green or gray tetrameric molecules. Center–center distances (arrows) between tetrad particles are drawn in the appropriate scale relative to RyR size, but the size of the particle is arbitrary. The RyR array in B has been directly observed; the others are modeled on the basis of the tetrad arrays.