Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8095-8100, July 1996 Physiology The whistle and the rattle: The design of sound producing muscles (rattlesnake/toadfish/calcium transients/muscle mechanics/swimbladder) LAWRENCE C. ROME*tt, DOUGLAS A. SYME*, STEPHEN HOLLINGWORTH§, STAN L. LINDSTEDT$, AND STEPHEN M. BAYLOR§ Departments of *Biology and §Physiology, University of Pennsylvania, Philadelphia, PA 19104; tMarine Biological Laboratories, Woods Hole, MA 02543; and IDepartment of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011 Communicated by Clara Franzini-Armstrong, University of Pennsylvania School ofMedicine, Philadelphia, PA, April 15, 1996 (received for review December 11, 1995) ABSTRACT Vertebrate sound producing muscles often sients-in fact the largest and fastest ever recorded. How- operate at frequencies exceeding 100 Hz, making them the ever, our results showed that a fast Ca2+ transient alone is fastest vertebrate muscles. Like other vertebrate muscle, these not sufficient for high frequency operation. By measuring sonic muscles are "synchronous," necessitating that calcium Vmax, an index of crossbridge detachment rate, and the be released and resequestered by the sarcoplasmic reticulum force-pCa relationship in skinned fibers, a possible index of during each contraction cycle. Thus to operate at such high troponin kinetics, we found that rapid activation and relax- frequencies, vertebrate sonic muscles require extreme adap- ation likely also require a modification of the crossbridge tations. We have found that to generate the "boatwhistle" kinetic rate, and probably a modification of the kinetics of mating call (-200 Hz), the swimbladder muscle fibers of Ca2+-troponin binding. In reaching these conclusions, we toadfish have evolved (i) a large and very fast calcium first compared the above measurements in three fiber types transient, (ii) a fast crossbridge detachment rate, and (iii) from toadfish, ranging from slow twitch swimming fibers to probably a fast kinetic off-rate of Ca2+ from troponin. The the superfast twitch swimbladder fibers. We then compared fi'bers of the shaker muscle ofrattlesnakes have independently the properties of rattlesnake shaker fibers with those of evolved similar traits, permitting tail rattling at '90 Hz. swimbladder. Skeletal muscle fibers perform a wide range of activities, and MATERIALS AND METHODS different fiber types are accordingly designed to operate at different speeds and frequencies (1). A number of modifica- In toadfish, the anatomical segregation of different fiber types tions appear to underlie this diversity. For example, in loco- permitted us to dissect small bundles of fibers (pure in fiber motory muscle, compared with slow twitch fibers, fast twitch type) from the slow twitch red and fast twitch white trunk fibers have a faster myosin with a higher maximum velocity of musculature, and the superfast twitch swimbladder muscles. In shortening (Vm,,) (2, 3), a greater content of sarcoplasmic rattlesnakes, small fiber bundles were dissected from shaker reticulum (SR), and its associated Ca2+ pumps (4, 5), a muscle, which is also composed of a single fiber type (18). Fiber different isoform of the SR Ca2+ pump (SERCAl in fast bundles were mounted in one of three chambers for different versus SERCA2 in slow) (6, 7) and a greater concentration of physiological measurements. parvalbumin (a soluble protein that binds both calcium and Myoplasmic [Ca2+] Transient. The change of myoplasmic magnesium) (5, 8). There is also evidence that fast fibers have free [Ca2+] was monitored during contractions by using furap- a briefer myoplasmic free Ca2+ concentration ([Ca2+]) tran- tra, a fluorescent calcium indicator with rapid reaction kinetics sient (9, 10) and less sensitive force-pCa relationship (11, 12). (22). The delay between a change in [Ca21] and a change in To understand the physiological modifications that underlie furaptra's fluorescence is not likely to be more than 0.2 ms very rapid contractions, we have studied two of the fastest (23). Furaptra was pressure injected into a single fiber within vertebrate muscles known. Both of these "sonic" muscles are a small bundle, and experiments and calibration were per- used to produce sounds at the frequency at which the muscle formed as in ref. 22. The fluorescence transients from swim- contracts. The "boatwhistle" mating call of the male toadfish bladder fibers were corrected for a small, Mg2+-dependent (Opsanus tau) is generated by '200 Hz contractions (25°C) of delayed decrease in fluorescence due to the exchange of Ca2+ the muscles encircling the fish's gas-filled swimbladder (13- for Mg2+ on parvalbumin (22). Additionally, in the case of swimbladder fibers, a correction to fluores- The familiar "rattle" of the venomous western diamond- furaptra's resting 15). cence was to account for the back rattlesnake is "90 Hz made fraction (0.28) of indicator (Crotalus atrox) generated by molecules located in the central core of the fiber (5), where the contractions of the shaker muscles at the base of the tail (35°C) change in [Ca2+] during a brief period of stimulation is likely (16-19). The operational frequencies of these sonic muscles to be negligible. To reduce tension and thus motion artifacts, are 1-2 orders of than magnitude higher those of the loco- fibers were stretched to sarcomere lengths (SL) of 2.7-3.0 ,um motory muscles in the same animals (0.5-5 Hz) (20). (swimbladder) and 3.3-4.0 ,um (other fiber types). Action For fibers to operate at such high frequencies, they must potentials were initiated with point stimulation, and fibers activate and relax rapidly. Based on morphological and bio- were studied only if they responded in all-or-none fashion. chemical evidence from swimbladder of a very large SR Ca2+ Muscle Mechanics. Muscle force was measured in all ex- pump density, a high density of SR Ca2+ release sites, and a perimental setups by means of a force transducer attached to large parvalbumin concentration, it has been proposed that an one end of a fiber bundle. In the muscle mechanics setup, the important modification of these sonic fibers is unusually rapid other end of the bundle was attached to a servomotor that Ca2+ cycling (5, 21). By measuring myoplasmic free [Ca2+], we imposed length changes on the muscle bundle. Bundles were found that these fibers do indeed have unusual Ca2+ tran- Abbreviations: SR, sarcoplasmic reticulum; [Ca2+], Ca2+ concentra- The publication costs of this article were defrayed in part by page charge tion; SL, sarcomere length; ML, muscle length. payment. This article must therefore be hereby marked "advertisement" in iTo whom reprint requests should be sent at the * address. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 8095 Downloaded by guest on September 26, 2021 8096 Physiology: Rome et aL Proc. Natl. Acad. Sci. USA 93 (1996) excited by field stimulation. The force-velocity curves of the ,um. The preparation was adjusted for the size of the fibers: for red and white toadfish fibers were determined by using force toadfish red muscle, single fibers were used; for toadfish white clamp measurements (24). The force-velocity curves of the and frog muscles, single fibers were split longitudinally to - 1/4 rattlesnake shaker fibers were determined by using length of their diameter; for swimbladder muscle, bundles of two or ramps (24). In all cases, Vmax was obtained by extrapolating the three fibers were used. Skinned fibers from rattlesnakes curve to zero force. For the swimbladder, Vmax was most proved unstable, and only the threshold for force generation accurately determined by using the slack test (24). Previous could be determined. Preparations were skinned in 3% Triton comparisons of these different techniques on other fiber types X-100 for 20 min and then exposed to 10 ,uM thapsigargin (an show that they all give the same Vmax (24); thus the results can SR calcium pump blocker) throughout the experiments. Both be compared directly. treatments abolish SR Ca2+ pumping, thereby preventing the The workloop technique (25, 26) was also used to determine SR from interfering with the measurements. if the muscle could generate mechanical work at high oscilla- Modeling. Ca2+ binding to troponin at 16°C was modeled tion frequencies. In this method, the muscle length was from the Ca2+ transient (as in ref. 28) with several different changed sinusoidally and the timing of the single stimulus values assumed for k0ff (the Ca2+ off-rate constant from during the length cycle was adjusted to maximize power troponin). Fractional troponin activation (equivalent to [tro- output. ponin occupancy]2) was estimated under the assumptions that Force-pCa Measurements. The force generated by (i) there are two equal and independent binding sites for Ca2+, "skinned" (permeablized) fibers was measured as a function of having a k10 for Ca2+ binding of 0.9 x 108 M-1s-1, and (ii) free [Ca2+], which was set with a Ca2+/EGTA buffer system both sites must be occupied to generate force. The relative (27). The solutions had a pH of 7.1 (15°C) and ionic strength rates of SR Ca2+ pumping in different fiber types of toadfish of 200 mM; the experiments were conducted at a SL of 2.2-2.3 were estimated from the Ca2+ transient as in ref. 28. A. Toadfish Fibers, 160C B. Sonic fibers (Toadfish and Rattlesnake) ,-rs16 s25 -,> ,rs35s 0 0 rs35 'U IlI 0.0 0.2 0.4 0.6 0.00 0.01 0.02 Time (s) FIG. 1. Twitch tension (Upper) and calcium transients (Lower) of three fiber types from toadfish at 16°C (A) and of sonic fibers at 16-35°C (B). In each case, the force and the calcium transient have been normalized tb their maximum value. (A) The twitch and calcium transientbecome briefer going from the slow-twitch red fiber (r), to the fast-twitch white fiber (w), and to the superfast-twitch swimbladder fiber (s).