Limited expression of slow tonic myosin heavy chain in human cranial muscles

Abstract

Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular, and developmental muscle with antibodies (Abs) ALD-58 and S46, considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal, and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots, Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform.

Figure 1

Serial sections from the adult human biceps brachii, monkey atrium, adult human medial rectus and human fetal tongue illustrating immunoreaction with Ab S46 at 1:5 dilution (A-D), Ab S46 at 1:25 dilution (E-H), Ab ALD-58 at 1:5 dilution (I-L), Ab ALD-58 at 1:25 dilution (M-P) and Abs A4.84 or BA-D5 (Q-T). At both dilutions, Ab S46 stongly labels intrafusal (muscle spindle) fibers in biceps brachii (A,E) and a subset of muscle fibers in the medial rectus (C,G) and fetal tongue (D,H, arrows), but does not label extrafusal fibers in biceps brachii or muscle fibers in monkey atrium. Inset in B shows tissue from biceps brachii reacted on same slide and photographed under the same optical conditions as monkey atrium in B; note the strong label of intrafusal but not extrafusal or atrial fibers. At strong dilution Ab ALD-58 strongly labels intrafusal fibers in a muscle spindle in biceps brachii (I), a subset of fibers in the medial rectus (K) and fibers in the monkey atrium (J). At strong dilution Ab ALD-58 moderately labels ‘slow’ fibers in biceps brachi (I, also inset in J). Inset in J shows tissue from biceps brachii reacted on the same slide and photographed under the same optical conditions as monkey atrium in J; note the strong label of both intrafusal and atrial fibers and the moderate label of extrafusal slow (“I”) fibers. At weak dilution, Ab ALD-58 does not label slow fibers, and either does not label, moderately labels or strongly labels slow tonic fibers in muscle spindle (M) and medial rectus (O). “I” denotes fibers positive for MHCI (labeled with Ab A4.84 or Ab BA-D5). “II” denotes fibers negative for MHCI and positive for MHCII (labeled with Ab MY-32, data not shown). Arrows in C, G, K. O, and S indicate same region in sections. Arrows in D and H indicate slow tonic fibers in human fetal tongue. For E, F, G, M, N and O, reaction solution also included the anti-laminin Ab D-18 to enable visualization of muscle fiber perimeters. Calibration bar = 100μm.

Figure 2

Serial sections of the adult human mylohyoid (A,B), adult human anterior digastric (C,D), adult human thyroarytenoid vocalis (E,F) and adult human hyoglossus (G,H) reacted with 1:5 dilutions of Ab S46 (A,C,E,G) and Ab ALD-58 (B,D,F,H). Inset in each figure shows medial rectus tissue reacted on the same slide and photographed under the same optical conditions (as a positive control for MHCst). Ab S46 strongly labels a subset of fibers in the medial rectus but labels few or no fibers in the other muscles (isolated labeled fibers marked with arrows in Figure E). Ab ALD-58 strongly labels a subset of fibers in the medial rectus and moderately to strongly labels slow muscle fibers in all other muscles. Symbols “#”, “+” and “*” identify the same fibers in sections. Calibration bar = 100μm.

Figure 3

Immunoblots of human thyroarytenoid vocalis (TAV), biceps brachi (BB), anterior digastric (AD), superior rectus (SR), medial rectus (MR) and chick anterior latissimus dorsi muscle (ALD) reacted with Ab S46 (top panel) and with Ab ALD-58 (bottom panel). Equivalent amounts of whole muscle protein were loaded for each human muscle tested. Note the strong reaction of Ab S46 to ALD and to the extraocular muscles SR and MR, the absence of Ab S46 reaction to TAV, BB and AD and the weak reaction of Ab ALD-58 to ALD only.