Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Abstract

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.

Fig. 1.

Identification of TnT, TnI and MHC isoforms in muscle homogenate. (A) Total protein extracts of adult rat EDL, soleus (SOL) and diaphragm (DPH) muscles were resolved by 14% SDS-PAGE with acrylamide:bisacrylamide = 180:1 and stained with Coomasie Brilliant Blue R250. (B) Dilutions of the same samples were analyzed by 8% SDS-PAGE with acrylamide:bisacrylamide = 50:1 and 30% glycerol followed by silver stain to reveal the four MHC isoforms. The samples were also analyzed by Western blots using anti-MHC-I mAbs FA2 (C), anti-slow skeletal muscle TnT mAb CT3 (D), anti-fast skeletal muscle TnT mAb T12 (E), and anti-TnI mAb TnI-1 (F). The results demonstrate an effective identification of the MHC, TnT and TnI isoforms by our experimental procedures. Similar to that seen in mouse soleus (), three alternatively spliced slow TnT bands were found in the rat soleus by the CT3 Western blot. The two high molecular weight and one low molecular weight rat slow TnT shown in this figure correspond to the recently sequenced rat slow TnT isoform 1, 2, and 4 ().

Fig. 2.

Expression of TnT, TnI and MHC isoforms in different parts of diaphragm. (A) The rat diaphragm (head view with the dorsal side on top) was dissected into twelve 30° sectors for the analysis of muscle protein contents. (B) Total protein extracts from the 12 diaphragm muscle samples were analyzed by 14% SDS-PAGE with acrylamide:bisacrylamide = 180:1 and stained with Coomasie Brilliant Blue R250 to examine the total protein contents, by 8% SDS-PAGE with acrylamide:bisacrylamide = 50:1 and 30% glycerol followed by silver stain to reveal the MHC isoforms contents, and by Western blots using anti-slow skeletal muscle TnT mAb CT3, anti-fast skeletal muscle TnT mAb T12, and anti-TnI mAb TnI-1 to examine the TnT and TnI isoforms. The results demonstrate that the 12 areas of diaphragm muscle had no significant difference in protein contents. Most areas of diaphragm muscle had similar patterns of MHC isoform expression except for the dorsal region (Sectors 1 and 12). Different sectors of the rat diaphragm muscle express similar ratios of slow and fast skeletal muscle TnT, and of slow and fast skeletal muscle TnI. These ratios were also similar to that detected in the total diaphragm muscle homogenate (Fig. 1).

Fig. 3.

Representative recording for the Ca²⁺-activated force development in wrapped EDL single fiber before and after gluing. The traces show the Ca²⁺-activated force development of a representative rat EDL single fiber. A series of measurements was first performed after securely mounting the fiber by the wrapping method. The spikes are artifact due to moving the fiber between troughs containing buffers with difference pCa. After recording the force development at four serial increases of [Ca²⁺], the fiber was washed in pCa8.5 buffer and the process was repeated. The results demonstrate that there was no significant change between the two rounds. In addition to show the reliable fiber mounting by the wrapping method, the results demonstrate that there was no run-down in fiber contractility under the experimental conditions. The wrapped ends of the fiber were then glued using a tissue adhesive composed of methoxypropyl cyanoacrylate monomer and polymeric modifiers (TISSUMEND II, Veterinary Products Laboratory, Phoenix, AZ) and another round of Ca²⁺ activation was measured. The results showed no difference for the Ca²⁺-activated forces recorded before and after gluing.

Fig. 4.

Rat single skeletal muscle fibers expressing representative combinations of MHC and TnT and TnI isoforms. The silver stained SDS-glycerol-gel (A), Western blots using a mixture of anti-slow TnT mAb CT3 and anti-TnI mAb TnI-1 (B), and Re-probing of the same nitrocellulose in (B) with anti-fast TnT mAb T12 (C) on extracts from single rat diaphragm (DPH) and EDL muscle fibers demonstrate four representative simple combinations of MHC, TnT and TnI isoforms. Total diaphragm muscle MHC were used as controls. Multiple fast TnT bands are present in every fast TnT single fibers, representing alternatively spliced variants with differences in the N-terminal variable region ().

Fig. 5.

Isometric force versus pCa relationship of representative diaphragm and gastrocnemius muscle fibers. (A) The diaphragm fibers containing MHC-I and slow troponin, MHC-IIa and fast troponin, or MHC-IIx and fast troponins were compared. The normalized force-pCa curves show that cooperativity of activation was higher in the MHC-II + fast troponin groups as compared to that of fibers expressing MHC-I and slow troponin. However, the MHC-I + slow troponin fibers were more sensitive to calcium (Table 2). No significant differences were detected between the MHC-IIa and MHC-IIx groups. (B) Gastrocnemius muscle fibers containing MHC-IIa + fast troponin or MHC-IIx + fast troponin also showed no significant differences in the contractile properties.

Fig. 6.

Isometric force versus pCa relationship of slow and fast fibers from different muscles. (A) Slow fibers (containing MCH-I and slow troponin) from diaphragm or soleus muscles showed very similar Ca₅₀ and cooperativity. (B) Fibers containing MCH-IIx and fast troponin from diaphragm or gastrocnemius muscles have very similar isometric contractile features. (C) Fibers containing MCH-IIb and fast troponin from gastrocnemius or EDL muscles also have very similar isometric contractile properties.