Origin of intrafusal fibers from a subset of primary myotubes in the rat

S46, a monoclonal antibody (mAb) specific for the SM-1 and SM-2 isoforms of avian slow myosin heavy chains (MHC), was used to study the earliest stages of development of intrafusal fibers in muscle spindles of the rat hindlimb. Spindles formed only in the regions of fetal muscles that contained primary myotubes reactive to mAb S46, such as the axial region of the tibialis anterior muscle. The first intrafusal fiber to form, the nuclear bag₂ fiber, originated from within the population of S46-reactive primary myotubes. Binding of mAb S46 by myotubes giving rise to the bag₂ fibers preceded the appearance of encapsulated spindles in the muscles by electron microscopy. However, reactivity to S46 intensified in the myotubes transforming into bag₂ fibers after the innervation of the fibers by afferents, and dissipated in myotubes differentiating into slow-twitch (type I) extrafusal fibers. Thus, afferents may enhance intrafusal expression of the MHC isoform reactive to mAb S46. The pattern of S46 binding to nuclear bag and chain intrafusal fibers in both developing and adult spindles was the same as that reported for the mAb ALD19, suggesting that both antibodies bind to the same MHC isoform. This isoform is probably a developmental form of slow myosin, because it was transiently expressed during the development of type I extrafusal fibers. The origin of bag₂ intrafusal and type I extrafusal fibers from a bipotential subpopulation of primary myotubes reactive to mAb S46 correlates with the location of muscle spindles in the slow regions of muscles in adult rat hindlimbs.     

Sequences of intrafusal fiber formation are muscle-dependent in rat hindlimbs

A rat muscle spindle typically contains four intrafusal fibers — one nuclear bag₂, one nuclear bag₁ and two nuclear chain fibers. We compared the sequence of formation of the three intrafusal fiber types among the tibialis anterior (TA), soleus (SOL) and medial gastrocnemius (MG) muscles using immunocytochemistry of spindle-specific myosin heavy chain isoforms. Spindles of the TA began to differentiate earlier and acquired the full complement of intrafusal fibers sooner than spindles of the SOL or MG muscles. At the onset of spindle assembly, the intrafusal myotubes expressed myosin heavy chains similar to those expressed by extrafusal myotubes. The first intrafusal myotube then differentiated into the bag₂ fiber regardless of the muscle. However, the fate of the second-forming intrafusal myotube varied among the muscles studied. It usually differentiated into a chain fiber in the TA, into a bag₁ fiber in the SOL, and into either a bag₁ or a chain in the MG. The fate of the third-forming intrafusal myotube was reciprocal to that of the second; i.e. in those spindles in which the bag₁ fiber was second to form, a chain was third, and vice versa. The fourth and last intrafusal myotube gave rise to a chain fiber. The inter- and intramuscular variability in the fate of intrafusal myotubes of the second and third generation argues against the existence of a program intrinsic to the myotubes that would mandate their differentiation along specific paths. Rather, an extrinsic regulatory factor, probably associated with the primary afferent neuron, may govern differentiation of pluripotential myotubes into particular types of intrafusal fiber. The fate of the intrafusal myotubes might then depend on the timing of the regulatory effect of afferents relative to the stage of development of the intrafusal bundle.     

Effects of 14 days of microgravity on fast hindlimb and diaphragm muscles of the rat

The purpose of the present study was to determine the effects of 14 days of microgravity on specific rat fast-twitch muscles, and to compare these data with previous data from rat fast-twitch muscles exposed to microgravity for 10 days (Kraemer et al. 2000). Hindlimb muscles containing predominately fast fibers [extensor digitorum longus (EDL), superficial “white” (GSW) and deep “red” (GDR) gastrocnemius] and the diaphragm (DIA) were removed from flight and ground-based control animals and analyzed for: muscle mass, fiber type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content. Gravitational unloading for 14 days caused significant decreases in muscle mass (8–9%) and cross-sectional area of almost all fiber types (10–35%) from both EDL and gastrocnemius muscles. However, microgravity had little effect on fiber type composition in these muscles with significant changes occurring only in the EDL type IID fiber population (9.5% decrease). Similarly, relative MHC isoform content was only slightly altered by exposure to microgravity (increased content of MHCIIa in flight EDL). No changes in area, fiber type percentages, or MHC isoform content were detected in the DIA following the 14-day spaceflight. Similar to data gathered following a 10-day spaceflight (Kraemer et al. 2000), the 14-day flight did not appear to cause significant slow-to-fast (I → IIA) or fast-to-faster (IIA → IID → IIB) transformations in hindlimb muscles containing predominantly fast-twitch fibers. However, the longer period of gravitational unloading did result in additional loss in muscle fiber cross-sectional area with involvement of more major fiber types.     

Differential effects of neonatal denervation on intrafusal muscle fibers in the rat

The response of developing muscle spindles to denervation was studied by sectioning the nerve to the medial gastrocnemius muscle of rats at birth. The denervated spindles were examined daily throughout the first postnatal week for changes in ultrastructure and expression of several isoforms of myosin heavy chain (MHC). Each of the three different types of intrafusal muscle fiber exhibited a different response to denervation. Within 5 days after the nerve section nuclear bag₂ fibers degenerated completely; nuclear bag₁ fibers persisted, but ceased to express the spindle-specific slow-tonic MHC isoform and thereby could not be differentiated from extrafusal fibers; nuclear chain fibers did not form. The capsules of spindles disassembled, hence spindles or their remnants could no longer be identified 1 week after denervation. Neonatal deefferentation has little effect on these features of developing spindles, so removal of afferent innervation is presumably the factor that induces the loss of spindles in denervated muscles. Degeneration of the bag₂ fiber, but not bag₁ or extrafusal fibers, reflects a greater dependence of the bag₂ fiber than the bag₁ fiber on afferent innervation for maintenance of its structural integrity. This difference in response of the two types of immature bag fiber to denervation might reflect an origin of the bag₂ fibers from a lineage of myogenic cells distinct from that giving rise to bag₁ or extrafusal fibers, or a difference in the length of contact with afferents between the two types of bag fiber prior to nerve section.     

Quantitative and histochemical aspects of the differentiation of muscle spindles in the anterior latissimus dorsi of the developing chick

Summary The differentiation of muscle spindles has been investigated both quantitatively and histochemically in the slow anterior latissimus dorsi (ALD) muscle during embryonic and post-hatching development of the chick. The first spindles are detected by the 13th day in ovo, and a rapid increase in number takes place until the 15th of embryonic life. Two histochemical fibre types of intrafusal fibres are distinguished as early as the 13th day of embryonic development when myofibrillar ATPase activity is demonstrated after acid preincubation. In ALD muscle from post-hatched animals, two intrafusal fibre types are also distinguished by histochemistry: one fibre type is characterized by an acid and alkali-stable myofibrillar ATPase activity while this activity is acid-labile and alkali-stable in the other type. As far as the properties of myofibrillar ATPase are concerned, such types of intrafusal fibres resemble the extrafusal and fibre types defined recently in ALD muscle.The development of spindles in the slow-tonic ALD muscle is compared to the differentiation of spindles in the fast-twitch posterior latissimus dorsi (PLD) muscle of the chick which has been previously described (Toutant et al. 1981).     

Morphology and morphometry of motor endings in primate intrafusal fibers

Summary The pre- and postsynaptic structure of 243 axon terminals of bag 1, bag 2, and chain fibers were studied in cynomolgus monkey skeletal muscle spindles. The motor endings of the biceps and gastrocnemius spindles (long limb muscle) were compared to the motor endings of lumbricals and opponens pollicis (intrinsic hand muscle) spindles. In both muscle groups the only significant difference observed in the presynaptic features was in the presynaptic membrane length. The postsynaptic features of bag 1, bag 2 and chain endings were similar in the long limb muscle spindles. In the intrinsic hand muscle, however, the bag 1 and chain endings showed complex postsynaptic structure which resembled the extra fusal endings while the postsynaptic structure of bag 2 endings was much simpler. From these studies we conclude that the postsynaptic structure of various intrafusal fiber types is dissimilar in different muscles.     

Axon contacts and acetylcholinesterase activity on chicken intrafusal muscle fiber types identified by their myosin heavy chain composition

Summary Muscle spindles of 8-week old chicken tibialis anterior muscles were examined to determine if specific intrafusal fiber types were also characterized by differences in motor innervation. Incubation with a monoclonal antibody against myosin heavy chains permitted the identification of strongly reactive, moderately reactive and unreactive intrafusal fibers. The innervation of each fiber type was evaluated in silver-impregnated sections, and in sections incubated with a monoclonal antibody against acetylcholinesterase. There was no acetylcholinesterase activity at the midequator of any fiber. At the juxtaequator and at the pole strongly reactive fibers typically exhibited fewer axon contacts and less acetylcholinesterase activity than unreactive and moderately reactive fibers. Differences were also recognized at neuromuscular junctions in the size and shape of acetylcholinesterase-positive sites. At the juxtaequator and at the pole strongly reactive fibers and moderately reactive fibers displayed significantly more small, dot-like acetylcholinesterase sites than unreactive fibers. On the contrary, the greatest number of larger, stout sites was found on unreactive fibers and the least number on strongly reactive fibers. Moderately reactive fibers took an intermediate position. The results indicate that myosin heavy chain-based chicken intrafusal fiber types are also set apart by differences in innervation.     

Histological study of an unusual cat muscle spindle deficient in motor innervation

Summary An unusual muscle spindle innervated by only one somatic motor axon and one primary sensory axon was encountered in a cat tenuissimus muscle cut in serial transverse sections and examined by light and electron microscopes. The motor axon branched to supply the distal poles of the nuclear bag₂ and nuclear chain intrafusal muscle fibers through motor terminals of several types. The proximal poles of the bag₂ and chain fibers and both poles of the nuclear bag₁ fiber were devoid of motor endings. In spite of the limited motor nerve supply the bag₁, bag₂ and chain fibers exhibited the usual morphologic characteristics in terms of the appearance of the equatorial region, relative fiber lengths and diameters, and the number of associated elastic fibers. It appears that motor axons may play only a limited role, if any, in the differentiation and maintenance of the three types of intrafusal muscle fiber in the cat.     

Exercise-induced fibre type transitions with regard to myosin,parvalbumin, and sarcoplasmic reticulum in muscles of the rat

Effects of a long-term, high intensity training program upon histochemically assessed myofibrillar actomyosin ATPase, myosin composition, peptide pattern of sarcoplasmic reticulum (SR), and parvalbumin content were analysed in muscles from the same rats which were used in a previous study (Green et al. 1983). Following 15 weeks of extreme training, an increase in type I and type IIA fibres and a decrease in type IIB fibres occurred both in plantaris and extensor digitorum longus (EDL) muscles. In the deep portion of vastus lateralis (VLD), there was a pronounced increase from 10±5% to 27±11% in type I fibres. No type I fibres were detected in the superficial portion of vastus lateralis (VLS) both in control and trained animals. An increase in slow type myosin light chains accompanied the histochemically observed fibre type transition in VLD. Changes in the peptide pattern of SR occurred both in VLS and VLD and suggested a complete transition from type IIB to IIA in VLS and from type IIA to I in VLD. A complete type IIA to I transition in the VLD was also suggested by the failure to detect parvalbumin in this muscle after 15 weeks of training. Changes in parvalbumin content and SR tended to precede the transitions in the myosin light chains. Obviously, high intensity endurance training is capable of transforming specific characteristics of muscle fibres beyond the commonly observed changes in the enzyme activity pattern of energy metabolism. The time courses of the various changes which are similar to those in chronic nerve stimulation experiments, indicate that various functional systems of the muscle fibre do not change simultaneously.     

Correlation between percentage fiber type area and myosin heavy chain content in human skeletal muscle

Histochemical methods are routinely used to delineate skeletal muscle fiber types. In the present investigation, this qualitative determination of fiber type composition was compared to the electrophoretically determined myosin heavy chain (MHC) content from a large number of human muscle biopsy samples. Biopsies were taken from the vastus lateralis muscle at the beginning and every 2 weeks during 8 weeks of highi-ntensity resistance training from men (n = 13) and woman (n = 8). Muscle was also extracted from nontraining men (n = 7) and women (n = 5) at the same periods. Six muscle fiber types (I, IC, IIAC, IIA, IIAB, and IIB) were determined using basic myofibrillar adenosine triphosphatase histochemistry. Cross-sectional areas were determined for the three major fiber types (I, IIA, and IIB) and used to calculate the percentage area of these types. Electrophoretic techniques were used to separate and quantify the percentage MHC content in these same biopsy samples, and these data were then used to compare with the percentage fiber type area. Correlation analyses suggest a relationship between the histochemically assessed percentage fiber type area and the electrophoretically assessed MHC content in human limb musculature. However, because of possible histochemical misclassification of some fibers (especially in trained muscle) both techniques may be essential in yielding important information about fiber type composition and possible fiber type transformations.     

An ultrastructure investigation of intrafusal muscle fibers in myotonic dystrophy

Summary Ultrastructure features of muscle spindles from two patients with myotonic dystrophy are described. Intrafusal muscle fibers exhibit extensive splitting with nuclear bag fibers affected more so than nuclear chain fibers. No sensory endings are present on nuclear chain fibers nor on one nuclear bag fiber throughout the equatorial and myotube regions. Small motor end plates are evident on various segments of split intrafusal fibers in the polar region and some of these extend into the myotube region. Satellite cells are numerous on both nuclear bag and nuclear chain fibers. These frequently occupy the cleft space between segments of split intrafusal fibers. The myotonic dystrophy muscle spindle ultrastructure features seem to closely resemble the appearance of developing mammalian muscle spindles as illustrated with opossum fetal tissue.Supported in part by federal grants 5TO1 AMO5401-12 and 5RO1 HDO2788-06     

Distribution patterns of muscle fibre types in major muscles of the bull (Bos taurus).

Summary The study describes the variations in distribution and cross-sectional area (fibre size) of three muscle fibre types (I, IIA, IIB) in 34 of the largest muscles of the bull (Bos taurus). The animals had been kept strictly unexercised for one year before slaughter. Representative sampling was done at 15 positions within each muscle, and from 2700 to 4500 fibres were analysed in each muscle. Different intermuscular patterns are described. The overall volume fraction (%) of type I fibres was about 10% higher in the forepart muscles than in the hindpart muscles (41% and 31%, respectively), while the mean content of type IIB fibres was similar. Type I fibres were particularly abundant in antigravity muscles. Of these, the hindlimb muscles contained 50% more type I fibres (by weight) than those of the forelimb. Typical antigravity antagonists contained very few type I fibres. In the thigh cross-section the proportion of type I fibres was highest in the anterior and medial parts, while the IIB fibres tended to be concentrated in the superficial and posterior parts. Intramuscular patterns were revealed, with type I fibres becoming gradually more abundant from superficial to deep regions, while IIB fibres had an opposite distribution. This was particularly evident in the thigh proper and in the scapular region. Within each fasciculus of all the muscles, the I fibres in the muscles of the forepart were on average about 15% larger than those of the muscles in the hindpart. The IIB fibres were on average about 10% larger in the hindpart than in the forepart muscles. A covariation between the proportion of type I and IIB fibres and their cross-sectional area was indicated.     

Differentiation of myosin in soleus and extensor digitorum longus muscle in different animal species during development

Summary Ca²⁺-ATPase activity and light chains of myosin prepared from fast and slow muscles of rat, guinea-pig and rabbit were studied during development from embryonic to old age to establish further correlation with the well-known developmental changes in contraction properties of these muscles. The changes involve the slow soleus muscle much more than the fast extensor digitorum longus muscle. Myosin-ATPase activity of the soleus muscle before or at birth is higher than in the muscle of adult animals. Myosin from the soleus muscle of embryos or newborn animals reveales light chains of myosin of both fast and slow type (with a preponderance of light chains of fast type in 26-days-old rabbit embryos). During postnatal development the amount of light chains of the fast type decreases, that of the slow type increases. Myosin from the soleus muscle of adult animals contains only light chains of the slow type. However, myosin from the soleus muscle of 30-months-old rats exhibits high myosin ATPase activity and contains light chains of myosin of both slow and fast type as in perinatal development. This is in agreement with the shortening of contraction time observed in this muscle in very old age. Thus developmental differentiation of myosin in the soleus muscle is followed by a trend of levelling out of the differences between fast and slow muscles of senescent animals. No such biphasic development is observed with respect to the fast extensor digitorum longus muscle.Abbreviations EDL extensor digitorum longus - SDS sodium dodecyl sulfate - LC light chain - LC₁ light chain 1     

Fiber differentiation of the human laryngeal muscles using the inhibition reactivation myofibrillar ATPase technique

Summary The aim of the present study was to further subdivide the type II fibers of the human thyroarytenoid and posterior cricoarytenoid muscles by means of a modified myosin ATPase reaction. In order to understand the functioning of these highly strained muscles better, it is important to know the respective percentage of fatigue-resistant type IIA fibers and fatigable type IIB fibers. The material comprised the larynges of seven laryngectomized males aged between 45 and 70 years and four laryngectomized females aged between 39 and 72 years. After having been frozen in nitrogen, 10-m-thick sections were cut from the laryngeal muscles in a cryostat. The pH-lability of the enzyme that can be utilized in a classical myosin ATPase reaction permits a differentiation between fiber types I, IIA and IIB. Evidently, this is not possible with every human muscle. The fiber types IIA and IIB of the thyroarytenoid and the posterior cricoarytenoid muscles could be clearly distinguished by means of the inhibition reactivation myofibrillar ATPase technique. Using this method, the myosin ATPase enzyme was initially inhibited by hydroxymer curibenzoate and subsequently reactivated by cysteine. Regarding the incidence of type I and IIA fibers, there was a statistically significant difference between the thyroarytenoid and the posterior cricoarytenoid muscles. The type IIA fiber content was statistically significantly higher in the arytenoid muscle than in the posterior cricoarytenoid muscle. The percentage of type IIB fibers was low, not only in the thyroarytenoid muscle and the posterior cricoarytenoid muscle but also in the other laryngeal muscles. The share of fiber types I, IIA and IIB in the thyroarytenoid muscles varied greatly from one patient to another. This was also true for the other laryngeal muscles. This aspect may be especially significant with regard to an individual's vocal character and vocal fatigability under stress.     

Muscular adaptations in response to three different resistance-training regimens: specificity of repetition maximum training zones

Thirty-two untrained men [mean (SD) age 22.5 (5.8) years, height 178.3 (7.2) cm, body mass 77.8 (11.9) kg] participated in an 8-week progressive resistance-training program to investigate the "strength–endurance continuum". Subjects were divided into four groups: a low repetition group (Low Rep, n=9) performing 3–5 repetitions maximum (RM) for four sets of each exercise with 3 min rest between sets and exercises, an intermediate repetition group (Int Rep, n=11) performing 9–11 RM for three sets with 2 min rest, a high repetition group (High Rep, n=7) performing 20–28 RM for two sets with 1 min rest, and a non-exercising control group (Con, n=5). Three exercises (leg press, squat, and knee extension) were performed 2 days/week for the first 4 weeks and 3 days/week for the final 4 weeks. Maximal strength [one repetition maximum, 1RM), local muscular endurance (maximal number of repetitions performed with 60% of 1RM), and various cardiorespiratory parameters (e.g., maximum oxygen consumption, pulmonary ventilation, maximal aerobic power, time to exhaustion) were assessed at the beginning and end of the study. In addition, pre- and post-training muscle biopsy samples were analyzed for fiber-type composition, cross-sectional area, myosin heavy chain (MHC) content, and capillarization. Maximal strength improved significantly more for the Low Rep group compared to the other training groups, and the maximal number of repetitions at 60% 1RM improved the most for the High Rep group. In addition, maximal aerobic power and time to exhaustion significantly increased at the end of the study for only the High Rep group. All three major fiber types (types I, IIA, and IIB) hypertrophied for the Low Rep and Int Rep groups, whereas no significant increases were demonstrated for either the High Rep or Con groups. However, the percentage of type IIB fibers decreased, with a concomitant increase in IIAB fibers for all three resistance-trained groups. These fiber-type conversions were supported by a significant decrease in MHCIIb accompanied by a significant increase in MHCIIa. No significant changes in fiber-type composition were found in the control samples. Although all three training regimens resulted in similar fiber-type transformations (IIB to IIA), the low to intermediate repetition resistance-training programs induced a greater hypertrophic effect compared to the high repetition regimen. The High Rep group, however, appeared better adapted for submaximal, prolonged contractions, with significant increases after training in aerobic power and time to exhaustion. Thus, low and intermediate RM training appears to induce similar muscular adaptations, at least after short-term training in previously untrained subjects. Overall, however, these data demonstrate that both physical performance and the associated physiological adaptations are linked to the intensity and number of repetitions performed, and thus lend support to the "strength–endurance continuum". Electronic Publication     

大鼠胸深肌肌纤维型的组成及分布

目的探讨SD大鼠胸深肌的肌纤维型组成和分布,借以了解该肌功能。方法采用Guth-Samaha肌球蛋白ATP酶组织化学染色法并稍做改良,对成年SD大鼠胸深肌冰冻切片进行肌纤维分型研究。结果大鼠胸深肌经肌球蛋白ATP酶组织化学染色后可明确分出2型肌纤维,即明亮色白的Ⅰ型纤维(慢缩纤维)和幽暗深褐的Ⅱ型纤维(快缩纤维),并且,两种纤维在肌内呈棋盘样均匀分布;图像分析仪下计数Ⅱ型纤维达到65%±6%,而Ⅰ纤维仅占35%±5%,前者明显高于后者(P<0.01)。结论大鼠胸深肌以Ⅱ型纤维为主,属于力量和速度型肌。     

Distribution of skeletofusimotor axons in lumbrical muscles of the monkey

Summary The nerve supply to 25 poles of muscle spindles in the monkey was reconstructed by light microscopy of serial 1-m thick transverse sections of lumbrical muscles. Twenty of 60 motor axons that supplied the spindle poles were identified as skeletofusimotor (). Twenty-eight percent of the spindle poles were innervated by axons, in addition to axons. Every -innervated spindle pole transected an endplate zone of extrafusal muscle. Most axons coinnervated extrafusal fibers rich in mitochondria and the nuclear bag₁ or nuclear chain intrafusal fibers. All but two axons innervated one type of intrafusal fiber only. The intramuscular organization of motor system in lumbrical muscles of the monkey was similar to that of the cat tenuissimus muscle. The function of -innervated spindles may be preferentially to monitor mechanical disturbances arising from the activity of extrafusal muscle units with which they share motor innervation.     

Morphological and histochemical differentiation of intrafusal fibres in the posterior latissimus dorsi muscle of the developing chick

Summary Morphological and histochemical differentiation of neuromuscular spindles was studied in the posterior latissimus dorsi (PLD) of the chick during embryonic and post-hatching development. A rapid increase in the number of spindles takes place between the 13th and 15th of embryonic life. By the 15th day in ovo, the spindle capsule appears filled with numerous contiguous cells. Large sensory endings and small primitive motor endings are observed on intrafusal fibres. Ultrastructural observations of the nerve supply of the spindles confirm that each developing spindle receives one thick Ia axon with one to three thin axons. The intracapsular space differentiates by the 17th day of embryonic development. All intrafusal fibres are morphologically of the nuclear-chain type, while two fibre types are distinguished as early as the 14th day of embryonic life, when myofibrillar ATPase activity is demonstrated after acid preincubation. These two histochemical types of intrafusal fibres are also described in the adult. The relation between these two histochemical types and different functional activity of intrafusal fibres is suggested.     

Tuning smooth muscle contraction by molecular motors

As in striated muscle, smooth muscle cells (SMC) contract by Ca²⁺ activated cyclic interaction between actin and type II myosin. However, smooth muscle maintains tone at basal activating Ca²⁺ and low energetic cost during sustained activation. This review analyzes the regulation of phasic and tonic contraction of SMC on the molecular level. Type II myosin is the molecular motor also of smooth muscle contraction. Six myosin heavy chain (MHC) isoenzymes (four smooth muscle, two nonmuscle) and five myosin light chain (MLC) isoforms (two 17 kDa, two 20 kDa, one 23 kDa) are expressed in SMC. These myosin subunits could be generated by alternative splicing or by differential gene expression. Thus different myosin isoenzymes are generated which may be modified posttranslationally by phosphorylation, affecting the contractile state of the SMC. Furthermore, they may be part of distinct contractile systems which are targeted by different second messenger cascades and are recruited differentially during activation, electromechanical, and pharmacomechanical coupling. Low energy consumption, shortening velocity, and MLC20 phosphorylation at low Ca²⁺ activation levels during tone maintenance ("latch") could be explained by a switch from smooth muscle myosin to nonmuscle myosin activation upon prolonged activation.Abbreviations MHC Myosin heavy chains - MLC Myosin light chains - MLCK Myosin light chain kinase - MLCP MLC20 phosphatase - NM Nonmuscle - nt Nucleotide - SM Smooth muscle - SMC Smooth muscle cells