Developmental effects on myonuclear domain size of rat diaphragm fibers.

During early postnatal development in rat diaphragm muscle (Diam), significant fiber growth and transitions in myosin heavy chain (MHC) isoform expression occur. Similar to other skeletal muscles, Diam fibers are multinucleated, and each myonucleus regulates the gene products within a finite volume: the myonuclear domain (MND). We hypothesized that postnatal changes in fiber cross-sectional area (CSA) are associated with increased number of myonuclei so that the MND size is maintained. The Diam was removed at postnatal days 14 (P-14) and 28 (P-28). MHC isoform expression was determined by SDS-PAGE. Fiber CSA, myonuclear number, and MND size were measured using confocal microscopy. By P-14, significant coexpression of MHC isoforms was present with no fiber displaying singular expression of MHCNeo. By P-28, singular expression was predominant. MND size was not different across fiber types at P-14. Significant fiber growth was evident by P-28 at all fiber types (fiber CSA increased by 61, 93, and 147% at fibers expressing MHCSlow, MHC2A, and MHC2X, respectively). The number of myonuclei per unit of fiber length was similar across fibers at P-14, but it was greater at fibers expressing MHC2X at P-28. The total number of myonuclei per fiber also increased between P-14 and P-28 at all fiber types. Accordingly, MND size increased significantly by P-28 at all fiber types, and it became larger at fibers expressing MHC2X compared with fibers expressing MHCSlow or MHC2A. These results suggest that MND size is not maintained during the considerable fiber growth associated with postnatal development of the Diam.     

Influence of corticosteroids on myonuclear domain size in the rat diaphragm muscle.

Skeletal muscle fibers are multinucleated. Each myonucleus regulates gene products and protein expression in only a restricted portion of the muscle fiber, the myonuclear domain (MND). In the rat diaphragm muscle (DIAm), corticosteroid (CoS) treatment causes atrophy of fibers containing myosin heavy chain (MHC): MHC2X and/or MHC2B. We hypothesized that DIAm fiber MND size is maintained during CoS-induced atrophy. Adult male rats received methylprednisolone for 11 days at 1 (CoS-Low, n = 8) or 8 mg x kg(-1) x day(-1) (CoS-High, n = 8). Age-matched (CTL-AgeM, n = 8), sham-operated (SHAM-AgeM, n = 8), and weight-matched (CTL-WtM, n = 8) animals served as controls. In single DIAm fibers, cross-sectional area (CSA), MND size, and MHC expression were determined. Fiber CSA and MND size were similar in CTL-AgeM and SHAM-AgeM groups. Only fibers containing MHCslow or MHC2A displayed smaller CSA in CTL-WtM than in CTL-AgeM and SHAM-AgeM groups, and MND size was reduced in all fibers. Thus fibers containing MHCslow and MHC2A maintain the number of myonuclei, whereas MHC2X or MHC2B fibers show loss of myonuclei during normal muscle growth. Both CoS groups displayed smaller CSA and MND size than CTL-AgeM and SHAM-AgeM groups. However, compared with CTL-WtM DIAm fibers, only fibers containing MHC2X or MHC2B displayed reduced CSA and MND size after CoS treatment. Thus little, if any, loss of myonuclei was associated with CoS-induced atrophy of MHC2X or MHC2B DIAm fibers. In summary, MND size does not appear to be regulated during CoS-induced DIAm atrophy.     

Maintenance of myonuclear domain size in rat soleus after overload and growth hormone/IGF-I treatment

The purpose ofthis study was to determine the effects of functional overload (FO)combined with growth hormone/insulin-like growth factor I (GH/IGF-I)administration on myonuclear number and domain size in rat soleusmuscle fibers. Adult female rats underwent bilateral ablation of theplantaris and gastrocnemius muscles and, after 7 days of recovery, wereinjected three times daily for 14 days with GH/IGF-I (1 mg/kg each; FO + GH/IGF-I group) or saline vehicle (FO group). Intact rats receivingsaline vehicle served as controls (Con group). Muscle wet weight was32% greater in the FO than in the Con group: 162 ± 8 vs. 123 ± 16 mg. Muscle weight in the FO + GH/IGF-I group (196 ± 14 mg) was59 and 21% larger than in the Con and FO groups, respectively. Meansoleus fiber cross-sectional area of the FO + GH/IGF-I group (2,826 ± 445 µm²) was increasedcompared with the Con (2,044 ± 108 µm²) and FO (2,267 ± 301 µm²) groups. The difference infiber size between the FO and Con groups was not significant. Meanmyonuclear number increased in FO (187 ± 15 myonuclei/mm) and FO + GH/IGF-I (217 ± 23 myonuclei/mm) rats compared with Con (155 ± 12 myonuclei/mm) rats, although the difference between FO and FO + GH/IGF-I animals was not significant. The mean cytoplasmic volume permyonucleus (myonuclear domain) was similar across groups. These resultsdemonstrate that the larger mean muscle weight and fibercross-sectional area occurred when FO was combined with GH/IGF-Iadministration and that myonuclear number increased concomitantly withfiber volume. Thus there appears to be some mechanism(s) that maintainsthe myonuclear domain when a fiber hypertrophies.

     

Modulation of myonuclear number in functionally overloaded and exercised rat plantaris fibers.

The effects of 10 wk of functional overload (FO), with and without daily treadmill endurance training, on the cross-sectional area, myonuclear number, and myonuclear domain size of mechanically isolated single fiber segments of the adult rat plantaris were determined. The fibers were typed on the basis of high-resolution gel electrophoresis for separation of specific myosin heavy chain (MHC) isoforms and grouped as type I(+) (containing some type I MHC with or without any combination of fast MHCs), type IIa(+) (containing some type IIa with or without some type IIx and/or IIb but no type I MHC), and type IIx/b (containing only type IIx and/or IIb MHCs). Type I(+) fibers had a higher myonuclear number than did both fast types of fibers in the control and FO, but not in the FO and treadmill trained, rats. All fiber types in both FO groups had a significantly larger (36-90%) cross-sectional area and a significantly higher (61-109%) myonuclear number than did control. The average myonuclear domain size of each fiber type was similar among the three groups, except for a smaller domain size in the type IIx/b fibers of the FO compared with control. In general, these data indicate that during hypertrophy the number of myonuclei increase proportionally to the increase in fiber volume. The maintenance of myonuclear domain size near control values suggests that regulatory mechanisms exist that ensure a tight coupling between the quantity of genetic machinery and the protein requirements of a fiber.     

Effects of inactivity on fiber size and myonuclear number in rat soleus muscle.

The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size, and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a midthoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared with control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus atrophy occurred earlier than the shift in myosin heavy chain (MHC) profile. The number of myonuclei was approximately 30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei per millimeter in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus myonuclei were eliminated from fibers containing only type I MHC. Because the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.     

Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle.

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Dia(m)) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (P(o)) and maximum unloaded shortening velocity (V(o)). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHC(neo) in the Dia(m) persisted, and emergence of MHC(2X) and MHC(2B) was delayed. By D-21 and D-28, relative expression of MHC(2A) and MHC(2B) was reduced in DNV compared with control (CTL) animals. Expression of MHC(neo) also reappeared in adult Dia(m) by 2-3 wk after DNV, and relative expression of MHC(2B) was reduced. At each age, P(o) was reduced and V(o) was slowed by DNV, compared with CTL. In CTL Dia(m), postnatal changes in P(o) and V(o) were associated with an increase in fast MHC isoform expression. In DNV Dia(m), no such association existed. We conclude that, in the Dia(m), DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.     

Effect of aminophylline on the contraction threshold of rat diaphragm fibers.

We studied the effect of aminophylline (0.1-1 mM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (x200) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT, in a dose-dependent manner, toward more negative values of the resting membrane potential (Vm). 2) Dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) shifts the CT, although this change is smaller than in the presence of xanthine. 3) Tetracaine (1 mM), a drug that diminishes Ca release from the sarcoplasmic reticulum, reduces the shift induced by 1 mM aminophylline; this is partially overcome by increasing aminophylline concentration to 5 mM. 4) Hyperpolarization of the fibers shifts the CT to more negative Vm. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca release from the sarcoplasmic reticulum.     

Effects of hypothyroidism on maximum specific force in rat diaphragm muscle fibers.

We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Dia(m)) fibers, 2) Hyp decreases the maximum specific force (F(max)) of Dia(m) fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and F(max) to a greater extent in fibers expressing MHC type 2X (MHC(2X)) and/or MHC type 2B (MHC(2B)). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC(2X) and/or MHC(2B). On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased F(max) across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (approximately 40 vs. approximately 20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC(2A) was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC(2X) and/or MHC(2B) in the Hyp Dia(m) than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC(2X) and/or MHC(2B) and that the reduction of F(max) with Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.     

Effect of unilateral denervation on maximum specific force in rat diaphragm muscle fibers.

We hypothesize that 1) the effect of denervation (DNV) is more pronounced in fibers expressing fast myosin heavy chain (MHC) isoforms and 2) the effect of DNV on maximum specific force reflects a reduction in MHC content per half sarcomere or the number of cross bridges in parallel. Studies were performed on single Triton X-100-permeabilized fibers activated at a pCa (-log Ca2+ concentration) of 4.0. MHC content per half sarcomere was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. After 2 of wk DNV, the maximum specific force of fibers expressing MHC2X was reduced by approximately 40% (MHC(2B) expression was absent), whereas the maximum specific force of fibers expressing MHC2A and MHC(slow) decreased by only approximately 20%. DNV also reduced the MHC content in fibers expressing MHC2X, with no effect on fibers expressing MHC2A and MHC(slow). When normalized for MHC content per half sarcomere, force generated by DNV fibers expressing MHC2X and MHC2A was decreased compared with control fibers. These results suggest the force per cross bridge is also affected by DNV.     

Maximum specific force depends on myosin heavy chain content in rat diaphragm muscle fibers.

In the present study, myosin heavy chain (MHC) content per half sarcomere, an estimate of the number of cross bridges available for force generation, was determined in rat diaphragm muscle (Dia(m)) fibers expressing different MHC isoforms. We hypothesize that fiber-type differences in maximum specific force [force per cross-sectional area (CSA)] reflect the number of cross bridges present per CSA. Studies were performed on single, Triton X-100-permeabilized rat Dia(m) fibers. Maximum specific force was determined by activation of single Dia(m) fibers in the presence of a high-calcium solution (pCa, -log Ca(2+) concentration of 4.0). SDS-PAGE and Western blot analyses were used to determine MHC isoform composition and MHC content per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC content. However, the force produced by slow fibers remained below that of fast fibers when normalized for the number of cross bridges available. On the basis of these results, the lower force produced by slow fibers may be due to less force per cross bridge compared with fast fibers.     

Adverse effects of myasthenia gravis on rat phrenic diaphragm contractile performance.

Myasthenia gravis has variable effects on the respiratory system, ranging from no abnormalities to life-threatening respiratory failure. Studies characterized diaphragm muscle contractile performance in rat autoimmune myasthenia gravis. Rats received monoclonal antibody that recognizes acetylcholine receptor determinants (or inactive antibody); 3 days later, phrenic nerve and diaphragm were studied in vitro. Myasthenic rats segregated into two groups, those with normal vs. impaired limb muscle function when tested in intact animals ("mild" and "severe" myasthenic). Baseline diaphragm twitch force was reduced for both severe (P < 0.01) and mild (P < 0.05) myasthenic compared with control animals (twitch force: normal 1,352 +/- 140, mild myasthenic 672 +/- 99, severe myasthenic 687 +/- 74 g/cm2). However, only severe myasthenic diaphragm had impaired diaphragm endurance, based on significantly (P < 0.05) accelerated rate of peak force decline during the initial period of stimulation (0.02 + 0.02, 0.03 +/- 0.01, and 0.09 +/- 0.01%/pulse for normal, mild myasthenic, and severe myasthenic, respectively, during continuous stimulation) and intratrain fatigue (up to 30.5 +/- 7.4% intratrain force drop in severe myasthenic vs. none in normal and mild myasthenic, P < 0.01). Furthermore, compared with continuous stimulation, intermittent stimulation had a protective effect on force of severe myasthenic diaphragm (force after 2,000 pulses was 31.4 +/- 2.0% of initial during intermittent stimulation vs. 13.0 +/- 2.1% of initial during continuous stimulation, P < 0.01) but not on normal diaphragm. These data indicate that baseline force and fatigue may be affected to different extents by varying severity of myasthenia gravis and furthermore provide a mechanism by which alterations in breathing pattern may worsen respiratory muscle function in neuromuscular diseases.     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

Hyperpolarizing effect of aminophylline, theophylline, and cAMP on rat diaphragm fibers

We studied the effect of aminophylline and theophylline (0.1-2 mM) on the resting membrane potential (Vm) of rat diaphragm fibers in vitro (25 degrees C). The main findings are the following. 1) Aminophylline and theophylline hyperpolarize the fibers in a dose-dependent manner. This effect is present with 0.1 and 0.25 mM of aminophylline and theophylline, respectively, and the maximum effect is reached with 1 mM of the drug (approximately 5-8 mV in comparison to the normal values). This effect is reversible by washing out the preparation with normal solution. 2) Dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 2 mM) produces a similar increment in the Vm. 3) The hyperpolarizing action observed in the presence of aminophylline, theophylline, and DBcAMP is suppressed by 5 X 10(-4) M ouabain or by lowering the bath temperature to 5 degrees C. These results suggest that the xanthines may directly or indirectly stimulate a Na-K pump. Two possibilities may be considered: 1) an electrogenic effect of the Na-K pump and 2) a reduction in the extracellular K+ concentration in the solution contacting the external side of the cell as a consequence of the activity of the Na-K pump. Alternative mechanisms such as a reduction in Na permeability or an increment in K permeability might collaborate in the hyperpolarizing effect of the drugs tested.     

The in vitro effects of benzodiazepines on the rat diaphragm. Structure-activity relationships

The effect of three different subgroups of benzodiazepines on the indirectly evoked twitch tension was investigated in the in vitro rat phrenic nerve - hemidiaphragm preparation. Two effects were observed: an initial increase in twitch tension at lower concentrations with some benzodiazepines, and a concentration-dependent depression at higher concentrations with all benzodiazepines. Significant differences for these effects were observed among the three subgroups of benzodiazepines and additionally within the subgroup of the 1,4-benzodiazepine-2-ketones. Structural requirements for both effects were different. For the increase of twitch tension a --CH3 substitution at R1 and a --F substitution at R2' were beneficial. For the twitch depression an --OH substitution at R3 and a --C1 substitution at R2' were optimal. An interaction between substituents at different substitution sites occurred. The potency of twitch depression showed a good correlation with literature reports of pKa values and a poor-to-inverse correlation with lipophilicity indices. A benzodiazepines antagonist, Ro 15-1788, caused no change in twitch tension in the concentration range of the investigated benzodiazepines nor did it prevent the twitch depression caused by benzodiazepines.     

Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle

Van Balkom, Roland H. H., Wen-Zhi Zhan, Y. S. Prakash, P. N. Richard Dekhuijzen, and Gary C. Sieck. Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle. J. Appl. Physiol. 83(4):1062-1067, 1997.The effects of corticosteroids (CS) on diaphragmmuscle (Dia_m) fiber morphologyand contractile properties were evaluated in three groups of rats:controls (Ctl), surgical sham and weight-matched controls (Sham), andCS-treated (6 mg · kg¹ · day¹prednisolone at 2.5 ml/h for 3 wk). In the CS-treatedDia_m, there was a selectiveatrophy of type IIx and IIb fibers, compared with a generalized atrophyof all fibers in the Sham group. Maximum isometric force was reduced by20% in the CS group compared with both Ctl and Sham. Maximumshortening velocity in the CS Dia_m was slowed by ~20% compared with Ctl and Sham. Peak power output ofthe CS Dia_m was only 60% of Ctland 70% of Sham. Endurance to repeated isotonic contractions improvedin the CS-treated Dia_m comparedwith Ctl. We conclude that the atrophy of type IIx and IIb fibers inthe Dia_m can only partiallyaccount for the CS-induced changes in isotonic contractile properties.Other factors such as reduced myofibrillar density or alteredcross-bridge cycling kinetics are also likely to contribute to theeffects of CS treatment.

     

Absence of regional differences in the size and oxidative capacity of diaphragm muscle fibers

The oxidative capacity and cross-sectional area of muscle fibers were compared between the costal and crural regions of the cat diaphragm and across the abdominal-thoracic extent of the muscle. Succinate dehydrogenase (SDH) activity of individual fibers was quantified using a microphotometric procedure implemented on an image-processing system. In both costal and crural regions, population distributions of SDH activities were unimodal for both type I and II fibers. The continuous distribution of SDH activities for type II fibers indicated that no clear threshold exists for the subclassification of fibers based on differences in oxidative capacity (e.g., the classification of fast-twitch glycolytic and fast-twitch oxidative glycolytic fiber types). No differences in either SDH activity or cross-sectional area were noted between fiber populations of the costal and crural regions. Differences in SDH activity and cross-sectional area were noted, however, between fiber populations located on the abdominal and thoracic sides of the costal region. Both type I and II fibers on the abdominal side of the costal diaphragm were larger and more oxidative than comparable fibers on the thoracic side.     

Nitric oxide effects on force-velocity characteristics of the rat diaphragm

The present experiments tested nitric oxide (NO) effects on shortening velocity and power production in maximally activated rat diaphragm at 37 degrees C. Diaphragm fiber bundles (n = 10/group) were incubated at 37 degrees C in Krebs-Ringer solution containing no added drug (control), the NO synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10 mM), the NO donor sodium nitroprusside (SNP; 1 mM), or a combination (L-NNA + SNP). Loaded shortening velocity was measured via the load-clamp technique over a range of afterloads. Unloaded shortening velocity (Vo) was measured in control and L-NNA-treated bundles (n = 12/group) by using the slack test. Force-velocity data fitted to the Hill equation determined a Vmax of 13.7+/-0.4 lengths/s, contradicting the notion that rat diaphragm Vmax declines at temperatures > 35 degrees C. In contrast, L-NNA decreased Vmax (P < 0.05), loaded shortening velocity (P < 0.001), and power production (P < 0.001), but did not change Vo or maximal isometric force. All L-NNA effects were prevented by coincubating fiber bundles with L-NNA + SNP. SNP alone had no effect on any variable. These data indicate that endogenous NO is essential for optimal myofilament function during active shortening.