Sieck, Gary C.

The present study examined Ca2+sensitivity of diaphragm muscle (Diam) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Diamfibers expressing the MHCslowisoform have greater Ca2+sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca2+sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Diamfibers. The Ca2+concentration at which 50% maximal force was generated (pCa50) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa50for Diamfibers expressing MHCslowwas significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca2+sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Diamfibers expressing MHCslowcontained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca2+sensitivity in Diamfibers.

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 diaphragm muscle (Diam) fiber morphology and contractile properties were evaluated in three groups of rats: controls (Ctl), surgical sham and weight-matched controls (Sham), and CS-treated (6 mg ⋅ kg−1 ⋅ day−1prednisolone at 2.5 ml/h for 3 wk). In the CS-treated Diam, there was a selective atrophy of type IIx and IIb fibers, compared with a generalized atrophy of all fibers in the Sham group. Maximum isometric force was reduced by 20% in the CS group compared with both Ctl and Sham. Maximum shortening velocity in the CS Diamwas slowed by ∼20% compared with Ctl and Sham. Peak power output of the CS Diam was only 60% of Ctl and 70% of Sham. Endurance to repeated isotonic contractions improved in the CS-treated Diam compared with Ctl. We conclude that the atrophy of type IIx and IIb fibers in the Diam can only partially account for the CS-induced changes in isotonic contractile properties. Other factors such as reduced myofibrillar density or altered cross-bridge cycling kinetics are also likely to contribute to the effects of CS treatment.

The effects of corticosteroid (CS) treatment (prednisolone continuously administered subcutaneously at a flow rate of 2.5 μl/h, daily dose 5.6 mg/kg, for 3 wk) on neuromuscular junction (NMJ) morphology and neuromuscular transmission in rat diaphragm muscle (Dimus) were compared with weight-matched (Sham) and ad libitum fed control (Ctl) groups. Fibers were classified on the basis of myosin heavy chain (MHC) isoform expression. CS treatment caused significant atrophy of fibers expressing MHC2X(type IIx), either alone or with MHC2B(type IIx/b). Fibers expressing MHCslow(type I) and MHC2A(type IIa) were unaffected by CS. The planar areas of nerve terminals and motor endplates at type IIx/b fibers were smaller in CS-treated Dimuscompared with Sham and Ctl. However, CS-induced atrophy of type IIx/b fibers exceeded changes in NMJ morphology. Thus, when normalized for fiber diameter, NMJs were relatively larger in the CS-treated group compared with Ctl. Neuromuscular transmission failure, assessed in vitro by comparing force loss during repetitive (40 Hz) nerve vs. direct muscle stimulation, was less in CS-treated Dimus. These results indicate that alterations in NMJ morphology after CS treatment are dependent on fiber type and may contribute to improved neuromuscular transmission.

We hypothesized that adaptations of the rabbit diaphragm (Dia) after unilateral denervation (DNV) result from removal of a neural influence rather than from passive stress. Length changes of midcostal and sternal Dia regions were measured before and after DNV by using sonomicrometry. Midcostal fibers passively lengthened after DNV, whereas sternal fibers shortened. In both regions, these length changes were associated with minimal stress, as estimated from passive force-length relationships. Morphological and contractile adaptions of midcostal and sternal Dia regions were examined after 1 and 4 wk of DNV. In both Dia regions, type I fibers progressively hypertrophied, whereas type IIb fibers atrophied. After DNV, changes in isometric contraction were similar in both Dia regions. Twitch contraction and half-relaxation times increased, force-frequency relationships shifted leftward, and maximum tetanic force decreased. We conclude that passive length changes and mechanical stress are not the main determinants of the morphological and contractile adaptations of the Dia after unilateral DNV but that these adaptations result from DNV itself.

The aim of this study was to evaluate the potential mechanisms underlying the improved contractility of the diaphragm (Dia) in adult intact male hamsters after nandrolone (Nan) administration, given subcutaneously over 4 wk via a controlled-release capsule (initial dose: 4.5 mg ⋅ kg−1 ⋅ day−1; with weight gain, final dose: 2.7 mg ⋅ kg−1 ⋅ day−1). Control (Ctl) animals received blank capsules. Isometric contractile properties of the Dia were determined in vitro after 4 wk. The maximum velocity of unloaded shortening ( V o) was determined in vitro by means of the slack test. Dia fibers were classified histochemically on the basis of myofibrillar ATPase staining and fiber cross-sectional area (CSA), and the relative interstitial space was quantitated. Ca2+-activated myosin ATPase activity was determined by quantitative histochemistry in individual diaphragm fibers. Myosin heavy chain (MHC) isoforms were identified electrophoretically, and their proportions were determined by using scanning densitometry. Peak twitch and tetanic forces, as well as V o, were significantly greater in Nan animals compared with Ctl. The proportion of type IIa Dia fibers was significantly increased in Nan animals. Nan increased the CSA of all fiber types (26–47%), whereas the relative interstitial space decreased. The relative contribution of fiber types to total costal Dia area was preserved between the groups. Proportions of MHC isoforms were similar between the groups. There was a tendency for increased expression of MHC2B with Nan. Ca2+-activated myosin ATPase activity was increased 35–39% in all fiber types in Nan animals. We conclude that, after Nan administration, the increase in Dia specific force results from the relatively greater Dia CSA occupied by hypertrophied muscle fibers, whereas the increased ATPase activity promotes a higher rate of cross-bridge turnover and thus increased V o. We speculate that Nan in supraphysiological doses have the potential to offset or ameliorate conditions associated with enhanced proteolysis and disordered protein turnover.

Prakash, Y. S., H. F. M. van der Heijden, M. S. Kannan, and G. C. Sieck. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J. Appl. Physiol. 82(6): 1836–1843, 1997.—Relaxation of airway smooth muscle (ASM) by β-adrenoceptor agonists involves reduction of intracellular Ca2+concentration ([Ca2+]i). In porcine ASM cells, acetylcholine induces [Ca2+]ioscillations that display frequency modulation by agonist concentration and basal [Ca2+]i. We used real-time confocal microscopy to examine the effect of salbutamol (1 nM to 1 μM), a β2-adrenoceptor agonist, on [Ca2+]ioscillations in freshly dissociated porcine ASM cells. Salbutamol decreased the frequency of [Ca2+]ioscillations in a concentration-dependent fashion, completely inhibiting the oscillations at 1 μM. These effects were mimicked by a cell-permeant analog of adenosine 3′,5′-cyclic monophosphate. The inhibitory effect of salbutamol was partially reversed by BAY K 8644. Salbutamol reduced [Ca2+]ieven when sarcoplasmic reticulum (SR) Ca2+reuptake and Ca2+influx were blocked. Lanthanum blockade of Ca2+efflux attenuated the inhibitory effect of salbutamol on [Ca2+]i. The [Ca2+]iresponse to caffeine was unaffected by salbutamol. On the basis of these results, we conclude that β2-adrenoceptor agonists have little effect on SR Ca2+release in ASM cells but reduce [Ca2+]iby inhibiting Ca2+influx through voltage-gated channels and by enhancing Ca2+efflux.

Watchko, Jon F., Monica J. Daood, Gary C. Sieck, John J. LaBella, Bill T. Ameredes, Alan P. Koretsky, and Be Wieringa. Combined myofibrillar and mitochondrial creatine kinase deficiency impairs mouse diaphragm isotonic function. J. Appl. Physiol. 82(5): 1416–1423, 1997.—Creatine kinase (CK) is an enzyme central to cellular high-energy phosphate metabolism in muscle. To characterize the physiological role of CK in respiratory muscle during dynamic contractions, we compared the force-velocity relationships, power, and work output characteristics of the diaphragm (Dia) from mice with combined myofibrillar and sarcomeric mitochondrial CK deficiency (CK[−/−]) with CK-sufficient controls (Ctl). Maximum velocity of shortening was significantly lower in CK[−/−] Dia (14.1 ± 0.9 Lo/s, where Lois optimal fiber length) compared with Ctl Dia (17.5 ± 1.1 Lo/s) ( P < 0.01). Maximum power was obtained at 0.4–0.5 tetanic force in both groups; absolute maximum power (2,293 ± 138 W/m2) and work (201 ± 9 J/m2) were lower in CK[−/−] Dia compared with Ctl Dia (2,744 ± 146 W/m2and 284 ± 26 J/m2, respectively) ( P < 0.05). The ability of CK[−/−] Dia to sustain shortening during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load) was markedly impaired, with CK[−/−] Dia power and work declining to zero by 37 ± 4 s, compared with 61 ± 5 s in Ctl Dia. We conclude that combined myofibrillar and sarcomeric mitochondrial CK deficiency profoundly impairs Dia power and work output, underscoring the functional importance of CK during dynamic contractions in skeletal muscle.

Fredberg, J. J., K. A. Jones, M. Nathan, S. Raboudi, Y. S. Prakash, S. A. Shore, J. P. Butler, and G. C. Sieck. Friction in airway smooth muscle: mechanism, latch, and implications in asthma. J. Appl. Physiol. 81(6): 2703–2712, 1996.—In muscle, active force and stiffness reflect numbers of actin-myosin interactions and shortening velocity reflects their turnover rates, but the molecular basis of mechanical friction is somewhat less clear. To better characterize molecular mechanisms that govern mechanical friction, we measured the rate of mechanical energy dissipation and the rate of actomyosin ATP utilization simultaneously in activated canine airway smooth muscle subjected to small periodic stretches as occur in breathing. The amplitude of the frictional stress is proportional to ηE, where E is the tissue stiffness defined by the slope of the resulting force vs. displacement loop and η is the hysteresivity defined by the fatness of that loop. From contractile stimulus onset, the time course of frictional stress amplitude followed a biphasic pattern that tracked that of the rate of actomyosin ATP consumption. The time course of hysteresivity, however, followed a different biphasic pattern that tracked that of shortening velocity. Taken together with an analysis of mechanical energy storage and dissipation in the cross-bridge cycle, these results indicate, first, that like shortening velocity and the rate of actomyosin ATP utilization, mechanical friction in airway smooth muscle is also governed by the rate of cross-bridge cycling; second, that changes in cycling rate associated with conversion of rapidly cycling cross bridges to slowly cycling latch bridges can be assessed from changes of hysteresivity of the force vs. displacement loop; and third, that steady-state force maintenance (latch) is a low-friction contractile state. This last finding may account for the unique inability of asthmatic patients to reverse spontaneous airways obstruction with a deep inspiration.

Howell, Sandra, Wen-Zhi Zhan, and Gary C. Sieck.Diaphragm disuse reduces Ca2+uptake capacity of sarcoplasmic reticulum. J. Appl. Physiol. 82(1): 164–171, 1997.—Chronic phrenic tetrodotoxin (TTX) blockade and phrenic denervation (Dnv) of hamster diaphragm result in decreased maximum specific tension, prolonged contraction time, and improved fatigue resistance (W. Z. Zhan and G. C. Sieck. J. Appl. Physiol. 72: 1445–1453, 1992). An underlying increased relative contribution of type I fibers to total muscle mass appears to be consistent with, but does not completely account for, changes in contractile and fatigue properties. The present study was designed to evaluate a potential role for altered cellular Ca2+metabolism in the adaptive response of the diaphragm to chronic disuse. An analytic method based on simulation and modeling of long-term45Ca2+efflux data was used to estimate Ca2+ contents (nmol Ca2+/g wet wt tissue) and exchange fluxes (nmol Ca2+ ⋅ min−1 ⋅ g−1) for extracellular and intracellular compartments in the in vitro hamster hemidiaphragm after prolonged disuse. Three groups were compared: control (Con, n = 5), phrenic TTX blockade (TTX, n = 5), and phrenic denervation (Dnv, n = 5). Experimental muscles were loaded with45Ca2+for 1 h, and efflux data were collected for 8 h by using a flow-through tissue chamber. Compartmental analysis of efflux data estimated that the Ca2+ contents and Ca2+ exchange fluxes of the largest and slowest intracellular compartment (putative longitudinal reticulum) were reduced by ∼50% in TTX and Dnv muscle groups compared with Con. In addition, the kinetic model predicted significant decreases in total intracellular Ca2+ and total diaphragm Ca2+ in TTX and Dnv muscles. We conclude that the data support the hypothesis that the capac- ity of the sarcoplasmic reticulum for Ca2+ sequestration is reduced in chronic diaphragm disuse. The impact of this effect on diaphragm contractile and fatigue properties is discussed.