Effect of acute nutritional deprivation on diaphragm structure and function in adolescent rats.

The influence of 90 h of acute nutritional deprivation (ND; water ad libitum only) on in vitro contractile and fatigue properties, muscle fiber type proportions, and cross-sectional areas (CSA) of the adolescent rat diaphragm was determined. Diaphragm muscle properties in the ND rats were compared with those in control rats (CTL; food and water ad libitum). Acute ND resulted in a 32% reduction in body mass, whereas the body mass of CTL rats increased by 29%. Acute ND resulted in a significant reduction in the mass of the diaphragm (costal, 36%; crural, 43%), soleus (36%), and medial gastrocnemius (45%) muscles. Isometric twitch characteristics of the diaphragm muscle (contraction and half-relaxation times) were prolonged in the ND animals. Peak twitch and maximum tetanic forces were unaffected by ND. Fatigue resistance of the diaphragm muscle was improved in ND animals. Diaphragm muscle fiber type proportions were similar in ND and CTL groups. The CSA of type I and II diaphragm muscle fibers were reduced by 22 and 40%, respectively, in ND animals compared with CTL. We conclude that, whereas an identical protocol of acute ND had no significant effects on diaphragm muscle structure and function in adult rats, adolescent animals exhibit significantly less nutritional reserve. These differences may be due to curtailment of the rapid anabolic rate in growing animals.     

IGF-I and/or growth hormone preserve diaphragm fiber size with moderate malnutrition

Resistance to theanabolic effects of growth hormone (GH) occurs with severe caloricdeficit. This study examined whether moderate caloric deficit (50% ofdaily intake for 7 days) in the adolescent rat exceeds a criticalthreshold for GH action and whether a combination of GH andinsulin-like growth factor I (IGF-I) would have enhanced anaboliceffects on the diaphragm (Dia). Five groups of rats (4 wk old) werestudied: 1) control (Ctl),2) nutritionally deprived (ND),3) ND + GH,4) ND + IGF-I, and5) ND + GH + IGF-I. IGF-I was givenby continuous infusion (200 µg/day). GH was injected subcutaneously(250 µg every 12 h). Contractile and fatigue properties of the Diawere determined in vitro. Quantitative histochemical methods were usedto determine Dia fiber type proportions, cross-sectional areas, andsuccinate dehydrogenase activities. The body weight of Ctl ratsincreased 46% compared with 7% in ND animals, whereas that of ND ratsreceiving growth factors was intermediate. Serum IGF-I levels werereduced 54% in ND animals and maintained with the provision of growthfactors. Dia fatigue resistance was improved in ND animals receivinggrowth factors. There were no differences in Dia contractileproperties, fiber type proportions, or succinate dehydrogenaseactivities across groups. ND resulted in atrophy/growth arrest of allDia fibers (20-32%) compared with Ctl. Administration of IGF-Iand/or GH completely prevented atrophy/growth arrest of all Diafibers. No additive or synergistic effects were noted. We propose thatthese growth factors may provide useful short-term adjunctivenutritional support in circumstances in which the provision of optimalnutrition may be delayed or inadequate.

     

Effects of undernutrition on diaphragm fiber size, SDH activity, and fatigue resistance

The influence of prolonged nutritional deprivation on the succinate dehydrogenase (SDH) activity and cross-sectional areas of individual fibers in the rat diaphragm and deep portion of the medial gastrocnemius (MGr) muscles was determined. Fatigue resistance of the diaphragm was measured by means of an in vitro nerve-muscle strip preparation. Fiber SDH activity and cross-sectional area were quantified by means of an image processing system. Diaphragm fatigue resistance was significantly improved in the nutritionally deprived (ND) group. In both muscles, nutritional deprivation resulted in a significant decrease in fiber cross-sectional area (both type I and II), type II fibers showing greater atrophy. The SDH activities of type I and II fibers in the diaphragm were not affected by nutritional deprivation. This contrasted with a significant decrease in the SDH activity of both type I and II fibers in the MGr of ND animals. An assessment of the interrelationships between fiber atrophy and fiber SDH activity revealed a greater effect of malnutrition on those diaphragm type II fibers that had the lowest relative SDH activities and the largest cross-sectional areas. By comparison, the effect of malnutrition on type I and II fibers in the MGr was nonselective with regard to fiber SDH activity. We conclude that the enhanced diaphragm fatigue resistance in the ND animals does not result from an increase in the oxidative capacity of muscle fibers and is best explained by the pattern of diaphragm muscle fiber atrophy.     

Tumor necrosis factor-alpha and malnutrition-induced inhibition of diaphragm fiber growth in young rats.

Tumor necrosis factor (TNF)-alpha has been implicated in several muscle-wasting disorders, with increased levels of the cytokine reported in malnourished children. The role of TNF-alpha in mediating malnutrition-induced inhibition of diaphragm (DIA) muscle growth in young growing rats was evaluated. Three groups of rats were studied: 1) control (CTL); 2) nutritional deprivation (ND; 50% of normal food intake for 7 days); and 3) ND + rat specific anti-TNF-alpha antibody. DIA fiber cross-sectional areas were determined. Serum and muscle TNF-alpha levels were measured by real-time PCR, ELISA, and immunohistochemistry. Body weights decreased 20% in ND rats and increased 46% in CTL animals. Anti-TNF-alpha had no effect on body weight or on DIA mass in ND animals. ND significantly reduced cross-sectional areas of all fiber types (33-46%). Anti-TNF-alpha failed to attenuate ND-induced inhibition of DIA fiber growth. Serum TNF-alpha levels increased 2.6-fold in ND animals, with levels suppressed to below CTL values with anti-TNF-alpha. DIA TNF-alpha mRNA and protein levels increased two- to threefold in ND rats. Anti-TNF-alpha antibodies suppressed muscle levels of the cytokine in ND animals to near CTL values. TNF-alpha immunoreactivity in all DIA fibers revealed similar directions of change in both ND groups. Direction and magnitude of change in DIA phosphorylated p38 MAPK (a likely second messenger of TNF-alpha) tracked those of TNF-alpha. Muscle levels of IGF-I mRNA and phosphorylated Akt were markedly reduced in ND animals with no change following anti-TNF-alpha therapy. Thus rat anti-TNF-alpha at a dose known to neutralize the cytokine failed to attenuate or reverse ND-induced inhibition of DIA fiber growth in our model.     

Effect of severe short-term malnutrition on diaphragm muscle signal transduction pathways influencing protein turnover.

The aim of this study was to evaluate the effect of nutritional deprivation (ND) on signal transduction pathways influencing the translational apparatus in the diaphragm muscle. Male rats were divided into two groups: 1) 20% of usual food intake for 4 days (ND) with water provided at libitum and 2) free-eating control (Ctl). Total protein and RNA were extracted from the diaphragm. Insulin-like growth factor I mRNA was analyzed by RT-PCR. Protein analyses of key cytoplasmic proteins for three signaling pathways deemed important in influencing protein turnover [phosphatidylinositol 3-kinase- Akt-mammalian target of rapamycin, P13K/Akt/glycogen synthase kinase (GSK)-3, and MAPK-ERK] were performed by Western blot. Body weight decreased 30% in ND and increased 17% in Ctl animals. Diaphragm mass decreased 29% in ND animals. Muscle insulin-like growth factor I mRNA abundance was reduced 63% in ND animals. ND resulted in a 55% reduction in phosphorylated (Ser473) Akt. Phosphorylation of mammalian target of rapamycin at Ser2448 was reduced by 85% in ND animals. Downstream effectors important in translation initiation were also affected by ND. Phosphorylated (Thr389) 70-kDa ribosomal protein S6 kinase was significantly reduced (35%) by ND. ND also resulted in significant dephosphorylation of the translational repressor initiation factor 4E-binding protein 1. Phosphorylation of GSK-3alpha (Ser21) and GSK-3beta (Ser9) was increased 55 and 45%, respectively, with ND. Phosphorylation of ERK1 (Thr202) and ERK2 (Tyr204), p44 and p42, respectively, was reduced 64 and 55%, respectively, with ND. Total protein concentration for all signaling intermediates of the three pathways was preserved. We conclude that short-term ND altered the phosphorylation states of key proteins of several pathways involved in protein turnover. This forms the framework for future studies aimed at identifying therapeutic targets in the management of short-term nutritionally induced cachectic states.     

Effect of insulin-like growth factor I and/or growth hormone on diaphragm of malnourished adolescent rats

Lewis, Michael I., Thomas J. LoRusso, and Mario Fournier.Effect of insulin-like growth factor I and/or growthhormone on diaphragm of malnourished adolescent rats.J. Appl. Physiol. 82(4):1064-1070, 1997.Young growing animals appear to havesignificantly reduced "nutritional reserve" to short periods ofunstressed starvation compared with adults, with resultant growtharrest and/or atrophy of diaphragm (Dia) muscle fibers. The aimof this study was to assess in an adolescent rat model of acutenutritional deprivation (ND; 72 h) the impact of insulin-like growthfactor I (IGF-I), with or without added growth hormone (GH), on thecross-sectional areas (CSA) of individual Dia muscle fibers. Fivegroups were studied: 1) control(Ctr); 2) ND;3) ND given IGF-I (ND/IGF-I); 4) ND given GH (ND/GH); and5) ND given a combination of IGF-I and GH (ND/IGF-I/GH). IGF-I was given by a subcutaneously implanted osmotic minipump (200 µg/day), whereas GH was administered twice daily by a subcutaneous injection (250 µg every 12 h). Isometric contractile and fatigue properties of the Dia were determined in vitro.Forces were normalized for muscle CSA (i.e., specific force). Dia fibertype proportions were determined histochemically, and fiber CSA wasquantified by using a computer-based image-processing system. Totalserum IGF-I concentrations were significantly reduced in ND and ND/GHanimals, compared with Ctr, and elevated in the groups receiving IGF-I.The provision of growth factors did not alter the contractile orfatigue properties of ND animals. Dia fiber type proportions weresimilar among the groups. In ND animals, there was a significantreduction in the CSA of types I, IIa, IIx, and IIc Dia fibers comparedwith Ctr. The administration of IGF-I alone or in combination with GHto ND animals significantly diminished the reduction in Dia fiber size.GH alone had no effect on Dia fiber size in ND animals. We concludethat with acute ND the peripheral resistance to the action of GHappears to be bypassed by the administration of IGF-I alone or incombination with GH.

     

Effect of nutritional deprivation on diaphragm contractility and muscle fiber size

The influence of nutritional deprivation on the contractile and fatigue properties of the diaphragm was studied in adult rats. Food access was restricted to one-third of normal daily intake until the body weight of nutritionally deprived (ND) animals was approximately 50% of controls (CTL). Isometric contractile properties were studied in an in vitro nerve muscle strip preparation. Both twitch (Pt) and tetanic (Po) tensions of diaphragms from the ND animals were markedly reduced compared with CTL; however, Pt/Po was higher for the ND group. The shape of the force-frequency curve (normalized to Po) was generally similar between the two groups, except at 5 and 10 pulses/s stimulation, where greater relative tensions were produced in diaphragms from the ND animals. Diaphragm fatigue was induced by repetitive stimulation at either 20 or 100 pulses/s. Endurance time (defined as the time required for tension to fall to 50% of initial) of diaphragms from ND animals was prolonged at both 20 and 100 pulses/s. Immediately after induction of fatigue, force-frequency curves for both ND and CTL diaphragms were shifted to the right. However, this rightward shift was attenuated in the ND group compared with CTL. Nutritional deprivation had no effect on the proportions of different fiber types within the diaphragm but did result in a significant decrease in the cross-sectional area of both fast-and slow-twitch fibers. This decrease in cross-sectional area was significantly greater for fast-twitch fibers. We conclude that these changes in diaphragm contractile and fatigue properties occur as a result of the influence of malnutrition on muscle fiber cross-sectional area.     

Effects of undernutrition on respiratory mechanics and lung parenchyma remodeling.

Undernutrition thwarts lung structure and function, but there are disagreements about the behavior of lung mechanics in malnourished animals. To clarify this issue, lung and chest wall mechanical properties were subdivided into their resistive, elastic, and viscoelastic properties in nutritionally deprived (ND) rats and correlated with the data gathered from histology (light and electron microscopy and elastic fiber content), and bronchoalveolar lavage fluid analysis (lipid and protein content). Twenty-four Wistar rats were assigned into two groups. In the control (Ctrl) group the animals received food ad libitum. In the ND group, rats received one-third of their usual daily food consumption until they lost 40% of their initial body weight. Lung static elastance, viscoelastic and resistive pressures (normalized by functional residual capacity), and chest wall pressures were higher in the ND group than in the Ctrl group. The ND group exhibited patchy atelectasis, areas of emphysema, interstitial edema, and reduced elastic fiber content. The amount of lipid and protein in bronchoalveolar lavage fluid was significantly reduced in the ND group. Electron microscopy showed 1) type II pneumocytes with a reduction in lamellar body content, multilamellated structures, membrane vesicles, granular debris, and structurally aberrant mitochondria; and 2) diaphragm and intercostals with atrophy, disarrangement of the myofibrils, and deposition of collagen type I fibers. In conclusion, undernutrition led to lung and chest wall mechanical changes that were the result from a balance among the following modifications: 1) distorted structure of diaphragm and intercostals, 2) surfactant content reduction, and 3) decrease in elastic fiber content.     

Insulin-like growth factor I prevents corticosteroid-induced diaphragm muscle atrophy in emphysematous hamsters

The aim of this study was to evaluate whether recombinant human insulin-like growth factor I (rhIGF-I) could attenuate or prevent diaphragm (DIA) fiber atrophy with corticosteroid (CS) administration to emphysematous (EMP) hamsters. DIA muscle IGF-I responses to CS administration with and without exogenous rhIGF-I administration were evaluated. Three groups were studied: 1) EMP; 2) EMP + triamcinolone (T; 0.4 mg.kg-1.day-1 im); and 3) EMP + T + IGF-I (600 microg/day by constant infusion). After 4 wk, the DIA was analyzed histochemically and biochemically (IGF-I mRNA levels by RT-PCR and endogenous and exogenous IGF-I peptide levels immunochemically). Body weights of EMP-T progressively decreased, while those of EMP and EMP-T-IGF-I remained stable despite similarly reduced food intake in both T groups. DIA weight was reduced with T but preserved with rhIGF-I infusion. DIA fiber proportions were similar among the groups. The cross-sectional areas of types I, IIa, and IIx fibers were reduced (17 to 31%) with T administration but unchanged with rhIGF-I infusion. DIA IGF-I mRNA levels were similar across all groups. By contrast, the endogenous DIA IGF-I levels were reduced (41%) in the EMP-T-IGF-I animals. Total DIA IGF-I levels (endogenous + exogenous) were still significantly reduced. IGF-I immunoreactivity confirmed this reduction in all DIA fibers. We conclude that DIA fiber atrophy with T was completely prevented by exogenous rhIGF-I administration. This effect was likely mediated by the pharmacological influences of exogenously administered rhIGF-I. We speculate that this results from increased bioavailability of free IGF-I to react with muscle receptors. Reduced endogenous IGF-I levels in the DIA likely reflect a negative-feedback influence. These results may have important clinical implications for treatment options to offset the adverse effects of CS on the respiratory muscles in patients with chronic lung disorders.     

Anabolic influences of insulin-like growth factor I and/or growth hormone on the diaphragm of young rats

Lewis, Michael I., Thomas J. LoRusso, and Mario Fournier.Anabolic influences of insulin-like growth factor I and/or growth hormone on the diaphragm of young rats. J. Appl. Physiol. 82(6): 1972-1978, 1997.It iscontroversial whether insulin-like growth factor I (IGF-I), growthhormone (GH), or their combination might enhance body growthand/or tissue anabolism in the well-fed animal with an intactsomatotrophic axis. To assess this further, we studied four groups ofadolescent rats: 1) control (Ctr),2) GH,3) IGF-I, and4) GH/IGF-I. IGF-I was given via anosmotic minipump, whereas GH was injected subcutaneously for a period of 72 h. Diaphragm (Dia) contractile and fatigue properties were determined in vitro. Quantitative histochemical and morphometric analyses were performed on Dia fibers. Total serum IGF-I levels weresignificantly increased in the groups receiving growth factors. Although body weight increased to a greater extent in the animals receiving growth factors, a further synergistic effect was noted in theGH/IGF-I animals compared with either GH or IGF-I groups. Costal Diamass was greater in the groups receiving growth factors. The Dia ofGH/IGF-I animals was more fatigue resistant than the Dia in Ctr. Thecross-sectional area of types IIa and IIx fibers were increased to asimilar extent in all groups receiving growth factors compared withCtr. Succinate dehydrogenase activity of type IIa fibers wassignificantly greater in the GH/IGF-I animals compared with the othergroups. We conclude that the short-term provision of growth factors towell-nourished, normally growing adolescent rats can accelerate bodygrowth and promote selective hypertrophy of predominantly type II Diafibers.

     

Functional, cellular, and biochemical adaptations to elastase-induced emphysema in hamster medial scalene.

The scalene has been reported to be an accessory inspiratory muscle in the hamster. We hypothesize that with the chronic loads and/or dynamic hyperinflation associated with emphysema (Emp), the scalene will be actively recruited, resulting in functional, cellular, and biochemical adaptations. Emp was induced in adult hamsters. Inspiratory electromyogram (EMG) activity was recorded from the medial scalene and costal diaphragm. Isometric contractile and fatigue properties were evaluated in vitro. Muscle fibers were classified histochemically and immunohistochemically. Individual fiber cross-sectional areas (CSA) and succinate dehydrogenase (SDH) activities were determined quantitatively. Myosin heavy chain (MHC) isoforms were identified by SDS-PAGE, and their proportions were determined by scanning densitometry. All Emp animals exhibited spontaneous scalene inspiratory EMG activity during quiet breathing, whereas the scalene muscles of controls (Ctl) were silent. There were no differences in contractile and fatigue properties of the scalene between Ctl and Emp. In Emp, the relative amount of MHC(2A) was 15% higher whereas that of MHC(2X) was 14% lower compared with Ctl. Similarly, the proportion of type IIa fibers increased significantly in Emp animals with a concomitant decrease in IIx fibers. CSA of type IIx fibers were significantly smaller in Emp compared with Ctl. SDH activities of all fiber types were significantly increased by 53 to 63% in Emp. We conclude that with Emp the actively recruited scalene exhibits primary-like inspiratory activity in the hamster. Adaptations of the scalene with Emp likely relate both to increased loads and to factors intrinsic to muscle architecture and chest mechanics.     

Effect of acute nutritional deprivation on diaphragm structure and function

The influence of 90 h of acute nutritional deprivation (ND) on the cross-sectional areas of muscle fibers and the contractile and fatigue properties of the adult rat diaphragm were determined. Isometric contractile properties and fatigue resistance of the diaphragm were measured by means of an in vitro nerve-muscle strip preparation. Contractions were evoked by using phrenic nerve stimulation (left hemidiaphragm) or direct muscle stimulation (right hemidiaphragm) in the presence of curare. Acute ND resulted in a 20% reduction in body weight. No significant decrements in diaphragm or soleus weights were noted in the ND animals compared with controls (CTL), whereas the weight of the medial gastrocnemius was reduced by 20% in the ND animals. Peak twitch and tetanic tensions (normalized for the weight of the diaphragm strip) were not reduced in ND compared with CTL animals after either nerve or muscle stimulation. The fatigue index of the diaphragm was significantly reduced in ND animals only after nerve stimulation. After the fatigue test, there was rapid recovery of the additional fatigue noted with nerve stimulation. The proportions of type I and II muscle fibers of the diaphragm were similar in the CTL and ND animals. No differences in diaphragm cross-sectional areas were noted for either type I or II muscle fibers in the CTL and ND animals. It is concluded that acute ND has no effect on diaphragm contractility or morphometry and only an inconsequential influence on diaphragm fatigue.     

Diaphragm muscle fiber function and structure in humans with hemidiaphragm paralysis

Recent studies proposed that mechanical inactivity of the human diaphragm during mechanical ventilation rapidly causes diaphragm atrophy and weakness. However, conclusive evidence for the notion that diaphragm weakness is a direct consequence of mechanical inactivity is lacking. To study the effect of hemidiaphragm paralysis on diaphragm muscle fiber function and structure in humans, biopsies were obtained from the paralyzed hemidiaphragm in eight patients with hemidiaphragm paralysis. All patients had unilateral paralysis of known duration, caused by en bloc resection of the phrenic nerve with a tumor. Furthermore, diaphragm biopsies were obtained from three control subjects. The contractile performance of demembranated muscle fibers was determined, as well as fiber ultrastructure and morphology. Finally, expression of E3 ligases and proteasome activity was determined to evaluate activation of the ubiquitin-proteasome pathway. The force-generating capacity, as well as myofibrillar ultrastructure, of diaphragm muscle fibers was preserved up to 8 wk of paralysis. The cross-sectional area of slow fibers was reduced after 2 wk of paralysis; that of fast fibers was preserved up to 8 wk. The expression of the E3 ligases MAFbx and MuRF-1 and proteasome activity was not significantly upregulated in diaphragm fibers following paralysis, not even after 72 and 88 wk of paralysis, at which time marked atrophy of slow and fast diaphragm fibers had occurred. Diaphragm muscle fiber atrophy and weakness following hemidiaphragm paralysis develops slowly and takes months to occur.     

Diaphragm single-fiber weakness and loss of myosin in congestive heart failure rats

Diaphragm weakness commonly occurs in patients with congestive heart failure (CHF) and is an independent predictor of mortality. However, the pathophysiology of diaphragm weakness is poorly understood. We hypothesized that CHF induces diaphragm weakness at the single-fiber level by decreasing myosin content. In addition, we hypothesized that myofibrillar Ca(2+) sensitivity is decreased and cross-bridge kinetics are slower in CHF diaphragm fibers. Finally, we hypothesized that loss of myosin in CHF diaphragm weakness is associated with increased proteolytic activities of caspase-3 and the proteasome. In skinned diaphragm single fibers of rats with CHF, induced by left coronary artery ligation, maximum force generation was reduced by approximately 35% (P < 0.01) compared with sham-operated animals for slow, 2a, and 2x fibers. In these CHF diaphragm fibers, myosin heavy chain content per half-sarcomere was concomitantly decreased (P < 0.01). Ca(2+) sensitivity of force generation and the rate constant of tension redevelopment were significantly reduced in CHF diaphragm fibers compared with sham-operated animals for all fiber types. The cleavage activity of the proteolytic enzyme caspase-3 and the proteasome were approximately 30% (P < 0.05) and approximately 60% (P < 0.05) higher, respectively, in diaphragm homogenates from CHF rats than from sham-operated rats. The present study demonstrates diaphragm weakness at the single-fiber level in a myocardial infarct model of CHF. The reduced maximal force generation can be explained by a loss of myosin content in all fiber types and is associated with activation of caspase-3 and the proteasome. Furthermore, CHF decreases myofibrillar Ca(2+) sensitivity and slows cross-bridge cycling kinetics in diaphragm fibers.     

Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming

This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 9x10(-7) M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin-eosin (HE); nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA (p<0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3+/-4.7% vs. S=75.7+/-2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81+/-3.1% vs. Sw=71.0+/-4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. On the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise.     

Alterations in diaphragm contractility after nandrolone administration: an analysis of potential mechanisms.

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 (Vo) 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 Vo, 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 Vo. We speculate that Nan in supraphysiological doses have the potential to offset or ameliorate conditions associated with enhanced proteolysis and disordered protein turnover.     

Effects of prolonged undernutrition on structure and function of the diaphragm

The present study examined the effect of prolonged undernutrition on diaphragmatic structure and force-generating ability. Studies were performed on 58 Syrian hamsters in which the feed was reduced by 33% for a 4-wk period. Sixty animals fed a similar diet ad libitum served as controls. Diaphragm muscle structure was assessed from its mass (wet and dry weight), thickness, fiber composition, and fiber size. Isometric force produced in vitro by isolated muscle strips in response to electrical stimulation of the phrenic nerve was examined over a range of muscle lengths (length-tension relationship). In undernourished animals, body weight decreased 25 +/- 5%. Diaphragm wet and dry weight, muscle thickness, and the cross-sectional area of fast-glycolytic (FG) and fast-oxidative (FO) fibers were significantly less in undernourished than control animals and correlated with reductions in body weight. The cross-sectional area of slow-oxidative (SO) fibers was the same in the two groups. The percentage of FG fibers in undernourished animals was decreased slightly and the percentage of SO fibers increased. Maximum isometric tension was reduced in undernourished animals as compared with controls, but the position and shape of the length-tension relationship was the same in the two groups. Reductions in muscle force appeared to be explained by decreases in muscle mass, since tension corrected for cross-sectional area or tissue weight was the same in the two groups. Therefore muscle mechanical efficiency appeared to be unaffected by undernutrition. These data indicate that prolonged undernutrition causes deleterious changes in diaphragm muscle structure that impair its ability to generate force.     

Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy

Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy.