Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats.

Insulin-like growth factor I (IGF-I) peptide levels have been shown to increase in overloaded skeletal muscles (G. R. Adams and F. Haddad. J. Appl. Physiol. 81: 2509-2516, 1996). In that study, the increase in IGF-I was found to precede measurable increases in muscle protein and was correlated with an increase in muscle DNA content. The present study was undertaken to test the hypothesis that direct IGF-I infusion would result in an increase in muscle DNA as well as in various measurements of muscle size. Either 0.9% saline or nonsystemic doses of IGF-I were infused directly into a non-weight-bearing muscle of rats, the tibialis anterior (TA), via a fenestrated catheter attached to a subcutaneous miniosmotic pump. Saline infusion had no effect on the mass, protein content, or DNA content of TA muscles. Local IGF-I infusion had no effect on body or heart weight. The absolute weight of the infused TA muscles was approximately 9% greater (P < 0.05) than that of the contralateral TA muscles. IGF-I infusion resulted in significant increases in the total protein and DNA content of TA muscles (P < 0.05). As a result of these coordinated changes, the DNA-to-protein ratio of the hypertrophied TA was similar to that of the contralateral muscles. These results suggest that IGF-I may be acting to directly stimulate processes such as protein synthesis and satellite cell proliferation, which result in skeletal muscle hypertrophy.

Substrate oxidation capacity in rodent skeletal muscle: effects of exposure to zero gravity.

A study was conducted, as part of the integrated National Aeronautics and Space Administration Space Life Sciences 1 mission flown in June of 1991, to ascertain the effects of 9 days of exposure to zero gravity on the capacity of rodent skeletal muscle fiber types to oxidize either [14C]pyruvate or [14C]palmitate under state 3 metabolic conditions, i.e., nonlimiting amounts of substrate and cofactors. In addition, activity levels of marker enzymes of the tricarboxylic acid cycle, malate shuttle, and beta-oxidation were measured. Results showed that significant differences in muscle weight occurred in both the predominantly slow vastus intermedius and predominantly fast vastus lateralis of flight vs. control groups (P < 0.05). Total protein content of the muscle samples was similar between groups. Both pyruvate oxidation capacity and the marker oxidative enzymes were not altered in the flight relative to control animals. However, the capacity to oxidize long-chain fatty acids was significantly reduced by 37% in both the high- and low-oxidative regions of the vastus muscle (P < 0.05). Although these findings of a selective reduction in fatty acid oxidation capacity in response to spaceflight are surprising, they are consistent with previous findings showing 1) an increased capacity to take up glucose and upregulate glucose transporter proteins and 2) a marked accumulation of triglycerides in the skeletal muscles of rats subjected to states of unloading. Thus, skeletal muscle of animals exposed to non-weight-bearing environments undergo subcellular transformations that may preferentially bias energy utilization to carbohydrates.

Food restriction-induced transformations in cardiac functional and biochemical properties in rats.

The primary objective of this study was to ascertain if various degrees of marked chronic food restriction (FR) as well as the combination of FR and exercise training of moderate intensity induce changes in the functional properties of the heart that are consistent with previously reported findings indicative of downregulation of high-adenosinetriphosphatase V1 isomyosin expression. Adult female rodents were randomly assigned to one of four experimental groups: 1) free eating, 2) 50% food restricted, 3) 75% food restricted, or 4) 50% food restricted plus treadmill trained. Results show that FR induced significant depression in the functional properties (heart rate, left ventricular pressure, rate of pressure development, and double product) of the heart in all FR groups and that this depression in functional capacity corresponded to the degree of FR. These functional changes were accompanied by significant downregulation of the alpha- and upregulation of the beta-myosin heavy chain gene expressions, as studied at both the mRNA and protein levels. The exercise training induced further alterations in cardiac function; however, these alterations occurred independently of any shifts in isomyosin composition. These results suggest that although severe FR is a potent stimulus to transform both the biochemical and functional properties of the rodent heart, the underlying mechanism(s) concerning these adaptations remains unresolved.

Activity-induced regulation of myosin isoform distribution: comparison of two contractile activity programs.

This study examined the role of specific types of contractile activity in regulating myosin heavy chain (MHC) isoform expression in rodent soleus. A combination of hindlimb suspension (SN) and two programmed contractile training activity paradigms, either isometric contractile activity (ST-IM) or high-load slowly shortening isovelocity activity, were utilized. Both training paradigms increased muscle mass compared with SN alone. However, only ST-IM resulted in a partial prevention of the suspension-induced decrease in type I MHC. With the use of a fluorescently labeled antibody to type IIa MHC, the distribution of MHCs among fibers was examined immunohistochemically. In SN, the percentage of cells staining positive for type IIa MHC was increased but the staining intensity of the positively staining cells was unchanged compared with control cells. In the ST-IM soleus, the percentage of positively staining fibers was unchanged but the intensity of the positively staining cells was decreased compared with SN values. These results suggest that: 1) isometric contractile activity is more effective than isovelocity activity in preventing suspension-induced shifts in soleus MHC distribution and 2) changes associated with both suspension and training occur in only a small number of fibers, with the majority of fibers apparently unresponsive to these interventions.

A new animal model for modulating myosin isoform expression by altered mechanical activity.

The purpose of this study was to develop a new rodent model that is capable of delineating the importance of mechanical loading on myosin heavy chain (MHC) isoform expression of the plantar and dorsi flexor muscles of the ankle. The essential components of this system include 1) stimulating electrodes that are chronically implanted into a muscle, allowing for the control of the activation pattern of the target muscle(s); 2) a training apparatus that translates the moment of the ankle into a linear force; and 3) a computer-controlled Cambridge 310 ergometer. The isovelocity profile of the ergometer ensured that the medial gastrocnemius (MG) produced forces that were > 90% of maximal isometric force (Po), and the eccentric contractions of the tibialis anterior (TA) were typically 120% of Po. Both the concentric and eccentric training programs produced statistically significant increases in the muscle mass of the MG (approximately 15%) and TA (approximately 7%) as well as a decrease in myofibrillar adenosinetriphosphatase activity. Both the white and red regions of the MG and TA exhibited significant increases in the relative content of the type IIa MHC and concomitant decreases in type IIb MHC expression. Although the red regions of the MG and red TA contained approximately 10% type I MHC, the training programs did not affect this isoform. It appears that when a fast-twitch muscle is stimulated at a high frequency (100 Hz) and required to contract either concentrically or eccentrically under high loading conditions, the expression of the type IIa MHC isoform will be upregulated, whereas that of the type IIb MHC will be concomitantly downregulated.

Role of denervation in modulating IIb MHC gene expression in response to T(3) plus unloading state.

Previously, we have reported that the combination of hindlimb suspension (HS) and thyroid hormone [triiodothyronine (T(3))] treatment induces the de novo expression of the fast IIb myosin heavy chain (MHC) gene in the soleus. Thus we tested the hypotheses that the induction of IIb MHC expression with HS + T(3) is prevented with denervation and that this IIb induction is regulated by transcriptional processes. Adult female rats were subjected to 2 wk of combined HS + T(3) in which one side of the lower leg was simultaneously denervated. HS + T(3) caused decreased expression of the slow type I MHC and concomitant increases in both the fast type IIx and IIb MHC isoforms in the intact soleus muscle. Denervation prevented the endogenous expression of the IIb MHC gene at both the protein and mRNA levels. Although HS + T(3) intervention was able to markedly increase the expression of the 2.6-kb IIb MHC promoter-reporter construct using direct gene transfer, this induction, however, was not inhibited by denervation. These findings collectively suggest that normal innervation is essential for inducing the unique expression of the IIb MHC in a slow muscle in response to HS + T(3); however, in the denervated muscle, there is a discordance between the regulation of the endogenous IIb MHC gene relative to the exogenous IIb MHC promoter-reporter construct.

Effects of spaceflight and thyroid deficiency on hindlimb development. I. Muscle mass and IGF-I expression.

Thyroid deficiency (TD) in neonatal rats causes reduced growth of skeletal muscle that is disproportionately greater than that for other tissues (G. R. Adams, S. A. McCue, M. Zeng, and K. M. Baldwin. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 276: R954-R961, 1999). TD depresses plasma insulin-like growth factor I (IGF-I) levels, suggesting a mechanism for this effect. We hypothesized that TD and exposure to spaceflight (SF) would interact to reduce skeletal muscle growth via a reduction in IGF-I levels. Neonatal rats were flown in space for 16 days. There was a similar, nonadditive reduction in the growth of the body ( approximately 50%) and muscle weight (fast muscles, approximately 60%) with either TD or SF. In the soleus muscle, either SF or TD alone resulted in growth reductions that were augmented by SF-TD interactions. There were strong correlations between 1) muscle mass and muscle IGF-I levels and 2) circulating IGF-I and body weight. These results indicate that either hypothyroidism or exposure to SF will limit the somatic and muscle-specific growth of neonatal rats. The impact of these perturbations on skeletal muscle growth is relatively greater than the effect on somatic growth. The mechanisms by which either TD or SF impact growth appear to have a common pathway involving the control of plasma and muscle IGF-I concentrations.

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression.

We examined the novel interaction of hyperthyroidism and hindlimb suspension on the pattern of myosin heavy chain (MHC) expression (mRNA and protein) in skeletal muscles. Female Sprague-Dawley rats were assigned to four groups: 1) normal control (Con); 2) thyroid hormone treated [150 micrograms 3,5,3'-triiodothyronine (T3). kg-1. day-1] (T3); 3) hindlimb suspension (HS); or 4) T3-treated and HS (T3 + HS). Results show for the first time the novel observation that the combination T3 + HS induces a rapid and sustained, marked (80-90%) downregulation of type I MHC gene expression that is mirrored temporally by concomitant marked upregulation of type IIb MHC gene expression, as evidenced by the de novo synthesis of type IIb MHC protein in the soleus. The fast type IIx MHC isoform showed a differential response among the experimental groups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing in the plantaris muscles. The fast type IIa MHC was the least responsive to suspension of the MHCs and reflected its greatest responsiveness to T3 treatment while also undergoing differential adaptations in slow vs. fast muscle (increases vs. decreases, respectively). These results confirm previous findings that all four adult MHC genes are sensitive to T3 and suspension in a muscle-specific manner. In addition, we show that T3 + HS can interact synergistically to create novel adaptations in MHC expression that could not be observed when each factor was imposed separately.

Effects of isometric training on skeletal myosin heavy chain expression.

This study tested the hypothesis that an isometric resistance-training program induces upregulation of slow myosin heavy chain (MHC) expression in a fast-twitch skeletal muscle. Thus we studied the effects of two resistance-training programs on rodent medial gastrocnemius (MG) muscle that were designed to elicit repetitive isometric contractions (10-12 per set; 4 sets per session) of different duration (8 vs. 5 s) and activation frequency (100 vs. 60 Hz) per contraction during each training session (total of 6 and 12 sessions). Results showed that both training paradigms produced significant increases in muscle weight ( approximately 11-13%) after completion of training (P < 0.05). Significant transformations in MHC expression occurred and involved specifically a decrease in the relative expression of the fast type IIb MHC and concomitant increased expression of the fast type IIx MHC. These adaptations were observed in both the "white" and "red" regions of the MG, and they occurred at both the mRNA and protein levels. These adaptations were detected after only six training sessions. Neither of the training programs produced any change in the relative expression of either the slow type I MHC or the moderately fast type IIa MHC, which can be upregulated in the red MG by chronic functional overload. These findings show that the isometric protocols used in this investigation were not sufficient to induce the hypothesized changes in the myosin heavy chain isoform expression in rodent skeletal muscle.

Effects of spaceflight and thyroid deficiency on rat hindlimb development. II. Expression of MHC isoforms.

Both slow-twitch and fast-twitch muscles are undifferentiated after birth as to their contractile protein phenotype. Thus we examined the separate and combined effects of spaceflight (SF) and thyroid deficiency (TD) on myosin heavy chain (MHC) gene expression (protein and mRNA) in muscles of neonatal rats (7 and 14 days of age at launch) exposed to SF for 16 days. Spaceflight markedly reduced expression of the slow, type I MHC gene by approximately 55%, whereas it augmented expression of the fast IIx and IIb MHCs in antigravity skeletal muscles. In fast muscles, SF caused subtle increases in the fast IIb MHC relative to the other adult MHCs. In contrast, TD prevented the normal expression of the fast MHC phenotype, particularly the IIb MHC, whereas TD maintained expression of the embryonic/neonatal MHC isoforms; this response occurred independently of gravity. Collectively, these results suggest that normal expression of the type I MHC gene requires signals associated with weight-bearing activity, whereas normal expression of the IIb MHC requires an intact thyroid state acting independently of the weight-bearing activities typically encountered during neonatal development of laboratory rodents. Finally, MHC expression in developing muscles is chiefly regulated by pretranslational processes based on the tight relationship between the MHC protein and mRNA data.

Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression.

This study examined the interaction of exogenous thyroid hormone 3,5,3'-triiodothyronine (T3) and functional overload on skeletal muscle myosin heavy chain (MHC) expression, studied at both the protein and mRNA level of analysis. Animals were allocated to the following groups: 1) normal control, 2) overload control, 3) hyperthyroid control, and 4) hyperthyroid+overload. Overload of the rat plantaris was accomplished by surgical removal of its synergists (soleus and gastrocnemius), and the animals were made hyperthyroid by injections of T3 (350 micrograms/kg every other day). After overload of 8 wk, muscle enlargement occurred by 53% for both overload groups (P < 0.05). This was accompanied by a 330 and 82% increase in the relative content of type I and IIa MHC, respectively, and a corresponding decrease by 16 and 44% in type IIx and IIb MHC, respectively, in the overload control group (P < 0.05 vs. normal control). Changes in the relative and absolute content of mRNA for these MHCs paralleled the protein response. Exogenous T3 completely reversed the upregulation of type I MHC and the downregulation of type IIx associated with overload at both the protein and mRNA level (P < 0.05). However, T3 was only partially effective in blunting the downregulation of IIb MHC and the upregulation of IIa MHC (protein and mRNA) accompanying the overload.(ABSTRACT TRUNCATED AT 250 WORDS)

Choriocarcinoma with cerebral metastases coexistent with a first pregnancy.

A case is presented of choriocarcinoma with cerebral metastases which was unsuspected until a life-threatening life-threatening situation occurred. A viable infant was born and there was dramatic response to treatment with probable cure.

Effects of diabetes on rodent cardiac thyroid hormone receptor and isomyosin expression.

Previous studies show that diabetes induces marked transformations in cardiac myosin heavy chain (MHC) gene expression that are somehow linked to the cellular action of thyroid hormone 3,5,3'-triiodothyronine (T3). In this study, we tested the hypothesis that diabetes induces a reduced expression of thyroid hormone receptors (TRs), which are known to be important transcription factors interacting with thyroid response elements (TREs) in the promoter region of both alpha- and beta-MHC genes. Adult female rats were randomly assigned to either a normal control (NC) or diabetic (D) group. Three and/or six weeks after induction of diabetes via streptozotocin injection, the hearts of the animals were analyzed for MHC and TR mRNA isoforms expression, nuclear T3 binding, and nuclear extract interaction with a palindromic TRE. Results showed that diabetes induced significant alteration in alpha- and beta-MHC expression. Northern blot analyses indicated no diabetes-associated differences in TR isoform mRNA signals. Cardiac nuclear T3 binding studies suggested no differences in either the binding capacity or the equilibrium binding constant among the two groups, indicating no changes in either the number of nuclear TRs or their affinity for T3. Furthermore, gel mobility shift assays detected no difference between NC and D groups for cardiac nuclear extract binding to palindromic TRE. Collectively, these findings suggest that, whereas diabetes exerts a profound effect on cardiac isomyosin gene expression, the underlying mechanism, although dependent on factors linked to T3 function, does not involve alterations in TR expression.

Single-fiber and whole muscle analyses of MHC isoform plasticity: interaction between T3 and unloading.

Previous data suggest that separate interventions of hyperthyroidism (T3) and hindlimb suspension (HS) act on some but not all slow type I fibers in the soleus muscle. This may be due to the presence of "refractory" fibers that are unresponsive to either of these interventions. Alternatively, T3 and HS might act on different populations of slow type I fibers in the soleus muscle. Adult female Sprague-Dawley rats were assigned to 1) control, 2) T3, 3) HS, or 4) T3+HS. Nine animals were assigned to each group. Single-fiber electrophoretic analyses (n = 40 per muscle) of the soleus muscle demonstrated that the HS reduced the percentage of slow type I fibers from approximately 80% (control) to approximately 40% (HS) of the fiber population. Although hyperthyroidism affected a greater percentage of slow type I fibers than HS, a small population (approximately 10% of the slow type I fibers) were unaffected by T3. The combined intervention, in contrast, transformed all slow type I fibers into fibers expressing various combinations of fast myosin heavy chain (MHC) isoforms. These findings demonstrate that the soleus muscle does not contain so-called refractory fibers. They further suggest that the soleus muscle contains different populations of slow type I fibers that vary in their sensitivity to altered physiological conditions.

Time course of myosin heavy chain transitions in neonatal rats: importance of innervation and thyroid state.

During the postnatal period, rat limb muscles adapt to weight bearing via the replacement of embryonic (Emb) and neonatal (Neo) myosin heavy chains (MHCs) by the adult isoforms. Our aim was to characterize this transition in terms of the six MHC isoforms expressed in skeletal muscle and to determine the importance of innervation and thyroid hormone status on the attainment of the adult MHC phenotype. Neonatal rats were made hypothyroid via propylthiouracil (PTU) injection. In normal and PTU subgroups, leg muscles were unilaterally denervated at 15 days of age. The MHC profiles of plantaris (PLN) and soleus (Sol) muscles were determined at 7, 14, 23, and 30 days postpartum. At day 7, the Sol MHC profile was 55% type I, 30% Emb, and 10% Neo; in the PLN, the pattern was 60% Neo and 25% Emb. By day 30 the Sol and PLN had essentially attained an adult MHC profile in the controls. PTU augmented slow MHC expression in the Sol, whereas in the PLN it markedly repressed IIb MHC by retaining neonatal MHC expression. Denervation blunted the upregulation of IIb in the PLN and of Type I in the Sol and shifted the pattern to greater expression of IIa and IIx MHCs in both muscles. In contrast to previous observations, these findings collectively suggest that both an intact thyroid and innervation state are obligatory for the attainment of the adult MHC phenotype, particularly in fast-twitch muscles.

Thyroid receptor plasticity in striated muscle types: effects of altered thyroid state.

This study examined nuclear thyroid receptor (TR) maximum binding capacity (Bmax), dissociation constant (Kd), and TR isoform (alpha1, alpha2, beta1) mRNA expression in rodent cardiac, "fast-twitch white," "fast-twitch red," and "slow-twitch red" muscle types as a function of thyroid state. These analyses were performed in the context of slow-twitch type I myosin heavy-chain (MHC) expression, a 3,5,3'-triiodothyronine (T3)-regulated gene that displays varying responsiveness to T3 in the above tissues. Nuclear T3 binding analyses show that the skeletal muscle types express more TRs per unit DNA than cardiac muscle, whereas the latter has a lower Kd than the former. Altered thyroid state had little effect on either cardiac Bmax or Kd, whereas hypothyroidism increased Bmax in the skeletal muscle types without affecting its Kd. Cardiac muscle demonstrated the greatest mRNA signal of TR-beta1 compared with the other muscle types, whereas the TR-alpha1 mRNA signals were more abundant in the skeletal muscle types, especially fast-twitch red. Hyperthyroidism increased the ratio of beta1 to alpha1 and decreased the ratio of alpha2- to alpha1+beta1-mRNA signal across the muscle types, whereas hypothyroidism caused the opposite effects. The nuclear T3 affinity correlated significantly with the TR-beta1 mRNA expression but not with TR-alpha1 mRNA expression. Collectively, these findings suggest that, despite a divergent pattern of TR mRNA expression in the different muscle types, these patterns follow similar qualitative changes under altered thyroid state. Furthermore, TR expression pattern cannot account for the quantitative and qualitative changes in type I MHC expression that occur in the different muscle types.

A longitudinal analysis of lymphocyte proliferative responses to mitogens and antigens during human pregnancy.

T-cell number and mitogen- and antigen-induced lymphocyte proliferative were assessed longitudinally in 18 normal human pregnancies to examine the effects of pregnancy on cellular immunity. The T-cell percentage and mitogen-induced responses did not change significantly in pregnant women as compared to nonpregnant, non-postpartum control adults. However, cell-mediated immune responses to three antigens were dramatically depressed during the third trimester and then returned to early pregnancy levels by 90 days post partum. This reduction in antigen-specific cellular immunity may be necessary to prevent rejection of the histoincompatible fetus by the mother and at the same time may render women in late gestation more susceptible to infection.

Cytomegalovirus-specific humoral and cellular immune responses in human pregnancy.

Cytomegalovirus (CMV)-specific humoral and cellular immunity was evaluated prospectively during and after 19 normal human pregnancies. Seropositive pregnant subjects had lymphocyte proliferative responses to purified CMV antigen that were markedly depressed by the end of the third trimester of pregnancy despite persistent levels of complement-fixing and immunofluorescent antibodies to CMV. These reduced lymphocyte proliferative responses returned to levels detected early in pregnancy by one year after delivery. None of the subjects excreted CMV during the study period. General parameters of cellular immunity, including thymus derived-cell counts as determined by formation of erythrocyte rosettes and mitogen-induced lymphocyte proliferation, were unaffected. Reactivation of latent CMV during pregnancy might be related to transient depression of CMV-specific cellular immunity.

Role of thyroid hormone and insulin in control of cardiac isomyosin expression.

Previous studies show that both diabetes and thyroid state exert a strong regulatory influence on the pattern of rodent cardiac isomyosin expression. Therefore, we determined the separate and combined effects of thyroid hormone (T3) and insulin treatment on rodent cardiac myosin heavy chain (MHC) expression using a model of combined thyroid deficiency (Tx) and diabetes (D). The combination of Tx and D completely transformed cardiac isomyosin expression such that the fast alpha-myosin heavy chain (MHC) was completely repressed at both the protein and mRNA level of expression; whereas, the slow beta-MHC was upregulated to constitute 100% of the total MHC pool, based on both protein and mRNA analyses. Daily low doses of exogenous T3 treatment (3 micrograms/kg b.w. i.p.). in the absence of insulin treatment, partially restored expression of the alpha-MHC, while inhibiting expression of the beta-isoform. In contrast daily insulin treatment (4 U/rat), in the absence of exogenous T3 treatment, failed to exert any significant influence on the pattern of isomyosin expression in the thyroid-deficient diabetic rat model. Furthermore, when exogenous T3 was administered in conjunction with insulin, the effect on MHC mRNA expression was greater than that of T3 alone, thus suggesting the existence of interaction between T3 and insulin action in the regulation of MHC mRNA expression. Collectively, these findings suggest that: (a) thyroid state is a dominant regulator of cardiac isomyosin phenotype: and (b) insulin does not exert any regulatory influence on cardiac MHC expression in a severe thyroid deficient state, instead it requires a critical level of circulating T3 in order to be effective in blunting MHC transformation associated with diabetes. It is thus concluded that the regulation of cardiac MHC by insulin is a complex mechanism involving interaction of insulin with subcellular factors likely to have impact on the specific action of T3. This interaction is disrupted in the absence of sufficient thyroid hormone.