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1.  Alterations in lung gene expression in streptozotocin-induced diabetic rats 
Diabetes profoundly affects gene expression in organs such as heart, skeletal muscle, kidney and liver, with areas of perturbation including carbohydrate and lipid metabolism, oxidative stress, and protein ubiquitination. Type 1 diabetes impairs lung function, but whether gene expression alterations in the lung parallel those of other tissue types is largely unexplored.
Lung from a rat model of diabetes mellitus induced by streptozotocin was subjected to gene expression microarray analysis.
Glucose levels were 67 and 260 mg/dl (p < 0.001) in control and diabetic rats, respectively. There were 46 genes with at least ± 1.5-fold significantly altered expression (19 increases, 27 decreases). Gene ontology groups with significant over-representation among genes with altered expression included apoptosis, response to stress (p = 0.03), regulation of protein kinase activity (p = 0.04), ion transporter activity (p = 0.01) and collagen (p = 0.01). All genes assigned to the apoptosis and response to stress groups had increased expression whereas all genes assigned to the collagen group had decreased expression. In contrast, the protein kinase activity and ion transporter activity groups had genes with both increased and decreased expression.
Gene expression in the lung is affected by type 1 diabetes in several specific areas, including apoptosis. However, the lung is resistant to changes in gene expression related to lipid and carbohydrate metabolism and oxidative stress that occur in other tissue types such as heart, skeletal muscle and kidney.
PMCID: PMC3945062  PMID: 24423257
Type 1 diabetes; Lung; Gene expression
2.  Gene expression of sternohyoid and diaphragm muscles in type 2 diabetic rats 
Type 2 diabetes differs from type 1 diabetes in its pathogenesis. Type 1 diabetic diaphragm has altered gene expression which includes lipid and carbohydrate metabolism, ubiquitination and oxidoreductase activity. The objectives of the present study were to assess respiratory muscle gene expression changes in type 2 diabetes and to determine whether they are greater for the diaphragm than an upper airway muscle.
Diaphragm and sternohyoid muscle from Zucker diabetic fatty (ZDF) rats were analyzed with Affymetrix gene expression arrays.
The two muscles had 97 and 102 genes, respectively, with at least ± 1.5-fold significantly changed expression with diabetes, and these were assigned to gene ontology groups based on over-representation analysis. Several significantly changed groups were common to both muscles, including lipid metabolism, carbohydrate metabolism, muscle contraction, ion transport and collagen, although the number of genes and the specific genes involved differed considerably for the two muscles. In both muscles there was a shift in metabolism gene expression from carbohydrate metabolism toward lipid metabolism, but the shift was greater and involved more genes in diabetic diaphragm than diabetic sternohyoid muscle. Groups present in only diaphragm were blood circulation and oxidoreductase activity. Groups present in only sternohyoid were immune & inflammation and response to stress & wounding, with complement genes being a prominent component.
Type 2 diabetes-induced gene expression changes in respiratory muscles has both similarities and differences relative to previous data on type 1 diabetes gene expression. Furthermore, the diabetic alterations in gene expression differ between diaphragm and sternohyoid.
PMCID: PMC3851765  PMID: 24199937
3.  The Effects of K+ Channel Blockade on Eccentric and Isotonic Twitch and Fatiguing Contractions in situ 
K+ channel blockers like 3,4-diaminopyridine (DAP) can double isometric muscle force. Functional movements require more complex concentric and eccentric contractions, however the effects of K+ channel blockade on these types of contractions in situ are unknown. Extensor digitorum longus (EDL) muscles were stimulated in situ with and without DAP in anesthetized rats and fatigability was addressed using a series of either concentric or eccentric contractions. During isotonic protocols (5–100% load), DAP significantly shifted shortening- and maximum shortening velocity-load curves upward and to the right and increased power and work. Maximum shortening, maximum shortening velocity, and power doubled while work increased by ∼250% during isotonic contraction at 50% load. During isotonic fatigue, DAP significantly augmented maximum shortening, work, shortening velocity, and power. During constant velocity eccentric protocols (2–12 mm/s), DAP increased muscle force during eccentric contractions at 6, 8, 10, and 12 mm/s. During eccentric contraction at a constant velocity of 6 mm/s while varying the stimulation frequency, DAP significantly increased muscle force during 20, 40, and 70 Hz. The effects of DAP on muscle contractile performance during eccentric fatigue varied with level of fatigue. DAP-induced contractile increases during isotonic contractions were similar to those produced during previously studied isometric contractions, while the DAP effect during eccentric contractions was more modest. These findings are especially important in attempting to optimize functional electrical stimulation parameters for spinal cord injury patients while also preventing rapid fatigue of those muscles.
PMCID: PMC3460371  PMID: 23060809
3,4-diaminopyridine; eccentric contraction; isotonic contraction; EDL; fatigue
4.  Impaired Wheel Running Exercise in CLC-1 Chloride Channel-Deficient Myotonic Mice 
Background: Genetic deficiency of the muscle CLC-1 chloride channel leads to myotonia, which is manifested most prominently by slowing of muscle relaxation. Humans experience this as muscle stiffness upon initiation of contraction, although this can be overcome with repeated efforts (the “warm-up” phenomenon). The extent to which CLC-1 deficiency impairs exercise activity is controversial. We hypothesized that skeletal muscle CLC-1 chloride channel deficiency leads to severe reductions in spontaneous exercise. Methodology/Principal Findings: To examine this quantitatively, myotonic CLC-1 deficient mice were provided access to running wheels, and their spontaneous running activity was quantified subsequently. Differences between myotonic and normal mice in running were not present soon after introduction to the running wheels, but were fully established during week 2. During the eighth week, myotonic mice were running significantly less than normal mice (322 ± 177 vs 5058 ± 1253 m/day, P = 0.025). Furthermore, there were considerable reductions in consecutive running times (18.8 ± 1.5 vs 59.0 ± 3.7 min, P < 0.001) and in the distance per consecutive running period (58 ± 38 vs 601 ± 174 m, P = 0.048) in myotonic compared with normal animals. Conclusion/Significance: These findings indicate that CLC-1 chloride deficient myotonia in mice markedly impairs spontaneous exercise activity, with reductions in both total distance and consecutive running times.
PMCID: PMC3152724  PMID: 21886624
myotonia congenita; exercise; genetic; CLC-1 chloride channel; skeletal muscle
5.  Expression of a Dominant Negative CELF Protein In Vivo Leads to Altered Muscle Organization, Fiber Size, and Subtype 
PLoS ONE  2011;6(4):e19274.
CUG-BP and ETR-3-like factor (CELF) proteins regulate tissue- and developmental stage-specific alternative splicing in striated muscle. We previously demonstrated that heart muscle-specific expression of a nuclear dominant negative CELF protein in transgenic mice (MHC-CELFΔ) effectively disrupts endogenous CELF activity in the heart in vivo, resulting in impaired cardiac function. In this study, transgenic mice that express the dominant negative protein under a skeletal muscle-specific promoter (Myo-CELFΔ) were generated to investigate the role of CELF-mediated alternative splicing programs in normal skeletal muscle.
Methodology/Principal Findings
Myo-CELFΔ mice exhibit modest changes in CELF-mediated alternative splicing in skeletal muscle, accompanied by a reduction of endomysial and perimysial spaces, an increase in fiber size variability, and an increase in slow twitch muscle fibers. Weight gain and mean body weight, total number of muscle fibers, and overall muscle strength were not affected.
Although these findings demonstrate that CELF activity contributes to the normal alternative splicing of a subset of muscle transcripts in vivo, the mildness of the effects in Myo-CELFΔ muscles compared to those in MHC-CELFΔ hearts suggests CELF activity may be less determinative for alternative splicing in skeletal muscle than in heart muscle. Nonetheless, even these small changes in CELF-mediated splicing regulation were sufficient to alter muscle organization and muscle fiber properties affected in myotonic dystrophy. This lends further evidence to the hypothesis that dysregulation of CELF-mediated alternative splicing programs may be responsible for the disruption of these properties during muscle pathogenesis.
PMCID: PMC3082560  PMID: 21541285
6.  Fatigue-inducing stimulation resolves myotonia in a drug-induced model 
BMC Physiology  2011;11:5.
Slowed muscle relaxation is the contractile hallmark of myotonia congenita, a disease caused by genetic CLC-1 chloride channel deficiency, which improves with antecedent brief contractions ("warm-up phenomenon"). It is unclear to what extent the myotonia continues to dissipate during continued repetitive contractions and how this relates temporally to muscle fatigue. Diaphragm, EDL, and soleus muscles were examined in vitro during repetitive 20 Hz and 50 Hz train stimulation in a drug-induced (9-AC) rat myotonia model.
At the onset of stimulation, 9-AC treated diaphragm and EDL muscle had markedly prolonged half relaxation and late relaxation times (range 147 to 884 ms, 894 to 1324 ms). Half relaxation and late relaxation times reached near-normal values over the 5-10 and 10-40 subsequent contractions, respectively. In both muscles myotonia declined faster during repetitive 50 Hz than 20 Hz stimulation, and much faster than the rate of force loss during fatigue at both frequencies. Soleus muscle was resistant to the myotonic effects of 9-AC.
In a drug-induced model of mechanical myotonia, fatigue-inducing stimulation resolves the myotonia, which furthermore appears to be independent from the development of muscle fatigue.
PMCID: PMC3052176  PMID: 21356096
7.  Improvement of diaphragm and limb muscle isotonic contractile performance by K+ channel blockade 
The K+ channel blocking aminopyridines greatly improve skeletal muscle isometric contractile performance during low to intermediate stimulation frequencies, making them potentially useful as inotropic agents for functional neuromuscular stimulation applications. Most restorative applications involve muscle shortening; however, previous studies on the effects of aminopyridines have involved muscle being held at constant length. Isotonic contractions differ substantially from isometric contractions at a cellular level with regards to factors such as cross-bridge formation and energetic requirements. The present study tested effects of 3,4-diaminopyridine (DAP) on isotonic contractile performance of diaphragm, extensor digitorum longus (EDL) and soleus muscles from rats. During contractions elicited during 20 Hz stimulation, DAP improved work over a range of loads for all three muscles. In contrast, peak power was augmented for the diaphragm and EDL but not the soleus. Maintenance of increased work and peak power was tested during repetitive fatigue-inducing stimulation using a single load of 40% and a stimulation frequency of 20 Hz. Work and peak power of both diaphragm and EDL were augmented by DAP for considerable periods of time, whereas that of soleus muscle was not affected significantly. These results demonstrate that DAP greatly improves both work and peak power of the diaphragm and EDL muscle during isotonic contractions, which combined with previous data on isometric contractions indicates that this agent is suitable for enhancing muscle performance during a range of contractile modalities.
PMCID: PMC2821379  PMID: 20064261
8.  Gene Expression Profiling in the Type 1 Diabetes Rat Diaphragm 
PLoS ONE  2009;4(11):e7832.
Respiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression.
Methodology/Principal Findings
Diaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least ±2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change −2.0 to −8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change −2.2 to −3.7) and organogenesis (P = 0.032, n = 7, fold change −2.1 to −3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested.
These data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in carbohydrate and lipid metabolism may change the availability of energetic substrates and thereby directly modulate fatigue resistance, an important issue for a muscle like the diaphragm which needs to contract without rest for the entire lifetime of the organism.
PMCID: PMC2773011  PMID: 19915678

Results 1-8 (8)