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Biomedical subjects

R W Tsika

Publications and source records attributed to R W Tsika.

At least 19 recordsLinked to original sources
PubMed2001

Multiprotein complex formation at the beta myosin heavy chain distal muscle CAT element correlates with slow muscle expression but not mechanical overload responsiveness.

To examine the role of the beta-myosin heavy chain (betaMyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.

Animals↗
PubMed2001

Magnetic resonance microscopy of morphological alterations in mouse trabecular bone structure under conditions of simulated microgravity.

This work describes the use of magnetic resonance (MR) microscopy to examine changes in tibial trabecular bone structure in mice following 28 days of hindlimb suspension, a model simulating the effects of microgravity in rodents. In this first MR study involving mice, analysis of 3D images showed that apparent bone volume fraction, trabecular number, and trabecular thickness were decreased, and apparent trabecular spacing increased, significantly (P < 0.05) in hindlimb-suspended mice compared to controls. These changes agreed well with light microscopy measurements from an independent study and also with actual spaceflight experiments with rats.

Animals↗
PubMed1999

Nuclear protein binding at the beta-myosin heavy chain A/T-rich element is enriched following increased skeletal muscle activity.

In adult mouse skeletal muscle, beta-myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow-type I fibers but can be induced in fast-type II fibers by mechanical overload (MOV). Our previous transgenic analyses have delimited an 89-base pair (bp) MOV-responsive region (-293 to -205), and shown that mutation of the MCAT and C-rich elements within this region did not abolish betaMyHC transgene induction by MOV. In this study we describe an A/T-rich element (betaA/T-rich; -269 5'-GGAGATATTTTT-3' -258) located within this 89-bp region that, only under MOV conditions, revealed enriched binding as characterized by electrophoretic mobility shift assays and dimethyl sulfate and diethyl pyrocarbonate interference footprinting. Direct, competition, and supershift electrophoretic mobility shift assays revealed highly enriched specific binding activity at the betaA/T-rich element that was antigenically distinct from GATA-4, MEF2A-D, SRF, and Oct-1, nuclear proteins that were previously shown to bind A/T-rich elements. In vitro translated GATA-4, MEF2C, SRF, and Oct-1 bound to consensus GATA, MEF2, SRE, and Oct-1 elements, respectively, but not to the betaA/T-rich element. Two-dimensional UV cross-linking of the bromodeoxyuridine-substituted betaA/T-rich element with mechanically overloaded plantaris (MOV-P) nuclear extract detected two proteins (44 and 48 kDa). Our results indicate that the betaA/T-rich element may function in vivo as a betaMyHC MOV-inducible element during hypertrophy of adult skeletal muscle by binding two distinct proteins identified only in MOV-P nuclear extract.

AT Rich Sequence↗
PubMed1999

Segregated regulatory elements direct beta-myosin heavy chain expression in response to altered muscle activity.

Our previous transgenic analyses revealed that a 600-base pair beta-myosin heavy chain (betaMyHC) promoter conferred mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct betaMyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair betaMyHC promoter fused to chloramphenicol acetyltransferase (beta293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the betaMyHC negative regulatory element (-332 to -300; betaNRE) within transgene beta350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the betaNRE sense strand (dbetaNRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the betaNRE sense strand was antigenically distinct from cellular nucleic acid-binding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV cross-linking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated betaMyHC promoter elements transcriptionally regulate betaMyHC transgene expression in response to two diverse modes of neuromuscular activity.

Animals↗
PubMed1997

beta-MHC transgene expression in suspended and mechanically overloaded/suspended soleus muscle of transgenic mice.

Non-weight-bearing (NWB) activity [space flight and hindlimb suspension (HS)] results in the loss of soleus muscle mass, a slow-to-fast fiber-type conversion, and decreased beta-myosin heavy chain (beta-MHC) protein and mRNA expression. To identify beta-MHC promoter sequences required for decreased beta-MHC expression in response to HS, we have modified an existing noninvasive hindlimb unweighting model to accommodate the use of (transgenic) mice. After 2 wk of HS, body and muscle (soleus > gastrocnemius > plantaris) weights were decreased as was the proportion of histochemically classified type I fibers in HS soleus muscle. Northern blot analysis revealed decreases in endogenous mRNA representing beta-MHC, slow myosin light chain 1 and 2, and cardiac/slow troponin C, whereas those representing skeletal troponin C, muscle creatine kinase, and glyceraldehyde-3-phosphate dehydrogenase increased. Protein extracts prepared from HS soleus (SS) muscle of mice harboring transgenes comprised of 5.6 or 0.6 kilobase of wild type (wt) mouse beta-MHC promoter (beta 5.6 wt, beta 0.6wt) and those carrying the simultaneous mutation (mut) of the MCAT, C-rich, and beta e3 subregions (beta 5.6mut3, beta 0.6mut3) revealed decreases in chloramphenicol acetyltransferase (CAT) specific activity relative to respective controls. Decreased CAT mRNA was observed for transgene beta 5.6mut3, line 85. Two weeks of the simultaneous imposition of mechanical overload (synergist ablation) and HS (MOV/HS) countermanded the loss in absolute and normalized SS weight but did not decrease beta 0.6wt transgene expression. These transgenic results demonstrate that regulatory sequences within a 600-base pair beta-MHC promoter are sufficient to direct decreased transcription of beta-MHC transgenes after 2 wk of HS.

Animals↗
PubMed1996

Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice.

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.

Animals↗
PubMed1996

Induction of beta-MHC transgene in overloaded skeletal muscle is not eliminated by mutation of conserved elements.

Mechanical overload leads to hypertrophy, increased type I fiber composition, and beta-myosin heavy chain (beta-MHC) induction in the fast-twitch plantaris muscle. To better understand the mechanism(s) involved in beta-MHC induction, we have examined inducible expression of transgenes carrying the simultaneous mutation of three DNA regulatory subregions [muscle CAT (MCAT), C-rich, and beta e3] in the context of either 5,600-base pair (bp; beta 5.6mut3) or 600-bp (beta 0.6mut3) beta-MHC promoter in overloaded plantaris muscles of transgenic mice. Protein extract from mechanically overloaded plantaris muscle of mice, harboring either mutant transgene beta 5.6mut3 or beta 0.6mut3, showed an unexpected 2.8- to 4.5-fold increase in chloramphenicol acetyltransferase (CAT) specific activity relative to their respective controls. Similar results were obtained with wild-type (wt) beta-MHC transgenes (beta 5.6wt, beta 0.6wt). Histochemical staining for both myofibrillar ATPase and CAT activity and CAT immunohistochemistry revealed a striking increase in type I fibers and that CAT expression was restricted to these fibers in overloaded plantaris muscle of beta 5.6mut3 transgenic mice. Our transgenic data suggest that beta-MHC transgenes, and perhaps the endogenous beta-MHC gene, are induced by mechanical overload via a mechanism(s) that does not exclusively require the MCAT, C-rich, or beta e3 subregions.

Animals↗
PubMed1996

Muscle-specific and inducible expression of 293-base pair beta-myosin heavy chain promoter in transgenic mice.

The DNA regulatory element(s) involved in beta-myosin heavy chain (beta-MHC) induction by the physiological stimulus of mechanical overload have not been identified as yet. To delineate regulatory sequences that are required for mechanical overload induction of the beta-MHC gene, transgenic mouse lines were generated that harbor transgenes containing serial deletions of the human beta-MHC promoter to nucleotides -293 (beta 293), -201 (beta 201), and -141 (beta 141) from the transcription start site (+1). Mechanically overloaded adult plantaris and soleus muscles contained 11- and 1.9-fold increases, respectively, in endogenous beta-MHC-specific mRNA transcripts (Northern blot) compared with sham-operated controls. Expression assays (chloramphenicol acetyltransferase specific activity) revealed that only transgene beta 293 expression was muscle specific in both fetal and adult mice and was induced in the plantaris (10- to 27-fold) and soleus (2- to 2.5-fold) muscles by mechanical overload. Histochemical staining for myosin adenosinetriphosphatase activity revealed a fiber-type transition of type II to type I in the overloaded plantaris and soleus muscles. These transgenic data suggest that sequences located between nucleotides -293 and +120 may be sufficient to regulate the endogenous beta-MHC gene in response to developmental signals and to the physiological signals generated by mechanical overload in fast- and slow-twitch muscles.

Adenosine Triphosphatases↗
PubMed1995

M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice.

The molecular pathways and regulatory molecules that underlie changes in gene transcription during mechanical overload of skeletal muscle remain obscure. To better understand this process, we have examined mouse muscle creatine kinase (MCK) gene expression in mechanically overloaded plantaris (OP) muscle of transgenic and nontransgenic mice. Northern blot analysis revealed that endogenous MCK-specific mRNA transcripts were decreased 150% in the OP muscles after 6 wk. To identify the MCK gene regions involved in the response to mechanical overload, three different mouse MCKCAT transgenes were studied by measuring chloramphenicol acetyltransferase (CAT assays) activity in OP and sham-operated (control plantaris) muscles. Mouse lines carrying (+enh206)117MCKCAT and -1256MCKCAT transgenes exhibited 30 and 40% lower CAT levels, whereas two mouse lines carrying -3300MCKCAT transgenes exhibited average decreases of 430%. Nearly identical results, including measurements of exogenous CAT mRNA, were obtained 2 days postoverload. Six weeks or 2 days of mechanical overload led to an average decrease in MM-CK isoprotein of 140%. These data provide evidence that mechanical overload induces changes in MCK gene expression that appear to be regulated by at least two portions of the MCK gene: the 206 base pair 5' enhancer and the -3,300 to -1,257 region.

Animals↗
PubMed1994

Transgenic animal models.

Exercise adaptations induce the differential expression of genes that encode components of the contractile apparatus, metabolic pathways, organelle systems, and membrane components. These changes in gene expression are presumably brought about by the activation of intracellular signaling pathways. The use of transgenic technology is particularly well suited for exercise studies because it uniquely allows investigators to evaluate gene regulatory mechanisms in the intact animal at all stages of the life cycle. Transgenic mice can be generated that harbor any of the varied genes involved in exercise adaptation, for the purpose of determining what sequences within this gene regulate the response to any exercise regimen. Once the corresponding transcriptional factor(s) have been identified and the gene(s) cloned, a series of similar studies (gene networking) could be undertaken to determine the signal transduction pathway. Alternatively, transgenics can be produced that overexpress a particular protein or express an isoform of a protein in a tissue where it is not normally expressed. The goal of these studies would be to determine if function is improved and if exercise adaptation is enhanced or facilitated. Ultimately, gene targeting can be used to either mutate or knock out a gene to determine whether its gene product is indispensable for function, exercise performance, and/or adaptation.

Adenoviruses, Human↗
PubMed1992

Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle.

Lipoprotein lipase (LPL) is anchored with high affinity to heparan sulphate proteoglycans on the luminal surface of the capillary endothelium. The levels of pre-heparin perfusate LPL activity increased from 16 +/- 1 to 145 +/- 6 U/hindlimb (nine-fold increase) in hindlimb muscle of exercise-trained rats measured immediately after the last bout of work. At the same time, post-heparin perfusate LPL activity decreased from 63 +/- 2 to 13 +/- 1 U/hindlimb (p less than 0.001). These results provide evidence that exercise-training has a heparin-like effect on capillary-bound LPL. The total amount of LPL (i.e., pre-heparin perfusate plus post-heparin perfusate) was twofold greater in the hindlimb of the trained animals versus the controls. The effect of exercise on muscle LPL activity appears to last for as long as 5 days after cessation of exercise. Serum triglycerides were reduced 38% and plasma free fatty acids increased fourfold. These results provide evidence that training increases the capacity to remove triglycerides from circulation.

Adipose Tissue↗
PubMed1990

An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter.

The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.

Animals↗
PubMed1990

Thyroid hormone regulates expression of a transfected human alpha-myosin heavy-chain fusion gene in fetal rat heart cells.

The rat alpha-myosin heavy-chain (alpha-MHC) gene is regulated by 3,5,3'-triiodo-L-thyronine (T3) in ventricular myocardium and is constitutively expressed in atrial tissue. Less is known about regulation of the human gene, but conservation of sequences in the 5'-flanking region between the rat and human alpha-MHC genes suggests that the human gene may be regulated similarly. Accordingly, T3-responsiveness and tissue-specific expression of human and rat alpha-MHC/chloramphenicol acetyltransferase fusion constructs have been compared in rat fetal heart cells, L6E9 myoblasts and myotubes, 3T3 fibroblasts, and HeLa cells. Transient transfection assays revealed a complex series of cis-regulatory elements in the 5'-flanking sequences in the human genes, including a basal promoter element with canonical TATAA and CAAT sequences, two positive regulatory element(s), and two negative regulatory elements, which markedly diminished both constitutive and T3-inducible activity. Interestingly, the human gene seemed to contain a proximal thyroid-hormone response element(s) not found in the rat gene. In L6E9 myoblasts and myotubes, the human constructs were constitutively expressed but not T3-regulated; none of the constructs were active in 3T3 or HeLa cells. We propose that interactions among the thyroid hormone responsive elements and other cis-acting elements in the human alpha-MHC 5'-flanking sequences may be sufficient to explain the characteristic features of expression of this gene in cardiac tissues.

Animals↗
PubMed1987

Interaction of compensatory overload and hindlimb suspension on myosin isoform expression.

The purpose of this study was to investigate the role of chronic weight-bearing activity as the primary inducer of compensatory muscle growth and changes in myosin isoform expression in rodent fast-twitch plantaris muscle. Thus, female rats were subjected to the independent and simultaneous exposure of functional overload (induced via synergist removal) and hindlimb unweighting (suspension) for 6 wk. Groups (n = 7/group) consisted of normal-control (NC); overload (OV); normal-suspension (N-SUS); and overload-suspension (OV-SUS). Body weight of both suspension groups was significantly less than both the NC and OV groups (P less than 0.001). Compared with the NC group, normalized plantaris weight (mg/g body wt) of both the OV and OV-SUS groups was greater, whereas that of the N-SUS was lower (P less than 0.001). However, normalized plantaris weight was greater in OV compared with OV-SUS by 35% (P less than 0.001). Myofibril protein content (mg/g) and Ca2+-regulated myofibril adenosinetriphosphatase (ATPase) specific activity were similar for all groups except that ATPase was lower in the OV group compared with the other groups (P less than 0.05). Native myosin isoform analysis revealed a significant increase in the expression of slow and intermediate myosin and the repression of fast myosin 1 (Fm1) in OV compared with NC. This shift in expression was not as pronounced in the OV-SUS group. Interestingly, only traces of slow myosin were observed in the N-SUS group compared with the other groups. These results suggest that weight bearing is an essential component of the overload model for inducing significant increases in both muscle mass and slow myosin isoform expression. Second, lack of weight bearing, while not markedly affecting fast myosins, appears to repress the expression of slow myosin.

Animals↗
PubMed1987

Subunit composition of rodent isomyosins and their distribution in hindlimb skeletal muscles.

Three adult skeletal muscle sarcomeric myosin heavy chain (MHC) genes have been identified in the rat, suggesting that the expressed native myosin isoforms can be differentiated, in part, on the basis of their MHC composition. This study was undertaken to ascertain whether the five major native isomyosins [3 fast (Fm1, Fm2, Fm3), 1 slow (Sm), and 1 intermediate (Im)], typically expressed in the spectrum of adult rat skeletal muscles comprising the hindlimb, could be further differentiated on the basis of their MHC profiles in addition to their light chain composition. Results show that in muscles comprised exclusively of fast-twitch glycolytic (FG) fibers and consisting of Fm1, Fm2, and Fm3, such as the tensor fasciae latae, only one MHC, designated as fast type IIb, could be resolved. In soleus muscle, comprised of both slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers and expressing Sm and Im, two MHC bands were resolved and designated as slow/cardiac beta-MHC and fast type IIa MHC. In muscles expressing a mixture of all three fiber types and a full complement of isomyosins, as seen in the plantaris, the MHC could be resolved into three bands. Light chain profiles were characterized for each muscle type, as well as for the purified isomyosins. These data suggest that Im (IIa) consists of a mixture of fast and slow light chains, whereas Fm (IIb) and Sm (beta) isoforms consist solely of fast- and slow-type light chains, respectively. Polypeptide mapping of denatured myosin extracted from muscles expressing contrasting isoform phenotypes suggests differences in the MHC primary structure between slow, intermediate, and fast myosin isotypes. These findings demonstrate that 1) Fm, Im, and Sm isoforms are differentiated on the bases of both their heavy and light chain components and 2) each isomyosin is distributed in a characteristic fashion among rat hindlimb skeletal muscles. Furthermore, these data suggest that the ratio of isomyosins in a given muscle or muscle region is of physiological importance to the function of that muscle during muscular activity.

Animals↗
PubMed1987

Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression.

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.

Adaptation, Physiological↗