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Biomedical subjects

K M Baldwin

Publications and source records attributed to K M Baldwin.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of antisense RNA expression during cardiac MHC gene switching in response to pressure overload.

Hypertension has been shown to cause cardiac hypertrophy and a shift in myosin heavy chain (MHC) gene expression from the faster alpha- to slower beta-MHC isoform. The expression of the beta- and alpha-MHC pre-mRNAs, mRNAs, as well as the newly discovered antisense beta-RNA were analyzed in three regions of the normal control (NC) and 12-day pressure-overloaded (AbCon) hearts: the left ventricle apex, left ventricle base, and the septum. The RNA analyses in the AbCon heart targeted both the 5' and the 3' ends of each RNA molecule. beta-MHC mRNA expression significantly increased relative to control in all three regions, regardless of the target site (5' or 3' end). In contrast, beta-MHC pre-mRNA expression in the AbCon heart depended on the site of the measurement (5' vs. 3' end). For example, whereas the pre-mRNA did not change when targeted at the 3' end (last intron), it increased significantly in the AbCon heart when measurement targeted the 5' end (2nd intron) of the 25-kb molecule. Analyses of the antisense beta-RNA revealed that its expression in the AbCon heart was significantly decreased relative to control regardless of its measurement site. A negative correlation was observed between the beta-mRNA expression and the antisense beta-RNA (P < 0.05), suggesting an inhibitory role of antisense RNA on the sense beta-MHC gene expression. In contrast, a positive correlation was observed between the antisense beta-RNA and the alpha-MHC pre-mRNA (P < 0.05). This latter observation along with the alpha-MHC gene position relative to that of the beta-antisense suggest that the alpha-MHC sense and beta-antisense transcription are coregulated likely via common intergenic regulatory sequences. Our results suggest that the increased beta-MHC expression in the AbCon heart not only is the result of increased beta-MHC transcription but also involves an antisense beta-RNA regulation scheme. Although the exact mechanism concerning antisense regulation is not clear, it could involve modulation of both transcriptional activity of the beta-MHC gene and posttranscriptional processing.

Animals↗
PubMed2005

Isometric resistance exercise fails to counteract skeletal muscle atrophy processes during the initial stages of unloading.

This study tested the hypothesis that an isometric resistance training paradigm targeting the medial gastrocnemius of adult rodents is effective in preventing muscle atrophy during the early stages of hindlimb unloading by maintaining normal activation of the insulin receptor substrate-1 (IRS-1)/phosphoinositide-3 kinase (PI3K)/Akt signaling pathway. This pathway has been shown to simultaneously create an anabolic response while inhibiting processes upregulating catabolic processes involving expression of key enzymes in the ubiquitination of proteins for degradation. The findings show that during the 5 days of unloading 1) absolute medial gastrocnemius muscle weight reduction occurred by approximately 20%, but muscle weight corrected to body weight was not different from normal weight-bearing controls (P < 0.05); 2) normalized myofibril fraction concentration and content were decreased; and 3) a robust isometric training program, known to induce a hypertrophy response, failed to maintain the myofibril protein content. This response occurred despite fully blunting the increases in the mRNA for of atrogin-1, MURF-1, and myostatin, e.g., sensitive gene markers of an activated catabolic state. Analyses of the IRS-1/PI3K/Akt markers indicated that abundance of IRS-1 and phosphorylation state of Akt and p70S6 kinase were decreased relative to normal control rats, and the resistance training failed to maintain these signaling markers at normal regulatory level. Our findings suggest that to fully prevent muscle atrophy responses affecting the myofibril system during unloading, the volume of mechanical stress must be augmented sufficiently to maintain optimal activity of the IRS-1/PI3K/Akt pathway to provide an effective anabolic stimulus on the muscle.

Animals↗
PubMed2004

Pretranslational markers of contractile protein expression in human skeletal muscle: effect of limb unloading plus resistance exercise.

Previously, it has been shown that the human ground-based model consisting of unilateral limb suspension (ULLS) induces atrophy and reduced strength of the affected quadriceps muscle group. Resistance exercise (RE) involving concentric-eccentric actions, in the face of ULLS, is effective in ameliorating these deficits. The goal of the present study was to determine whether alterations in contractile protein gene expression, e.g., myosin heavy chain and actin, as studied at the pretranslational level, provide molecular markers concerning the deficits that occur in muscle mass/volume during ULLS, as well as its maintenance in response to ULLS plus RE. Muscle biopsies were obtained from the vastus lateralis muscle of 31 middle-aged men and women before and after 5 wk of ULLS, ULLS plus RE, or RE only. The RE paradigm consisted of 12 sessions of 4 sets of 7 concentric-eccentric knee extensions. Our findings show that there were net deficits in total RNA, total mRNA, and actin and myosin heavy chain mRNA levels of expression after ULLS (P < 0.05), whereas these alterations were blunted in the two groups receiving RE. Additional observations involving IGF-I and its associated receptor and binding proteins suggest that RE postures the skeletal muscle for signaling processes that favor a greater anabolic state relative to that observed in the ULLS group. Collectively, these findings suggest that molecular markers of contractile protein gene expression serve as useful subcellular indicators for ascertaining the underlying mechanisms regulating alterations in muscle mass in human subjects in response to altered loading states.

Adult↗
PubMed2003

Transcriptional regulation of IGF-I expression in skeletal muscle.

The present study investigated the role of transcription in the regulation of insulin-like growth factor (IGF)-I expression in skeletal muscle. RT-PCR was used to determine endogenous expression of IGF-I pre-mRNA and mRNA in control (Con) and functionally overloaded (FO) rat plantaris. The transcriptional activities of five different-length IGF-I promoter fragments controlling transcription of a firefly luciferase (FLuc) reporter gene were tested in vitro by transfection of myoblasts or in vivo during FO by direct gene transfer into the plantaris. Increased endogenous IGF-I gene transcription during 7 days of plantaris FO was evidenced by an approximately 140-160% increase (P < 0.0001) in IGF-I pre-mRNA (a transcriptional marker). IGF-I mRNA expression also increased by approximately 90% (P < 0.0001), and it was correlated (R = 0.93; P < 0.0001) with the pre-mRNA increases. The three longest IGF-I exon 1 promoters induced reporter gene expression in proliferating C2C12 and L6E9 myoblasts. In differentiated L6E9 myotubes, promoter activity increased approximately two- to threefold over myoblasts. Overexpression of calcineurin and MyoD increased the activity of the -852/+192 promoter in C2C12 myotubes by approximately 5- and approximately 18-fold, respectively. However, FO did not induce these exogenous promoter fragments. Nevertheless, the present findings are consistent with the hypothesis that the IGF-I gene is transcriptionally regulated during muscle hypertrophy in vivo as evidenced by the induction of the endogenous IGF-I pre-mRNA during plantaris FO. The exon 1 promoter region of the IGF-I gene is sufficient to direct inducible expression in vitro; however, an in vivo response to FO may require elements outside the -852/+346 region of the exon 1 IGF-I promoter or features inherent to the endogenous IGF-I gene.

Animals↗
PubMed2003

Atrophy responses to muscle inactivity. I. Cellular markers of protein deficits.

The goal of this study was to use the model of spinal cord isolation (SI), which blocks nearly all neuromuscular activity while leaving the motoneuron muscle-fiber connections intact, to characterize the cellular processes linked to marked muscle atrophy. Rats randomly assigned to normal control and SI groups were studied at 0, 2, 4, 8, and 15 days after SI surgery. The slow soleus muscle atrophied by approximately 50%, with the greatest degree of loss occurring during the first 8 days. Throughout the SI duration, muscle protein concentration was maintained at the control level, whereas myofibrillar protein concentration steadily decreased between 4 and 15 days of SI, and this was associated with a 50% decrease in myosin heavy chain (MHC) normalized to total protein. Actin relative to the total protein was maintained at the control level. Marked reductions occurred in total RNA and DNA content and in total MHC and actin mRNA expressed relative to 18S ribosomal RNA. These findings suggest that two key factors contributing to the muscle atrophy in the SI model are 1). a reduction in ribosomal RNA that is consistent with a reduction in protein translational capacity, and 2). insufficient mRNA substrate for translating key sarcomeric proteins comprising the myofibril fraction, such as MHC and actin. In addition, the marked selective depletion of MHC protein in the muscles of SI rats suggests that this protein is more vulnerable to inactivity than actin protein. This selective MHC loss could be a major contributor for the previously reported loss in the functional integrity of SI muscles. Collectively, these data are consistent with the involvement of pretranslational and translational processes in muscle atrophy due to SI.

Actins↗
PubMed2003

Atrophy responses to muscle inactivity. II. Molecular markers of protein deficits.

We examined the expression of several molecular markers of protein balance in response to skeletal muscle atrophy induced by spinal cord isolation (SI; i.e., a complete transection of the spinal cord at both a midthoracic and a high sacral level plus complete deafferentation between the two transection sites). This treatment nearly eliminates neuromuscular activity (activation and loading) of the hindlimb muscles while maintaining neuromuscular connectivity. SI was associated with a reduced transcriptional activity (via pre-mRNA analyses) of myosin heavy chain (MHC) and actin. In addition, there was an increased gene expression of enzyme systems impacting protein degradation (calpain-1; plus enzymes associated with polyubquitination processes) that could further contribute to the protein deficits in the SI muscles via degradative pathways. IGF-I receptor and binding protein-5 mRNA expression was induced throughout the 15-day period of SI, whereas IGF-I mRNA was induced at 8 and 15 days. These responses occurred in the absence of an upregulation of translational regulatory proteins (p70 S6 kinase; eukaryotic 4E binding protein 1) to compensate for the decreased protein translational capacity. These data collectively demonstrate that 1). the molecular changes accompanying SI-induced muscle atrophy are not necessarily the reverse of those occurring during muscle hypertrophy, and 2). the rapid and marked atrophy that defines this model of muscle inactivity is likely the result of multifactorial processes affecting transcription, translation, and protein degradation.

Actins↗
PubMed2002

Transcriptional regulation of the type I myosin heavy chain gene in denervated rat soleus.

Denervation (DEN) of rat soleus is associated with a decreased expression of slow type I myosin heavy chain (MHC) and an increased expression of the faster MHC isoforms. The molecular mechanisms behind these shifts remain unclear. We first investigated endogenous transcriptional activity of the type I MHC gene in normal and denervated soleus muscles via pre-mRNA analysis. Our results suggest that the type I MHC gene is regulated via transcriptional processes in the denervated soleus. Deletion and mutational analysis of the rat type I MHC promoter was then used to identify cis elements or regions of the promoter involved in this response. DEN significantly decreased in vivo activity of the -3,500, -2,500, -914, -408, -299, and -215 bp type I MHC promoters, relative to the alpha-skeletal actin promoter. In contrast, normalized -171 promoter activity was unchanged. Mutation of the betae3 element (-214/-190) in the -215 promoter and deletion of this element (-171 promoter) blunted type I downregulation with DEN. In contrast, betae3 mutation in the -408 promoters was not effective in attenuating the DEN response, suggesting the existence of additional DEN-responsive sites between -408 and -215. Western blotting and gel mobility supershift assays demonstrated decreased expression and DNA binding of transcription enhancer factor 1 (TEF-1) with DEN, suggesting that this decrease may contribute to type I MHC downregulation in denervated muscle.

Animals↗
PubMed2001

Temporal effects of inactivty on myosin heavy chain gene expression in rat slow muscle.

Myosin heavy chain (MHC) mRNA and protein profiles in adult rat soleus and adductor longus were determined after 4, 8, 15, 30, 60, and 90 days of spinal cord isolation (SI). SI results in complete neuromuscular inactivity while leaving the motoneuron-muscle fiber connections intact. From 15 to 90 days, type I MHC mRNA was significantly decreased, whereas type I MHC protein did not significantly decrease until 30 and 60 days in the soleus and adductor longus, respectively. However, in both muscles, slow MHC downregulation was offset by significant upregulation of the faster MHC isoforms, primarily IIx. From 60 to 90 days, type I MHC was almost completely replaced with faster isoforms at the mRNA and protein levels. Thus, chronic inactivity and unloading of slow rat hindlimb muscles shifted the MHC profile from predominately type I to type IIx MHC mRNA and protein.

Animals↗
PubMed2001

The role of stem cells in midgut growth and regeneration.

The Manduca sexta (L.) [Lepidoptera: Sphingidae] and Heliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation.

Animals↗
PubMed2001

In vivo regulation of the beta-myosin heavy chain gene in hypertensive rodent heart.

The main goal of this study was to examine the transcriptional activity of different-length beta-myosin heavy chain (beta-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by approximately 45% relative to control. Results show that beta-MHC promoter activity of all tested wild-type constructs, i.e., -3500, -408, -299, -215, -171, and -71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the -71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting betae1, betae2, GATA, and betae3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the beta-MHC gene. Collectively, these results indicate that three separate regions on the beta-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between -408 and -3500 bp, 2) a proximal region between -299 and -215 bp, and 3) a basal region within -71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate beta-MHC transcriptional regulation in response to pressure overload.

Animals↗
PubMed2001

Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle.

The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of heterogeneity among the individual fibers would ensure a large functional diversity in performing complex movement patterns. Future studies must now focus on 1) the signaling pathways and the underlying mechanisms governing the transcriptional/translational machinery that control this marked degree of plasticity and 2) the morphological organization and functional implications of the muscle fiber's capacity to express such a diversity of motor proteins.

Animals↗
PubMed2000

MHC polymorphism in rodent plantaris muscle: effects of mechanical overload and hypothyroidism.

In a previous study, it was shown that a combined treatment of hyperthyroidism and hindlimb suspension effectively converted the slow-twitch soleus muscle to a fast-twitch muscle. The objective of this study was to test the hypothesis that hypothyroidism [absence of triiodothyronine (-T(3))] and mechanical overload (OV) would convert the plantaris (Plan) muscle from a fast- to a slow-twitch muscle. Single-fiber analyses demonstrated that the normal rodent Plan muscle was composed of approximately 13 different fiber types as defined by myosin heavy chain (MHC) isoform content. The largest proportion of fibers ( approximately 35%) coexpressed the fast type IIX and IIB MHC isoforms (i.e., type IIX/IIB fibers). In this context, the combined intervention of -T(3) and OV produced a significant reduction in the relative proportion of the fast type IIB MHC isoform and a concomitant increase in the slow type I MHC isoform. These transitions were manifested by a large decrease in the proportion of type IIX/IIB fibers and a large increase in fibers coexpressing all four MHC protein isoforms. The mechanical consequences of these transitions, however, were modest, producing a 15% decrease in maximal shortening velocity. The findings of this study demonstrate that -T(3) + OV does produce a partial shift toward a slower phenotype; however, the high degree of polymorphism found in the Plan muscle represents a unique design that appears to minimize the functional consequences of these significant MHC transitions.

Animals↗
PubMed2000

In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats.

In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

Animals↗
PubMed2000

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism.

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

Age Factors↗