HLA class II diversity in HIV-1 uninfected individuals from the placebo arm of the RV144 Thai vaccine efficacy trial.

The RV144 HIV vaccine trial in Thailand elicited antibody responses to the envelope of HIV-1, which correlated significantly with the risk of HIV-1 acquisition. Human leukocyte antigen (HLA) class II molecules are essential in antigen presentation to CD4 T cells for activation of B cells to produce antibodies. We genotyped the classical HLA-DRB1, DQB1, and DPB1 genes in 450 individuals from the placebo arm of the RV144 study to determine the background allele and haplotype frequencies of these genes in this cohort. High-resolution 4 and 6-digit class II HLA typing data was generated using sequencing-based methods. The observed diversity for the HLA loci was 33 HLA-DRB1, 15 HLA-DQB1, and 26 HLA-DPB1 alleles. Common alleles with frequencies greater than 10% were DRB1*07:01, DRB1*09:01, DRB1*12:02, DRB1*15:02, DQB1*02:01/02, DQB1*03:01, DQB1*03:03, DQB1*05:01, DQB1*05:02, DPB1*04:01:01, DPB1*05:01:01, and DPB1*13:01:01. We identified 28 rare alleles with frequencies of less than 1% in the Thai individuals. Ambiguity for HLA-DPB1*28:01 in exon 2 was resolved to DPB1*296:01 by next-generation sequencing of all exons. Multi-locus haplotypes including HLA class I and II loci were reported in this study. This is the first comprehensive report of allele and haplotype frequencies of all three HLA class II genes from a Thai population. A high-resolution genotyping method such as next-generation sequencing avoids missing rare alleles and resolves ambiguous calls. The HLA class II genotyping data generated in this study will be beneficial not only for future disease association/vaccine efficacy studies related to the RV144 study, but also for similar studies in other diseases in the Thai population, as well as population genetics and transplantation studies.

Cardiac myosin heavy chain gene regulation by thyroid hormone involves altered histone modifications.

The antithetical regulation of cardiac α- and ß-myosin heavy chain (MHC) genes by thyroid hormone (T(3)) is not well understood but appears to involve thyroid hormone interaction with its nuclear receptor and MHC promoters as well as cis-acting noncoding regulatory RNA (ncRNA). Both of these phenomena involve epigenetic regulations. This study investigated the extent that altered thyroid state induces histone modifications in the chromatin associated with the cardiac MHC genes. We hypothesized that specific epigenetic events could be identified and linked to cardiac MHC gene switching in response to a hypothyroid or hyperthyroid state. A hypothyroid state was induced in rats by propylthiouracil treatment (PTU), whereas a hyperthyroid (T(3)) was induced by T(3) treatment. The left ventricle was analyzed after 7 days for MHC pre-mRNA expression, and the chromatin was assessed for enrichment in specific histone modifications using chromatin immunoprecipitation quantitative PCR assays. At both the α-MHC promoter and the intergenic region, the enrichment in acetyl histone H3 at K9/14 (H3K9/14ac) and trimethyl histone H3 at K4 (H3K4me3) changed in a similar fashion. They were both decreased with PTU treatment but did not change under T(3), except at a location situated 5' to the antisense intergenic transcription start site. These same marks varied differently on the ß-MHC promoter. For example, H3K4me3 enrichment correlated with the ß-promoter activity in PTU and T(3) groups, whereas H3K9/14ac was repressed in the T(3) group but did not change under PTU. Histone H3K9me was enriched in chromatin of both the intergenic and α-MHC promoters in the PTU group, whereas histone H4K20me1 was enriched in chromatin of ß-MHC promoter in the normal control and T(3) groups. Collectively, these findings provide evidence that specific epigenetic phenomena modulate MHC gene expression in altered thyroid states.

Artificial gravity as a countermeasure to microgravity: a pilot study examining the effects on knee extensor and plantar flexor muscle groups.

The goal of this project was to examine the effects of artificial gravity (AG) on skeletal muscle strength and key anabolic/catabolic markers known to regulate muscle mass. Two groups of subjects were selected for study: 1) a 21 day-bed rest (BR) group (n = 7) and 2) an AG group (n = 8), which was subjected to 21 days of 6 degrees head-down tilt bed rest plus daily 1-h exposures to AG (2.5 G at the feet). Centrifugation was produced using a short-arm centrifuge with the foot plate approximately 220 cm from the center of rotation. The torque-velocity relationships of the knee extensors and plantar flexors of the ankle were determined pre- and posttreatment. Muscle biopsy samples obtained from the vastus lateralis and soleus muscles were used for a series of gene expression analyses (mRNA abundance) of key factors implicated in the anabolic vs. catabolic state of the muscle. Post/pre torque-velocity determinations revealed greater decrements in knee extensor performance in the BR vs. AG group (P < 0.04). The plantar flexors of the AG subjects actually demonstrated a net gain in the torque-velocity relationship, whereas in the BR group, the responses declined (AG vs. BR, P < 0.001). Muscle fiber cross-sectional area decreased by approximately 20% in the BR group, whereas no losses were evident in the AG group. RT-PCR analyses of muscle biopsy specimens demonstrated that markers of growth and cytoskeletal integrity were higher in the AG group, whereas catabolic markers were elevated in the BR group. Importantly, these patterns were seen in both muscles. We conclude that paradigms of AG have the potential to maintain the functional, biochemical, and structural homeostasis of skeletal muscle in the face of chronic unloading.

Effect of unloading followed by reloading on expression of collagen and related growth factors in rat tendon and muscle.

Tendon tissue and the extracellular matrix of skeletal muscle respond to mechanical loading by increased collagen expression and synthesis. This response is likely a secondary effect of a mechanically induced expression of growth factors, including transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-I (IGF-I). It is not known whether unloading of tendon tissue can reduce the expression of collagen and collagen-inducing growth factors. Furthermore, the coordinated response of tendon and muscle tissue to disuse, followed by reloading, is unclear. Female Sprague-Dawley rats were subjected to hindlimb suspension (HS) for 7 or 14 days, followed by 2, 4, 8, or 16 days of reload (RL) (n = 8 in each group). Age-matched controls were included for day 0, day 14 HS, and day 16 RL (n = 8). mRNA expression levels for collagen I (COL1A1), collagen III (COL3A1), TGF-beta1, connective tissue growth factor (CTGF), myostatin, and IGF-I isoforms were measured by real-time RT-PCR in Achilles tendon and soleus muscle. The tendon mass was unchanged, while the muscle mass was reduced by 50% after HS (P < 0.05) and returned to control levels during RL. Collagen I and III, TGF-beta1, and CTGF mRNA levels were unaltered by HS, although collagen III tended to decrease in muscle at day 7 HS. IGF-I isoforms were significantly induced in tendon after 7 days of HS (P < 0.001), and mechanogrowth factor increased in muscle at day 14 HS (P < 0.05). Reload increased muscle collagen I and III mRNA (>10-fold) (P < 0.001) and growth factor expression (P < 0.05), while the tendon response was limited to a moderate induction of collagen expression (2-fold) (P < 0.05). Unloading of tendon and muscle tissue did not reduce expression of collagen and collagen-inducing growth factors, indicating that the response to unloading is not opposite that of loading. Furthermore, the tendon response was clearly different and less pronounced than the muscle tissue response.

Combined isometric, concentric, and eccentric resistance exercise prevents unloading-induced muscle atrophy in rats.

Previously, we reported that an isometric resistance training program that was effective in stimulating muscle hypertrophy in ambulatory rats could not completely prevent muscle atrophy during unloading (Haddad F, Adams GR, Bodell PW, Baldwin KM. J Appl Physiol 100: 433-441, 2006). These results indicated that preventing muscle atrophy does not appear to be simply a function of providing an anabolic stimulus. The present study was undertaken to determine if resistance training, with increased volume (3-s contractions) and incorporating both static and dynamic components, would be effective in preventing unloading-induced muscle atrophy. Rats were exposed to 5 days of muscle unloading via tail suspension. During that time one leg received electrically stimulated resistance exercise (RE) that included an isometric, concentric, and eccentric phase. The results of this study indicate that this combined-mode RE provided an anabolic stimulus sufficient to maintain the mass and myofibril content of the trained but not the contralateral medial gastrocnemius (MG) muscle. Relative to the contralateral MG, the RE stimulus increased the amount of total RNA (indicative of translational capacity) as well as the mRNA for several anabolic/myogenic markers such as insulin-like growth factor-I, myogenin, myoferlin, and procollagen III-alpha-1 and decreased that of myostatin, a negative regulator of muscle size. The combined-mode RE protocol also increased the activity of anabolic signaling intermediates such as p70S6 kinase. These results indicate that a combination of static- and dynamic-mode RE of sufficient volume provides an effective stimulus to stimulate anabolic/myogenic mechanisms to counter the initial stages of unloading-induced muscle atrophy.

Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types.

Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF-beta-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-beta-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-beta-1 in loading-induced collagen synthesis in the muscle-tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

Short-term strength training and the expression of myostatin and IGF-I isoforms in rat muscle and tendon: differential effects of specific contraction types.

In skeletal muscle, an increased expression of insulin like growth factor-I isoforms IGF-IEa and mechano-growth factor (MGF) combined with downregulation of myostatin is thought to be essential for training-induced hypertrophy. However, the specific effects of different contraction types on regulation of these factors in muscle are still unclear, and in tendon the functions of myostatin, IGF-IEa, and MGF in relation to training are unknown. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric, or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve during general anesthesia. mRNA levels for myostatin, IGF-IEa, and MGF in muscle and Achilles' tendon were measured by real-time RT-PCR. Muscle myostatin mRNA decreased in response to all types of training (2- to 8-fold) (P < 0.05), but the effect of eccentric training was greater than concentric and isometric training (P < 0.05). In tendon, myostatin mRNA was detected, but no changes were seen after exercise. IGF-IEa and MGF increased in muscle (up to 15-fold) and tendon (up to 4-fold) in response to training (P < 0.01). In tendon no difference was seen between training types, but in muscle the effect of eccentric training was greater than concentric training for both IGF-IEa and MGF (P < 0.05), and for IGF-IEa isometric training had greater effect than concentric (P < 0.05). The results indicate a possible role for IGF-IEa and MGF in adaptation of tendon to training, and the combined changes in myostatin and IGF-IEa/MGF expression could explain the important effect of eccentric actions for muscle hypertrophy.

Similar acute molecular responses to equivalent volumes of isometric, lengthening, or shortening mode resistance exercise.

The present study was undertaken to test the hypothesis that the contraction mode of action [static-isometric (Iso), shortening-concentric (Con), or lengthening-eccentric (Ecc)] used to stress the muscle provides a differential mechanical stimulus eliciting greater or lesser degrees of anabolic response at the initiation of a resistance training program. We performed an acute resistance training study in which different groups of rodents completed four training sessions in either the Iso, Con, or Ecc mode of contraction under conditions of activation and movement specifically designed to elicit equivalent volumes of force accumulation. The results of this experiment indicate that the three modes of contraction produced nearly identical cell signaling, indicative of an anabolic response involving factors such as increased levels of mRNA for IGF-I, procollagen III alpha1, decreased myostatin mRNA, and increased total RNA concentration. The resulting profiles collectively provide evidence that pure mode of muscle action, in and of itself, does not appear to be a primary variable in determining the efficacy of increased loading paradigms with regard to the initiation of selected muscle anabolic responses.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.