Skeletal muscle satellite cells.

Evidence now suggests that satellite cells constitute a class of myogenic cells that differ distinctly from other embryonic myoblasts. Satellite cells arise from somites and first appear as a distinct myoblast type well before birth. Satellite cells from different muscles cannot be functionally distinguished from one another and are able to provide nuclei to all fibers without regard to phenotype. Thus, it is difficult to ascribe any significant function to establishing or stabilizing fiber type, even during regeneration. Within a muscle, satellite cells exhibit marked heterogeneity with respect to their proliferative behavior. The satellite cell population on a fiber can be partitioned into those that function as stem cells and those which are readily available for fusion. Recent studies have shown that the cells are not simply spindle shaped, but are very diverse in their morphology and have multiple branches emanating from the poles of the cells. This finding is consistent with other studies indicating that the cells have the capacity for extensive migration within, and perhaps between, muscles. Complexity of cell shape usually reflects increased cytoplasmic volume and organelles including a well developed Golgi, and is usually associated with growing postnatal muscle or muscles undergoing some form of induced adaptive change or repair. The appearance of activated satellite cells suggests some function of the cells in the adaptive process through elaboration and secretion of a product. Significant advances have been made in determining the potential secretion products that satellite cells make. The manner in which satellite cell proliferative and fusion behavior is controlled has also been studied. There seems to be little doubt that cellcell coupling is not how satellite cells and myofibers communicate. Rather satellite cell regulation is through a number of potential growth factors that arise from a number of sources. Critical to the understanding of this form of control is to determine which of the many growth factors that can alter satellite cell behavior in vitro are at work in vivo. Little work has been done to determine what controls are at work after a regeneration response has been initiated. It seems likely that, after injury, growth factors are liberated through proteolytic activity and initiate an activation process whereby cells enter into a proliferative phase. After myofibers are formed, it also seems likely that satellite cell behavior is regulated through diffusible factors arising from the fibers rather than continuous control by circulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Exercise-induced satellite cell activation in senescent soleus muscle.

The purposes of this study were 1) to determine satellite cell mitotic activity and myofiber nuclear density in the soleus muscle of aged rats and 2) to examine the effect of exercise training on these same parameters. Twenty-four-month-old specific pathogen-free female Fischer 344 rats were assigned to either a training or a control group. The trained group performed 10 wk of progressive treadmill running that resulted in a significant increase (P less than or equal to 0.05) in vastus lateralis muscle malate dehydrogenase activity compared with control rats. Training produced a doubling of soleus muscle satellite cell mitotic activity (trained 1.28 +/- 0.33, control 0.52 +/- 0.13 thymidine-labeled satellite cells per 1,000 nuclei; P less than or equal to 0.05). Training also resulted in a doubling in the number of damaged fibers in the soleus muscle (P less than or equal to 0.05). Mean myofiber nuclear density was unaltered by exercise training but varied as a function of soleus muscle fiber size. Nuclear density of a subpopulation of small fibers (cross-sectional area less than one standard deviation below the mean cross-sectional area of all fibers examined) was significantly higher (P less than or equal to 0.05) than in other fibers in the soleus muscle. A high nuclear density and small size suggest that these fibers were immature. In addition, the soleus muscle from trained rats had significantly more (P less than or equal to 0.05) small fibers with high nuclear density than muscle from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of adrenergic receptors in the reflex diuresis in rabbits during pulmonary lymphatic obstruction.

The role of adrenergic receptors in the reflex diuresis in response to pulmonary lymphatic drainage was examined in anaesthetized, artificially ventilated New Zealand White rabbits. Pulmonary lymphatic drainage was obstructed by raising the pressure in a pouch created from the right external jugular vein. This pulmonary lymphatic obstruction results in a reflex increase in urine flow and sodium excretion. This reflex is abolished by renal denervation and by administration of L-NAME, a non-selective inhibitor of nitric oxide synthase. Also, infusion of the relatively selective neuronal nitric oxide synthase blocker, 7-nitroindazole sodium salt, into the renal medulla abolished the reflex diuresis. In this study the effects of adrenergic receptor antagonists on the reflex increase in urine were observed. Both ureters were cannulated in order to determine urine flow from both kidneys separately. Prazosin, an alpha1 adrenergic receptor antagonist, was infused into the renal medulla of the right kidney, while the left kidney acted as control. Administration of prazosin in this manner did not block the reflex diuresis in response to pulmonary lymphatic obstruction in either kidney. However, rauwolscine, an alpha2 adrenergic receptor antagonist, abolished the reflex increase in urine and sodium excretion in the ipsilateral kidney while preserving it in the contralateral kidney. These findings suggest that the increase in urine flow in rabbits caused by pulmonary lymphatic obstruction is dependent upon activation of alpha2 adrenergic receptors within the renal medulla.

TGFbeta2 activation status during cardiac morphogenesis.

Transforming growth factor beta (TGFbeta) is secreted as a biologically inactive complex by many cell types in vitro, but little is known of TGFbeta's activation status in vivo. This study examined the in vivo expression of active and total (active + acid-activatable) TGFbeta2 in embryonic chicken hearts during cardiac morphogenesis (Hamburger-Hamilton stage 10-24). The concentration of TGFbeta2 was measured by an enzyme-linked immunoassay that recognized active TGFbeta2. Whole heart homogenates were either left untreated to measure active TGFbeta2 or treated with acid before assay to measure total (active + acid-activatable) TGFbeta2. Total TGFbeta2 concentration increased more than 16-fold between stage 10/11 and stage 24. Active TGFbeta2 concentration was highest at stage 14/15, but overall remained relatively constant varying at most by 2.8-fold. When expressed relative to total TGFbeta2, the amount of active TGFbeta2 progressively declined from 70% in stage 10/11 hearts to 7% in stage 24 hearts. The distribution of active and total TGFbeta2 was examined by immunostaining with an antibody against active TGFbeta2. Before immunostaining, sections were either treated with acid or left untreated to determine the distribution of total and active TGFbeta2, respectively. Active TGFbeta2 immunostaining was first detected in the endothelium, myocardium, and cardiac jelly of stage 14 hearts. Acid treatment had no effect on the distribution or intensity of immunostaining at this stage. Faint, active TGFbeta2 immunostaining was restricted to the ventricular myocardium in stage 18 hearts. Acid treatment resulted in a marked increase in staining intensity in the ventricle, but no staining was observed in the atrium or outflow tract. In stage 24 hearts, faint active TGFbeta2 staining was detected in the ventricle before acid treatment. After acid treatment, patches of intense punctate stain were found in all regions of the embryonic heart. Increases in TGFbeta2 concentration and immunostaining intensity after acidification suggest that a significant amount of TGFbeta2 is in the latent form. Stage-dependent differences in activation status suggest that activation may be a developmentally regulated process in the chick heart and support the notion that activation is an important step in regulating TGFbeta actions in vivo.

Mechanisms of nascent fiber formation during avian skeletal muscle hypertrophy.

This study examined two putative mechanisms of new fiber formation in postnatal skeletal muscle, namely longitudinal fragmentation of existing fibers and de novo formation. The relative contributions of these two mechanisms to fiber formation in hypertrophying anterior latissimus dorsi (ALD) muscle were assessed by quantitative analysis of their nuclear populations. Muscle hypertrophy was induced by wing-weighting for 1 week. All nuclei formed during the weighting period were labeled by continuous infusion of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, and embryonic-like fibers were identified using an antibody to ventricular-like embryonic (V-EMB) myosin. The number of BrdU-labeled and unlabeled nuclei in V-EMB-positive fibers were counted. Wing-weighting resulted in significant muscle enlargement and the appearance of many V-EMB+ fibers. The majority of V-EMB+ fibers were completely independent of mature fibers and had a nuclear density characteristics of developing fibers. Furthermore, nearly 100% of the nuclei in independent V-EMB+ fibers were labeled. These findings strongly suggest that most V-EMB+ fibers were nascent fibers formed de novo during the weighting period by satellite cell activation and fusion. Nascent fibers were found primarily in the space between fascicles where they formed a complex anastomosing network of fibers running at angles to one another. Although wing-weighting induced an increase in the number of branched fibers, there was no evidence that V-EMB+ fibers were formed by longitudinal fragmentation. The location of newly formed fibers in wing-weighted and regenerating ALD muscle was compared to determine whether satellite cells in the ALD muscle were unusual in that, if stimulated to divide, they would form fibers in the inter- and intrafascicular space. In contrast to wing-weighted muscle, nascent fibers were always found closely associated with necrotic fibers. These results suggest that wing-weighting is not simply another model of regeneration, but rather produces a unique environment which induces satellite cell migration and subsequent fiber formation in the interfascicular space. De novo fiber formation is apparently the principal mechanism for the hyperplasia reported to occur in the ALD muscle undergoing hypertrophy induced by wing-weighting.

Role of satellite cells in altering myosin expression during avian skeletal muscle hypertrophy.

This study examined whether satellite cells express an embryonic isoform of myosin upon fusion with hypertrophying muscle fibers. Anterior latissimus dorsi (ALD) muscle hypertrophy was induced in adult chickens by weighting one wing. One and 7 days of wing-weighting produced significant increases in ALD muscle wet weight and in the number of mature fibers expressing ventricular-like embryonic (V-EMB) myosin. V-EMB myosin expression could be an event during regeneration of fibers injured by overload or part of the hypertrophy process itself. Although there was an increase in both the number of damaged fibers and the number of mature fibers expressing embryonic myosin after wing-weighting, results from this study suggest that these two events were not necessarily related. The apparent health of fibers expressing V-EMB myosin and the lack of correlation between the numbers of damaged and V-EMB myosin positive fibers (r = 0.20) suggest that embryonic myosin expression in mature fibers was likely a feature of the hypertrophy process itself. The appearance of V-EMB myosin in mature fibers 1 day after wing-weighting suggests that the change in myosin expression did not involve satellite cells since 24 hr is too short a time to permit more than limited satellite cell fusion. The relationship between satellite cells and embryonic myosin expression was examined more closely by labeling dividing satellite cells and their progeny with 5-bromo-2-deoxyuridine, and then colocalizing labeled myofiber nuclei and embryonic myosin in consecutive transverse sections of hypertrophied ALD muscle. One week of wing-weighting resulted in marked increases in myofiber nuclear labeling index and myofiber nuclear density compared to contralateral control. V-EMB myosin was not expressed uniformly throughout individual fibers, but rather in discrete regions of varying length. Many V-EMB myosin positive regions had a higher labeled nuclear density than V-EMB myosin negative regions indicating that V-EMB myosin expression was associated with an accumulation of satellite cell progeny in a restricted area. However, it was also clear that satellite cell progeny were not the sole source of V-EMB myosin since labeled nuclei were completely absent from 41% of the V-EMB positive regions. Furthermore, the presence of new nuclei did not result in obligatory expression of embryonic myosin because many V-EMB negative regions had a high labeled nuclear density. Thus, recently incorporated nuclei arising by satellite cell division are implicated as one, but not the sole source of embryonic myosin in hypertrophying muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

The molecular basis of genetics and inheritance.

This article reviews the molecular basis of genetic disorders. It is presented at an introductory level, assuming that the reader has a good physiologic background but has little expertise in the fields of molecular biology and molecular genetics. It addresses the following questions: (1) What is DNA? (2) What are genes and chromosomes? (3) How are genes expressed and how is gene expression regulated? (4) How is DNA replicated? (5) How is genetic material inherited? (6) How is phenotype determined? (7) How are genetic diseases inherited? The goal of this article is to provide vocabulary and concepts that are key for understanding the substantive articles that follow on the subjects of clinical genetics, gene therapy, ethical issues in genetics, and the molecular genetics of cardiovascular disorders. Included in this article is a list of key terms with a corresponding page number where the term is defined or discussed. This should be used as a resource for reading the other articles. In addition, further readings, programmed CDs, and web sites in the areas of molecular biology and molecular genetics are suggested.

Effects of beta-adrenergic blockade on training-induced structural adaptations in rat left ventricle.

The study was designed to evaluate the effects of eight weeks of exercise training or training-beta-adrenergic blockade combination on gross and microscopic alterations of rat cardiac muscle and microvascular bed. Rats were randomly assigned to either sedentary control (C), trained (T), metoprolol-trained (MT), or propranolol-trained (PT) groups. The training protocol involved treadmill running for 8 weeks at 0.5 ms-1, 20% grade. Earlier experiments by us showed this training protocol to be effective in producing significant changes in selected skeletal muscle enzyme activities in all trained groups. In the current study an absolute reduction in left ventricular (LV) weight was observed in the PT compared to the C group (0.91 +/- 0.02 vs. 1.04 +/- 0.04 g, P less than 0.05). LV weight in the T and MT groups was no different from C so that LV to BW ratio (mg.g-1) was significantly increased (P less than 0.05) due to a similar reduction in body weight (BW) in all three training groups. Morphometric analysis of LV myocardium revealed no significant differences in myocyte mean cross-sectional area (micron 2) in any of the groups (289 +/- 16-C, 332 +/- 20-T, 281 +/- 44-MT, and 273 +/- 12-PT). Capillary density independently calculated by light and electron microscopy was unchanged by training or training-beta-blockade combination. It was concluded that training of sufficient intensity and duration to produce skeletal muscle enzyme adaptations does not necessarily produce myocyte hypertrophy or alter LV capillarity. Additionally functioning beta-adrenergic receptors appear to play a role in both the central and peripheral adaptations to endurance exercise training.

Coordinate changes in C protein and myosin expression during skeletal muscle hypertrophy.

In this study, two new C protein isoforms in adult rat skeletal muscle were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These isoforms migrated between previously identified fast (Cf) and slow (Cs) C protein isoforms; hence they were named intermediate C proteins (Ci1 and Ci2). Cyanogen bromide peptide mapping and Western blotting showed that the intermediate isoforms were more similar to Cs than Cf. The distribution of specific C protein and myosin heavy chain (MHC) isoforms was highly correlated in several hindlimb muscles, suggesting that the expression of these two thick-filament proteins is coordinated. This notion was tested by determining whether specific C protein and MHC isoforms change in parallel during muscle hypertrophy. Eight weeks after ablation of its synergists, the overloaded plantaris muscle showed significant increases in type IIa MHC and intermediate C protein, with corresponding decreases in type IIb MHC and Cf protein. These results indicate that C protein expression is linked to MHC expression during plantaris muscle hypertrophy.

Insulin-like growth factor-II/mannose-6-phosphate receptor expression during early heart development.

Expression of the insulin-like growth factor-II/mannose-6-phosphate (ICF-II/ M6P) receptor was examined during the major stages of heart morphogenesis in the chicken embryo. By using an affinity-purified antibody, Western blot analysis of total embryonic proteins from stages 5-24 revealed little if any IGF-II/M6P receptor protein until stage 7, approximately 8 hours prior to the appearance of the rudimentary myocardial tubes. Thereafter, receptor accumulation increased until stage 14, after which receptor protein levels remained constant, up to 7 days in ovo. Immunohistochemical localization revealed that, among all embryonic tissues at stages 10-24, the predominant site of receptor expression was the developing myocardium. Receptor expression was also immunohistochemically evaluated in a defined in vitro model of cardiogenesis in which explanted precardiac mesoderm is induced to undergo differentiation by co-explanted endoderm. In this system, as in vivo, IGF-II/M6P receptors were only detected after precardiac mesoderm had differentiated into a synchronously contractile multilayer which expressed cardiac alpha-actin. These findings indicate that the IGF-II/M6P receptor has an important role during early heart development.

Creatine kinase isoforms following isometric exercise.

The present study assessed creatine kinase (CK) activity, CK MM isoforms, and muscle soreness following an exercise regimen designed to induce skeletal muscle damage. Eight college-age subjects performed 40 maximal isometric contractions of the knee extensor muscles (10-second contraction/20-second rest). Serum samples and soreness ratings were taken prior to and 2, 6, 18, and 24 hours after the exercise. The CK MM1 and CK MM3 isoforms were determined by flatbed isoelectric focusing (IEF). In serum, the MM1 isoform (the pure gene product) is considered to be evidence of newly released CK from muscle, as upon entering the plasma, the CK MM1 is converted to MM2 and then MM3. A significant increase in serum CK activity was found at 6 hours and remained elevated at 24 hours. CK MM1 increased significantly at 2 hours, peaked at 6 hours, then approached baseline. Soreness did not increase significantly until 18 hours. Analysis of CK isoforms in serum can provide an earlier indicator of skeletal muscle damage than total CK or perception of soreness and may be useful in tracking the time course of skeletal muscle damage and repair.

Role of nitric oxide in the reflex diuresis in rabbits during pulmonary lymphatic obstruction.

The role of nitric oxide in the reflex diuresis in response to pulmonary lymphatic drainage was examined in anaesthetized, artificially ventilated New Zealand White rabbits. Pulmonary lymphatic drainage was obstructed by raising the pressure in a pouch created from the right external jugular vein. Pulmonary lymphatic obstruction resulted in a significant increase in urine flow from an initial control value of 8.9 +/- 0.5 ml (10 min)(-1) to 12.1 +/- 0.6 ml (10 min)(-1) during lymphatic obstruction (mean +/-s.e.m.; n= 17, P < 0.001). This increase in urine flow was accompanied by a significant increase in the excretion of sodium. Additionally, renal blood flow remained unchanged during the increase in urine flow caused by lymphatic obstruction. Intravenous infusion of L-NAME, a non-selective inhibitor of nitric oxide synthase (NOS), abolished the reflex diuresis. Furthermore, intraperitoneal administration of the relatively selective neuronal NOS blocker, 7-nitroindazole also abolished the response. It was observed that infusion of a more soluble neuronal NOS blocker, 7-nitroindazole sodium salt (7-NINA), into the renal medulla also abolished the reflex diuresis. These findings suggest that the increase in urine flow in rabbits caused by pulmonary lymphatic obstruction is dependent upon the integrity of neuronal NOS activity within the renal medulla.

Muscle soreness and serum creatine kinase activity following isometric, eccentric, and concentric exercise.

Serum creatine kinase (CK) activity and subjective ratings of muscle soreness were assessed in 28 college women following three different arm flexion exercise regimens. The subjects were randomly assigned to an eccentric, isometric, or concentric exercise regimen. Each regimen was equated for total work time and work-to-rest ratio. Blood samples for determination of serum CK activity and perceived soreness ratings were obtained prior to and 5, 10, and 25 h following each exercise. Significant increases in perceived soreness ratings were observed for each exercise regimen. The magnitude of the post-exercise increase in perceived soreness was greatest for the eccentric and the isometric exercises with minimal soreness following the concentric exercise. A small but significant increase in serum CK activity was observed following the three exercises (eccentric = 35.8%, concentric = 37.6%, isometric = 34.0%). The post-exercise serum CK increases did not differ significantly among the three regimens. The rise in serum CK activity suggests that muscle damage occurred during all three tasks. However, due to multiple factors which can affect serum CK levels, the increase in serum CK activity may not provide a sensitive indicator of the magnitude of the injury.