Functional characteristics of canine costal and crural diaphragm

We estimated the in situ force-generating capacity of the costal and crural portions of the canine diaphragm by relating in vitro contractile properties and diaphragmatic dimensions to in situ lengths. Piezoelectric crystals were implanted on right costal and left crural diaphragms of anesthetized dogs, via midline laparatomy. With the abdomen reclosed, diaphragm lengths were recorded at five lung volumes. Contractile properties of excised muscle bundles were then measured. In vitro force-frequency and length-tension characteristics of the costal and crural diaphragms were virtually identical; their optimal force values were 2.15 and 2.22 kg/cm2, respectively. In situ, at residual volume, functional residual capacity (FRC), and total lung capacity the costal diaphragm lay at 102, 95, and 60% of optimal length (Lo), whereas the crural diaphragm lay at 88, 84, and 66% of Lo. Muscle cross-sectional area was 40% greater in costal than in crural diaphragms. Considering in situ lengths, cross-sectional areas, and in vitro length-tension characteristics at FRC, the costal diaphragm could exert 60% more force than the crural diaphragm.     

Effect of chronic respiratory load on cytochrome oxidase activity in diaphragmatic fibers.

To determine whether the increase in oxidative capacity after respiratory muscle training with chronic inspiratory loads in sheep is specific to a particular fiber type, we measured cytochrome c oxidase (COX) activity in type I and type II fibers. COX activity in individual fibers was examined histochemically and measured as relative optical density by use of an image processing system. Fiber types were differentiated by the myosin adenosine-triphosphatase reaction. We found that COX activity was higher in both fiber types in the trained diaphragms than in the control diaphragms (P less than 0.01). The increase with training was greater in type II (39%) than in type I fibers (21%), resulting in relatively homogeneous COX activity in all diaphragmatic fibers. The proportion of type I fibers increased from 43.4 +/- 5.4% in the control diaphragm to 53.1 +/- 2.9% in the trained diaphragm, whereas the proportion of type II fibers decreased (P less than 0.001). We conclude that respiratory muscle training activates oxidative enzyme activity in both diaphragmatic fiber types; this activation is differentially more in type II fibers, which also decrease in proportion, and less in type I fibers, which increase in proportion.     

Effect of chronic obstructive pulmonary disease on calcium pump ATPase expression in human diaphragm.

We have previously demonstrated that human diaphragm remodeling elicited by severe chronic obstructive pulmonary disease (COPD) is characterized by a fast-to-slow myosin heavy chain isoform transformation. To test the hypothesis that COPD-induced diaphragm remodeling also elicits a fast-to-slow isoform shift in the sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), the other major ATPase in skeletal muscle, we obtained intraoperative biopsies of the costal diaphragm from 10 severe COPD patients and 10 control subjects. We then used isoform-specific monoclonal antibodies to characterize diaphragm fibers with respect to the expression of SERCA isoforms. Compared with control diaphragms, COPD diaphragms exhibited a 63% decrease in fibers expressing only fast SERCA (i.e., SERCA1; P < 0.001), a 190% increase in fibers containing both fast and slow SERCA isoforms (P < 0.01), and a 19% increase (P < 0.05) in fibers expressing only the slow SERCA isoform (i.e., SERCA2). Additionally, immunoblot experiments carried out on diaphragm homogenates indicated that COPD diaphragms expressed only one-third the SERCA1 content noted in control diaphragms; in contrast, COPD and control diaphragms did not differ with respect to SERCA2 content. The combination of these histological and immunoblot results is consistent with the hypothesis that diaphragm remodeling elicited by severe COPD is characterized by a fast-to-slow SERCA isoform transformation. Moreover, the combination of these SERCA data and our previously reported myosin heavy chain isoform data (Levine S, Nguyen T, Kaiser LR, Rubinstein NA, Maislin G, Gregory C, Rome LC, Dudley GA, Sieck GC, and Shrager JB. Am J Respir Crit Care Med 168: 706-713, 2003) suggests that diaphragm remodeling elicited by severe COPD should decrease ATP utilization by the diaphragm.     

Regenerative capacity of the dystrophic (mdx) diaphragm after induced injury

Duchenne muscular dystrophy is characterized by myofiber necrosis, muscle replacement by connective tissue, and crippling weakness. Although the mdx mouse also lacks dystrophin, most muscles show little myofiber loss or functional impairment. An exception is the mdx diaphragm, which is phenotypically similar to the human disease. Here we tested the hypothesis that the mdx diaphragm has a defective regenerative response to necrotic injury, which could account for its severe phenotype. Massive necrosis was induced in mdx and wild-type (C57BL10) mouse diaphragms in vivo by topical application of notexin, which destroys mature myofibers while leaving myogenic precursor satellite cells intact. At 4 h after acute exposure to notexin, >90% of diaphragm myofibers in both wild-type and mdx mice demonstrated pathological sarcolemmal leakiness, and there was a complete loss of isometric force-generating capacity. Both groups of mice showed strong expression of embryonic myosin within the diaphragm at 5 days, which was largely extinguished by 20 days after injury. At 60 days postinjury, wild-type diaphragms exhibited a persistent loss ( approximately 25%) of isometric force-generating capacity, associated with a trend toward increased connective tissue infiltration. In contrast, mdx diaphragms achieved complete functional recovery of force generation to noninjured values, and there was no increase in muscle connective tissue over baseline. These data argue against any loss of intrinsic regenerative capacity within the mdx diaphragm, despite characteristic features of major dystrophic pathology being present. Our findings support the concept that significant latent regenerative capacity resides within dystrophic muscles, which could potentially be exploited for therapeutic purposes.     

Multiple PV1 dimers reside in the same stomatal or fenestral diaphragm

Several of the endothelium-specific structures that have been involved in microvascular permeability [such as caveolae, transendothelial channels (TECs), vesiculovacuolar organelles (VVOs), and fenestrae] can be provided with either a stomatal or fenestral diaphragm. In the case of fenestrae, the diaphragm has the presumed function of creating a permselective barrier for solutes from blood plasma and interstitium. PV1 is an endothelium-specific integral membrane glycoprotein that is associated with both the stomatal diaphragms of caveolae, TECs, and VVOs as well as the diaphragms of endothelial fenestrae. The intimate structure of these diaphragms has been shown to consist of a meshwork formed by radial fibrils. We have recently shown that PV1 is a key structural element of both types of diaphragms, with its expression being sufficient to form de novo stomatal and fenestral diaphragms in both endothelial and nonendothelial cell types in culture. We have further tested the role of PV1 in the structure of the diaphragms and demonstrate here that multiple PV1 homodimers reside in close proximity within the same diaphragm. Our data bring further support to the paradigm by which PV1 dimers would form the fibrils of the diaphragms with a function in the microvascular permeability.     

Insulin and phorbol ester stimulate phosphorylation of a 15,000 dalton membrane protein in rat diaphragm in a similar manner

The effects of insulin on the phosphorylation of a 15 kilodalton (kDa) membrane protein in rat diaphragm in situ have been investigated. Incubation of the diaphragm with insulin or tumor-promoting phorbol ester increased the 32P-labelling of the 15 kDa protein at serine residues by 50 +/- 8% and 64 +/- 11%, (mean +/- S.E.), respectively. Thermolytic peptide mapping of the 15 kDa protein after insulin treatment of the diaphragm yielded two major phosphopeptides, one of which was absent from digests from control diaphragms. The same two phosphopeptides were identified after incubation of the diaphragm with phorbol ester and after phosphorylation of sarcolemma in vitro with [gamma-32P]ATP and protein kinase C. Additional experiments indicated that pretreatment of diaphragms with insulin or phorbol ester both increased the state of phosphorylation of the 15 kDa sarcolemma protein on phosphorylation sites regulated by protein kinase C. The stimulatory effect of insulin was decreased by staurosporine or by preincubation of the diaphragms with phorbol esters. These results indicate that the insulin-induced increases in protein kinase C activity previously found in rat diaphragm (Walaas et al. (1987) FEBS Lett. 220, 311-318) may be involved in insulin-mediated regulation of phosphorylation of the 15 kDa protein in situ.     

Chronic hypoxia modulates diaphragm function in the developing rat.

We studied the effect of chronic hypoxia on contractile properties and neuromuscular transmission in the developing rat diaphragm. We hypothesized that chronic hypoxia delays maturation of neuromuscular transmission. Phrenic nerve hemidiaphragm preparations were harvested from 3- to 26-day-old rats and littermates raised in 9.5% oxygen. Specific force, contraction time, and one-half relaxation time were measured. Each diaphragm was stimulated directly or via its nerve with 1-s trains at 10-100 Hz. Contraction time and one-half relaxation time decreased with advancing age in both groups, with a greater rate of decrease in hypoxic diaphragms. Specific force was lower for hypoxic diaphragms compared with controls. Diaphragms from the 3- to 10-day-old control and hypoxic groups generated less force in response to stimulation at frequencies >40 Hz but did so to a greater degree with nerve stimulation. Nerve stimulation of diaphragms from 11- to 18-day-old hypoxic rats showed a greater decrease in force with increasing frequency compared with age-matched controls. Diaphragms from 19- to 26-day-old rats showed no difference between the hypoxic and control groups. We conclude that chronic hypoxia leads to diaphragms that generate lower specific force as well as to a delayed maturation of mechanisms involved in neuromuscular transmission.     

Relationship of the reduction-oxidation state to protein degradation in skeletal and atrial muscle

Changes in proteolysis were correlated with the cell reduction-oxidation state in rat diaphragm and atrium. Protein degradation was measured in the presence of cycloheximide as the linear release of tyrosine into the medium. Intracellular ratios of lactate/pyruvate, total $\text{NADH}\text{NAD}$, and malate/pyruvate were used as indicators of the muscle reduction-oxidation state. Incubation of diaphragms with leucine (0.5–2.0 mm) or its transamination product, sodium α-ketoisocaproate (0.5 mm), resulted in a lower rate of proteolysis and a higher ratio of lactate/pyruvate and $\text{NADH}\text{NAD}$. These effects of leucine could be abolished by inhibiting its transamination with l-cycloserine. Unlike leucine, neither isoleucine nor valine alone produced any change in these parameters. Incubation of diaphragms with glucose (20 mm) or atria with sodium lactate (2 mm) produced a diminution of tyrosine release from the muscles and a rise in the ratio of total $\text{NADH}\text{NAD}$. Similarly, in incubated diaphragms of fasted rats, the anabolic effects of insulin, epinephrine and isoproterenol on protein degradation were associated with a higher malate/pyruvate ratio. In catabolic states, such as fasting, cortisol treatment of fasted, adrenalectomized rats or traumatization, enhanced muscle proteolysis was observed. Fresh-frozen diaphragms from these rats had both lower lactate/pyruvate and malate/pyruvate ratios than did muscles from control animals. These data show that diminution of proteolysis in diaphragm is accompanied by an increase of the $\text{NAD(P)H}\text{NAD(P)}$ ratios. In contrast to these findings, chymostatin and leupeptin, which inhibit directly muscle proteinases, caused a decrease of the lactate/pyruvate and malate/pyruvate ratios. These results suggest that protein degradation in diaphragm and atrium is linked to the cellular redox state.     

Effect of nutritional deprivation on diaphragm contractility and muscle fiber size

The influence of nutritional deprivation on the contractile and fatigue properties of the diaphragm was studied in adult rats. Food access was restricted to one-third of normal daily intake until the body weight of nutritionally deprived (ND) animals was approximately 50% of controls (CTL). Isometric contractile properties were studied in an in vitro nerve muscle strip preparation. Both twitch (Pt) and tetanic (Po) tensions of diaphragms from the ND animals were markedly reduced compared with CTL; however, Pt/Po was higher for the ND group. The shape of the force-frequency curve (normalized to Po) was generally similar between the two groups, except at 5 and 10 pulses/s stimulation, where greater relative tensions were produced in diaphragms from the ND animals. Diaphragm fatigue was induced by repetitive stimulation at either 20 or 100 pulses/s. Endurance time (defined as the time required for tension to fall to 50% of initial) of diaphragms from ND animals was prolonged at both 20 and 100 pulses/s. Immediately after induction of fatigue, force-frequency curves for both ND and CTL diaphragms were shifted to the right. However, this rightward shift was attenuated in the ND group compared with CTL. Nutritional deprivation had no effect on the proportions of different fiber types within the diaphragm but did result in a significant decrease in the cross-sectional area of both fast-and slow-twitch fibers. This decrease in cross-sectional area was significantly greater for fast-twitch fibers. We conclude that these changes in diaphragm contractile and fatigue properties occur as a result of the influence of malnutrition on muscle fiber cross-sectional area.     

Early mechanical dysfunction of the diaphragm in the muscular dystrophy with myositis (Ttnmdm) model

A complex rearrangement mutation in the mouse titin gene leads to an in-frame 83-amino acid deletion in the N2A region of titin. Autosomal recessive inheritance of the titin muscular dystrophy with myositis (Ttn(mdm/mdm)) mutation leads to a severe early-onset muscular dystrophy and premature death. We hypothesized that the N2A deletion would negatively impact the force-generating capacity and passive mechanical properties of the mdm diaphragm. We measured in vitro active isometric contractile and passive length-tension properties to assess muscle function at 2 and 6 wk of age. Micro-CT, myosin heavy chain Western blotting, and histology were used to assess diaphragm structure. Marked chest wall distortions began at 2 wk and progressively worsened until 5 wk. The percentage of myofibers with centrally located nuclei in mdm mice was significantly (P < 0.01) increased at 2 and 6 wk by 4% and 17%, respectively, compared with controls. At 6 wk, mdm diaphragm twitch stress was significantly (P < 0.01) reduced by 71%, time to peak twitch was significantly (P < 0.05) reduced by 52%, and half-relaxation time was significantly (P < 0.05) reduced by 57%. Isometric tetanic stress was significantly (P < 0.05) depressed in 2- and 6-wk mdm diaphragms by as much as 64%. Length-tension relationships of the 2- and 6-wk mdm diaphragms showed significantly (P < 0.05) decreased extensibility and increased stiffness. Slow myosin heavy chain expression was aberrantly favored in the mdm diaphragm at 6 wk. Our data strongly support early contractile and passive mechanical aberrations of the respiratory pump in mdm mice.     

Formation of intercellular gas space in the diaphragm during the development of aerenchyma in the leaf petiole of Sagittaria trifolia

The development of aerenchyma in the petiole of Sagittaria trifolia L. was studied by means of light-microscopy, scanning electron microscope, transmission electron microscope and immunofluorescence, focusing on the formation of intercellular spaces in diaphragms and its relationship with the organization of cortical microtubule arrays. A complex and organized honeycomb-like schizogenous aerenchyma formed by cylinders and vascular diaphragms was observed in the petiole of S. trifolia at different developmental stages. Cell division was the primary factor contributing to the increased volume of air spaces at early stages, while cell enlargement became the primary factor at later stages. The cortical microtubules localize at the sites where intercellular spaces and the secondary cell walls will be formed or deposited during the formation of intercellular spaces by the separation of diaphragm cells. Cortical microtubules were observed at the boundary of diaphragm cells and the fringes of intercellular spaces at later developmental stages where cell expansion occurs rapidly. These observations support the hypothesis that reorganization of cortical microtubule arrays might be related to the formation of air spaces in diaphragms and are involved in the deposition of secondary cell walls.     

In vitro functions of the rat diaphragm during postnatal development.

This study was designed to determine the developmental changes in the functional characteristics of the rat diaphragm. A total of 150 animals were studied at 1, 3, 5, 7 and 9 weeks of postnatal age. Body and diaphragm muscle weights were measured. Diaphragm strips were studied in an in vitro preparation to assess muscle contractile and endurance properties. Total diaphragm weight increased considerably, by a factor of 23 over the 9 week-period of study and was highly correlated with body weight (r = 0.93, P less than 0.01). However, the ratio of diaphragm-to-body weight decreased progressively with age. In comparison with those from older animals, diaphragms from 1 and 3 weeks old animals: (1) generated similar force normalized for muscle weight but a lower force normalized for fibre cross-sectional area (P less than 0.05), (2) had longer time-to-peak tension and one-half relaxation times (P less than 0.01) and (3) were more resistant to fatigue (P less than 0.01). The mechanisms underlying the diaphragm functional development were discussed.     

Diaphragmatic free radical generation increases in an animal model of heart failure.

Heart failure evokes diaphragm weakness, but the mechanism(s) by which this occurs are not known. We postulated that heart failure increases diaphragm free radical generation and that free radicals trigger diaphragm dysfunction in this condition. The purpose of the present study was to test this hypothesis. Experiments were performed using halothane-anesthetized sham-operated control rats and rats in which myocardial infarction was induced by ligation of the left anterior descending coronary artery. Animals were killed 6 wk after surgery, the diaphragms were removed, and the following were assessed: 1) mitochondrial hydrogen peroxide (H2O2) generation, 2) free radical generation in resting and contracting intact diaphragm using a fluorescent-indicator technique, 3) 8-isoprostane and protein carbonyls (indexes of free radical-induced lipid and protein oxidation), and 4) the diaphragm force-frequency relationship. In additional experiments, a group of coronary ligation animals were treated with polyethylene glycol-superoxide dismutase (PEG-SOD, 2,000 units x kg(-1) x day(-1)) for 4 wk. We found that coronary ligation evoked an increase in free radical formation by the intact diaphragm, increased diaphragm mitochondrial H2O2 generation, increased diaphragm protein carbonyl levels, and increased diaphragm 8-isoprostane levels compared with controls (P < 0.001 for the first 3 comparisons, P < 0.05 for 8-isoprostane levels). Force generated in response to 20-Hz stimulation was reduced by coronary ligation (P < 0.05); PEG-SOD administration restored force to control levels (P < 0.03). These findings indicate that cardiac dysfunction due to coronary ligation increases diaphragm free radical generation and that free radicals evoke reductions in diaphragm force generation.     

In vivo length-force relationship of canine diaphragm

Diaphragmatic length was measured by sonomicrometry and transdiaphragmatic pressure (Pdi) by conventional latex balloons in eight dogs anesthetized with pentobarbital sodium under passive conditions and during supramaximal phrenic stimulation. The passive length-pressure relationship indicates that the crural part of the diaphragm is more compliant than the costal part. With supramaximal stimulation the costal diaphragm showed a length-pressure relationship similar in shape to in vitro length-tension curves previously described for the canine diaphragm. The crural part has a smaller pressure-length slope than the costal part in the length range from 80% of optimum muscle length (Lo) to Lo. At supine functional residual capacity (FRC) the resting length (LFRC) of the costal and crural diaphragms are not at Lo. The costal part is distended to 105% of Lo, and crural is shortened to 92% of Lo. Tidal shortening will increase the force output of costal while decreasing that of the crural diaphragm. The major forces setting the passive supine LFRC are the abdominal weight (pressure) and the elastic recoil of the lungs. The equilibrium length (resting length of excised diaphragmatic strips) was 79 +/- 3.6% LFRC for the costal diaphragm and 87 +/- 3.9% LFRC for the crural diaphragm. Similar shortening was obtained in the upright position, indicating passive diaphragmatic stretch at supine LFRC.     

Effects of diaphragmatic ischemia on the inspiratory motor drive.

To assess the effect of diaphragmatic ischemia on the inspiratory motor drive, we studied the in situ isolated and innervated left diaphragm in anesthetized, vagotomized, and mechanically ventilated dogs. The arterial and venous vessels of the left diaphragm were catheterized and isolated from the systemic circulation. Inspiratory muscle activation was assessed by recording the integrated electromyographic (EMG) activity of the left and right costal diaphragms and parasternal intercostal and alae nasi muscles. Tension generated by the left diaphragm during spontaneous breathing attempts was also measured. In eight animals, left diaphragmatic ischemia was induced by occluding the phrenic artery for 20 min, followed by 10 min of reperfusion. This elicited a progressive increase in EMG activity of the left and right diaphragms and parasternal and alae nasi muscles to 170, 157, 152, and 128% of baseline values, respectively, an increase in the frequency of breathing efforts, and no change in left diaphragmatic spontaneous tension. Thus the ratio of left diaphragmatic EMG to tension rose progressively during ischemia. During reperfusion, only the frequency of breathing efforts and alae nasi EMG recovered completely. In four additional animals, left diaphragmatic ischemia was induced after the left phrenic nerve was sectioned. Neither EMG activity of inspiratory muscles nor respiratory timing changed significantly during ischemia. In conclusion, diaphragmatic ischemia increases inspiratory motor drive through activation of phrenic afferents. The changes in alae nasi activity and respiratory timing indicate that this influence is achieved through supraspinal pathways.     

Consequences of thyroxine treatment on diaphragm and EDL of normal and dystrophic hamsters

Previously administration of thyroxine (T4) to dystrophic hamsters improved ventilation and slowed the progression of the disease. We hypothesized that the normalization of ventilation in these animals was due to T4 improving structural and functional characteristics of the diaphragm. In the present study, contractile characteristics of the diaphragm and the extensor digitorum longus (EDL) from normal and dystrophic hamsters were evaluated after two months of T4 treatment. Compared to their placebo-treated counterparts, diaphragms and EDLs of T4-treated normal hamsters showed increased optimal muscle lengths and twitch tension, decreased contraction times and increased fatigability. T4-treatment in dystrophic hamsters showed only an increase in diaphragmatic twitch tension development. Force-frequency curves before treatment were generally higher for the normal compared to dystrophic diaphragms and EDLs. T4 administration only increased the force in normal diaphragms at the lower frequencies and in the EDLs at the higher frequencies. Although T4 serum levels were increased in both T4-treated groups, triiodothyronine (T3) was much lower in the dystrophic compared to normal hamsters, suggesting that conversion of T4 to T3 was reduced in dystrophic hamsters. We conclude that the limited functional changes in the diaphragms of T4-treated dystrophic hamsters cannot account for the marked improvement in ventilation previously reported.     

Stellate cells storing retinol in the liver of adult lamprey,Lampetra japonica

Summary Distribution, localization and fine structure of the stellate cells in the liver of lamprey, Lampetra japonica, were studied during the spawning migration by use of Kupffer's gold-chloride method, fluorescence microscopy for vitamin A (retinol) and electron microscopy. The stellate cells in the lamprey liver differ in some of their properties from those in mammalian livers. Stellate cells which store abundant retinol in lipid droplets, occur not only in the hepatic parenchyma, but also in the dense perivascular and capsular connective tissue of the liver and in the interstitium of pancreatic tissue. In the hepatic parenchyma these cells are located perisinusoidally or along thick bundles of collagen fibrils. The stellate cells display a number of large retinol-containing lipid droplets, granular endoplasmic reticulum, tubular structures, dense bodies, Golgi complex, microtubules, and microfilaments. In the space of Disse, the stellate cells and extracellular fibrilar components such as collagen fibrils and microfibrils (11–12 nm in diameter) are intervened between the two layers of basal laminae. Differentiation and possible functions of the stellate cells in the lamprey liver are discussed.     

Diaphragm muscle development in bottlenose dolphins (Tursiops truncatus)

Being born directly into the aquatic environment creates unique challenges for the breathing muscles of neonatal cetaceans. Not only must these muscles be active at the instant of birth to ventilate the lungs, but their activities must also be coordinated with those of the locomotor muscles such that breathing takes place only at the water's surface. At least one major locomotory muscle of bottlenose dolphins (Tursiops truncatus) has been demonstrated to be well developed and, therefore, able to power the neonatal dolphin's early movements (Dearolf et al. [2000] J Morphol 244:203-215). Thus, because of the demands for coordinated behavior with the locomotor muscles, it is hypothesized that the breathing muscles of bottlenose dolphins, represented in this study by the diaphragm, will also demonstrate adult morphology at birth. However, histochemical and biochemical analyses demonstrate that neonatal dolphins have immature diaphragms, with only 52% of the adult slow fiber-type profile (neonates: 34% slow-twitch fibers; adults: 66% slow-twitch fibers). The developmental state of the dolphin diaphragm is compared to those of other neonatal mammals, using a muscle development index (% slow-twitch fibers in neonatal muscle / % slow-twitch fibers in adult muscle). Fiber-type profiles reported in the literature are used to calculate index values for the diaphragms of altricial rats, rabbits, and cats, intermediate baboons and humans, and precocial sheep and horses. The dolphin is not unique in having an immature diaphragm at birth; however, there is a positive relationship between the developmental state of the diaphragm and the overall developmental state of the neonate. The presence of type IIc ("undifferentiated") fibers in the diaphragms of altricial developers (e.g., rats, rabbits, and cats) is correlated with the slow contraction speeds recorded from their diaphragms. The diaphragms of neonatal horses and dolphins express little to no type IIc fibers and, thus, may have the ability to contract at the speeds required for their increased ventilation rates. These results lead to the modification of the criterion for evaluating the developmental state of a muscle at birth. Thus, the developmental state of a neonatal muscle should be based on both its value of Dearolf et al.'s (2000) developmental index, as well as the percentage of type IIc fibers found in that muscle.     

PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms

PV1 is an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of caveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. Multiple PV1 homodimers are found within each stomatal and fenestral diaphragm. We investigated the function of PV1 within these diaphragms and their regulation and found that treatment of endothelial cells in culture with phorbol myristate acetate (PMA) led to upregulation of PV1. This correlated with de novo formation of stomatal diaphragms of caveolae and transendothelial channels as well as fenestrae upon PMA treatment. The newly formed diaphragms could be labeled with anti-PV1 antibodies. The upregulation of PV1 and formation of stomatal and fenestral diaphragms by PMA was endothelium specific and was the highest in microvascular endothelial cells compared with their large vessel counterparts. By using a siRNA approach, PV1 mRNA silencing prevented the de novo formation of the diaphragms of caveolae as well as fenestrae and transendothelial channels. Overexpression of PV1 in endothelial cells as well as in cell types that do not harbor caveolar diaphragms in situ induced de novo formation of caveolar stomatal diaphragms. Lastly, PV1 upregulation by PMA required the activation of Erk1/2 MAP kinase pathway and was protein kinase C independent. Taken together, these data show that PV1 is a key structural component, necessary for the biogenesis of the stomatal and fenestral diaphragms.     

Regional differences in myosin heavy chain isoforms and enzyme activities of the rat diaphragm.

Myosin heavy chain isoforms and enzyme activities were compared between the costal and crural regions of the rat diaphragm. The percentage of heavy chain (HC) IIb in the crural region of the diaphragm was significantly (P less than 0.05) higher than that in the costal region (mean 7.3 vs. 3.0%), and the percentage of HCI was significantly lower in the crural than in the costal diaphragm (22.7 vs. 27.9%). The distributions of HCIIa and HCIId were relatively homogeneous in both regions. Succinate dehydrogenase activity in the costal diaphragm was 21% greater (P less than 0.01) than in the crural diaphragm. In contrast, there was no significant difference in the activity of phosphofructokinase in the crural and costal diaphragms. These results demonstrate that a difference in myosin heavy chain isoforms and oxidative capacity exists between the costal and crural regions of the rat diaphragm.