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Voluntary exercise (VE) has a beneficial influence on the heart and mean lifespan. The present study evaluates structural adaptations of cardiomyocytes and their mitochondria due to VE by new, unbiased stereological methods. Female, 7-9-week-old mice were randomly assigned to a control (CG, n = 7) or VE group (EG, n = 7). EG animals were housed in cages with free access to a running wheel and had a mean running distance of 6.7 (1.8) km per day. After 4 weeks, the hearts of all mice were processed for light and electron microscopy. We estimated the number and volume of cardiomyocytes by the disector method and the number and volume of mitochondria by estimation of the Euler number. In comparison to CG, VE did not have an effect on the myocardial volume of the left ventricle (CG: 93 (10), EG: 103 (17) (mm(3))), the number of cardiomyocytes (CG: 2.81 (0.27), EG: 2.82 (0.43) (x10(6))) and their number-weighted mean volume. However, the composition of the cardiomyocytes changed due to VE. The total volume of mitochondria (CG: 21.8 (4.9), EG: 32.2 (4.3) (mm(3)), P < 0.01) and the total number (CG: 3.76 (0.44), EG: 7.02 (1.13) (x10(10)), P < 0.001) were significantly higher in EG than in CG. The mean number-weighted mitochondrial volume was smaller in EG than in CG (P < 0.05). In summary, VE does not alter ventricular volume nor cardiomyocyte volume or number but the oxidative capacity of cardiomyocytes by an increased mitochondrial number and total volume in the left ventricle. These structural changes may participate in the beneficial effects of VE.

Mammalian birth is accompanied by profound changes in metabolic rate that can be described in terms of body size relationship (Kleiber's rule). Whereas the fetus, probably as an adaptation to the low intrauterine pO2, exhibits an "inappropriately" low, adult-like specific metabolic rate, the term neonate undergoes a rapid metabolic increase up to the level to be expected from body size. A similar, albeit slowed, "switching-on" of metabolic size allometry is found in human preterm neonates whereas animals that are normally born in a very immature state are able to retard or even suppress the postnatal metabolic increase in favor of weight gain and O2 supply. Moreover, small immature mammalian neonates exhibit a temporary oxyconforming behavior which enhances their hypoxia tolerance, yet is lost to the extent by which the size-adjusted metabolic rate is "locked" by increasing mitochondrial density. Beyond the perinatal period, there are no other deviations from metabolic size allometry among mammals except in hibernation where the temporary "switching-off" of Kleiber's rule is accompanied by a deep reduction in tissue pO2. This gives support to the hypothesis that the postnatal metabolic increase represents an "escape from oxygen" similar to the evolutionary roots of mitochondrial respiration, and that the overall increase in specific metabolic rate with decreasing size might contribute to prevent tissues from O2 toxicity.

The penetration, translocation, and distribution of ultrafine and nanoparticles in tissues and cells are challenging issues in aerosol research. This article describes a set of novel quantitative microscopic methods for evaluating particle distributions within sectional images of tissues and cells by addressing the following questions: (1) is the observed distribution of particles between spatial compartments random? (2) Which compartments are preferentially targeted by particles? and (3) Does the observed particle distribution shift between different experimental groups? Each of these questions can be addressed by testing an appropriate null hypothesis. The methods all require observed particle distributions to be estimated by counting the number of particles associated with each defined compartment. For studying preferential labeling of compartments, the size of each of the compartments must also be estimated by counting the number of points of a randomly superimposed test grid that hit the different compartments. The latter provides information about the particle distribution that would be expected if the particles were randomly distributed, that is, the expected number of particles. From these data, we can calculate a relative deposition index (RDI) by dividing the observed number of particles by the expected number of particles. The RDI indicates whether the observed number of particles corresponds to that predicted solely by compartment size (for which RDI = 1). Within one group, the observed and expected particle distributions are compared by chi-squared analysis. The total chi-squared value indicates whether an observed distribution is random. If not, the partial chi-squared values help to identify those compartments that are preferential targets of the particles (RDI > 1). Particle distributions between different groups can be compared in a similar way by contingency table analysis. We first describe the preconditions and the way to implement these methods, then provide three worked examples, and finally discuss the advantages, pitfalls, and limitations of this method.

Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions.This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the distributions of nanoparticles in tissues and cells.This comprehensive article aims to provide a basis for scientists in nanoparticle research to integrate electron microscopic analyses into their study design and to select the appropriate microscopic strategy.

Background: Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency., Methods: SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test., Main Results: After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment., Conclusion: Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

Background: Experimental studies provide evidence that inhaled nanoparticles may translocate over the airspace epithelium and cause increased cellular inflammation. Little is known, however, about the dependence of particle size or material on translocation characteristics, inflammatory response and intracellular localization., Results: Using a triple cell co-culture model of the human airway wall composed of epithelial cells, macrophages and dendritic cells we quantified the entering of fine (1 mum) and nano-sized (0.078 mum) polystyrene particles by laser scanning microscopy. The number distribution of particles within the cell types was significantly different between fine and nano-sized particles suggesting different translocation characteristics. Analysis of the intracellular localization of gold (0.025 mum) and titanium dioxide (0.02-0.03 mum) nanoparticles by energy filtering transmission electron microscopy showed differences in intracellular localization depending on particle composition. Titanium dioxide nanoparticles were detected as single particles without membranes as well as in membrane-bound agglomerations. Gold nanoparticles were found inside the cells as free particles only. The potential of the different particle types (different sizes and different materials) to induce a cellular response was determined by measurements of the tumour necrosis factor-alpha in the supernatants. We measured a 2-3 fold increase of tumour necrosis factor-alpha in the supernatants after applying 1 mum polystyrene particles, gold nanoparticles, but not with polystyrene and titanium dioxide nanoparticles., Conclusion: Quantitative laser scanning microscopy provided evidence that the translocation and entering characteristics of particles are size-dependent. Energy filtering transmission electron microscopy showed that the intracellular localization of nanoparticles depends on the particle material. Both particle size and material affect the cellular responses to particle exposure as measured by the generation of tumour necrosis factor-alpha.

Background: Translocation of nanoparticles (NP) from the pulmonary airways into other pulmonary compartments or the systemic circulation is controversially discussed in the literature. In a previous study it was shown that titanium dioxide (TiO2) NP were "distributed in four lung compartments (air-filled spaces, epithelium/endothelium, connective tissue, capillary lumen) in correlation with compartment size". It was concluded that particles can move freely between these tissue compartments. To analyze whether the distribution of TiO2 NP in the lungs is really random or shows a preferential targeting we applied a newly developed method for comparing NP distributions., Methods: Rat lungs exposed to an aerosol containing TiO2 NP were prepared for light and electron microscopy at 1 h and at 24 h after exposure. Numbers of TiO2 NP associated with each compartment were counted using energy filtering transmission electron microscopy. Compartment size was estimated by unbiased stereology from systematically sampled light micrographs. Numbers of particles were related to compartment size using a relative deposition index and chi-squared analysis., Results: Nanoparticle distribution within the four compartments was not random at 1 h or at 24 h after exposure. At 1 h the connective tissue was the preferential target of the particles. At 24 h the NP were preferentially located in the capillary lumen., Conclusion: We conclude that TiO2 NP do not move freely between pulmonary tissue compartments, although they can pass from one compartment to another with relative ease. The residence time of NP in each tissue compartment of the respiratory system depends on the compartment and the time after exposure. It is suggested that a small fraction of TiO2 NP are rapidly transported from the airway lumen to the connective tissue and subsequently released into the systemic circulation.

Pulmonary surfactant prevents alveolar collapse via reduction of surface tension. In contrast to human neonates, rats are born with saccular lungs. Therefore, rat lungs serve as a model for investigation of the surfactant system during postnatal alveolar formation. We hypothesized that this process is associated with characteristic structural and biochemical surfactant alterations. We aimed to discriminate changes related to alveolarization from those being either invariable or follow continuous patterns of postnatal changes. Secreted active (mainly tubular myelin (tm)) and inactive (unilamellar vesicles (ulv)) surfactant subtypes as well as intracellular surfactant (lamellar bodies (lb)) in type II pneumocytes (PNII) were quantified before (day (d) 1), during (d 7), at the end of alveolarization (d 14), and after completion of lung maturation (d 42) using electron microscopic methods supplemented by biochemical analyses (phospholipid quantification, immunoblotting for SP-A). Immunoelectron microscopy determined the localization of surfactant protein A (SP-A). (1) At d 1 secreted surfactant was increased relative to d 7-42 and then decreased significantly. (2) Air spaces of neonatal lungs comprised lower fractions of tm and increased ulv, which correlated with low SP-A concentrations in lung lavage fluid (LLF) and increased respiratory rates, respectively. (3) Alveolarization (d 7-14) was associated with decreasing PNII size although volume and sizes of Lb continuously increased. (4) The volume fractions of Lb correlated well with the pool sizes of phospholipids in lavaged lungs. Our study emphasizes differential patterns of developmental changes of the surfactant system relative to postnatal alveolarization., ((c) 2007 Wiley-Liss, Inc.)

A major aim in lung transplantation is to prevent the loss of structural integrity due to ischemia and reperfusion (I/R) injury. Preservation solutions protect the lung against I/R injury to a variable extent. We compared the influence of two extracellular-type preservation solutions (Perfadex, or PX, and Celsior, or CE) on the morphological alterations induced by I/R. Pigs were randomly assigned to sham (n = 4), PX (n = 5), or CE (n = 2) group. After flush perfusion with PX or CE, donor lungs were excised and stored for 27 hr at 4 degrees C. The left donor lung was implanted into the recipient, reperfused for 6 hr, and, afterward, prepared for light and electron microscopy. Intra-alveolar, septal, and peribronchovascular edema as well as the integrity of the blood-air barrier were determined stereologically. Intra-alveolar edema was more pronounced in CE (219.80 +/- 207.55 ml) than in PX (31.46 +/- 15.75 ml). Peribronchovascular (sham: 13.20 +/- 4.99 ml; PX: 15.57 +/- 5.53 ml; CE: 31.56 +/- 5.78 ml) and septal edema (thickness of alveolar septal interstitium, sham: 98 +/- 33 nm; PX: 84 +/- 8 nm; CE: 249 +/- 85 nm) were only found in CE. The blood-air barrier was similarly well preserved in sham and PX but showed larger areas of swollen and fragmented epithelium or endothelium in CE. The present study shows that Perfadex effectively prevents intra-alveolar, septal, and peribronchovascular edema formation as well as injury of the blood-air barrier during I/R. Celsior was not effective in preserving the lung from morphological I/R injury., ((c) 2007 Wiley-Liss, Inc.)

Background: Lung retrieval from non-heart-beating donors (NHBD) has been introduced into clinical practice successfully. However, because of potentially deleterious effects of warm ischemia on microvascular integrity, use of NHBD lungs is limited by short tolerable time periods before preservation. Recently, improvement of NHBD graft function was demonstrated by donor pre-treatment using aerosolized Ventavis (Schering Inc., Berlin, Germany). Currently, there is no information whether additional application of this approach in reperfusion can further optimize immediate graft function., Material and Methods: Asystolic pigs (n = 5/group) were ventilated for 180-min of warm ischemia (groups 1-3). In groups 2 and 3, 100 microg Ventavis were aerosolized over 30-min using an ultrasonic nebulizer (Optineb). Lungs were then retrogradely preserved with Perfadex and stored for 3-h. After left lung transplantation and contralateral lung exclusion, grafts were reperfused for 6-h. Only in group 3, another dose of 100 microg Ventavis was aerosolized during the first 30-min of reperfusion. Hemodynamics, pO2/FiO2 and dynamic compliance were monitored continuously and compared to controls. Intraalveolar edema was quantified stereologically, and extravascular-lung-water-index (EVLWI) was measured. Statistics comprised ANOVA analysis with repeated measurements., Results: Dynamic compliance was significantly lower in both Ventavis groups, but additional administration did not result in further improvement. Oxygenation, pulmonary hemodynamics, EVLWI and intraalveolar edema formation were comparable between groups., Conclusions: Alveolar deposition of Ventavis in NHBD lungs before preservation significantly improves dynamic lung compliance and represents an important strategy for improvement of preservation quality and expansion of warm ischemic intervals. However, additional application of this method in early reperfusion is of no benefit.

The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.

Connexins are known to play an essential role in the ischemic preconditioning (IP) of the heart; their functional role in this process, however, has not been clearly defined. For this reason, anesthetized rats were subjected to regional myocardial ischemia, with or without IP or reperfusion. In frozen sections of hearts, fluorescence immunohistochemical staining for connexin43 (Cx43) was performed. In contrast to undisturbed zones, tissue that had been subjected to ischemia revealed Cx43 immunostaining not only in the gap junctions but also in a conspicuous pattern in the free cellular membranes of the myocytes. In myocardium that was exposed to IP only, the ratio of immunofluorescence intensity in the free cellular membrane to that in the interior of the cell was 1.22 +/- 0.04 (ratio in non-ischemia-exposed area = 1.04 +/- 0.01). When 15 or 45 min of permanent ischemia followed IP, the effect became more evident (ratio = 1.31 +/- 0.03 and 1.46 +/- 0.03, respectively) and proved to be significantly greater than in the corresponding non-IP groups (ratio = 1.16 +/- 0.03 and 1.30 +/- 0.03, respectively, P < 0.01). Reperfusion led to an overall weakening of fluorescence intensities and a disappearance of the observed IP-specific differences. We conclude that IP initiates a redistribution of Cx43 from its natural position in the gap junctions toward the free plasma membrane, thereby improving the cell's chance of survival during the subsequent phase of prolonged ischemia by an unknown, supposedly gap junction-independent, mechanism.

Objective: Cardiac surgery on cardiopulmonary bypass (CPB) results in progressive myocardial dysfunction, despite unimpaired coronary blood flow, and is associated with increased myocardial tumor necrosis factor-alpha (TNFalpha) expression. We investigated whether anti-inflammatory treatment prevents increased TNFalpha expression and myocardial dysfunction after CPB., Methods and Results: Baseline systemic hemodynamics, myocardial contractile function, aortic and coronary blood flow were measured in anesthetized pigs. Then, placebo (PLA; saline; n=7) or methylprednisolone (MP; 30 mg/kg; n=6) was infused intravenously and CPB was instituted. Global ischemia was induced for 10 min by aortic cross-clamping, followed by 1 h of cardioplegic cardiac arrest. After declamping and reperfusion, CPB was terminated after a total of 3 h. Measurements were repeated at 15 min, 4 h, and 8 h following termination of CPB. Systemic TNFalpha-plasma concentrations and left ventricular TNFalpha expression were analyzed. With unchanged coronary blood flow in both groups, a progressive loss of myocardial contractile function to 38+/-2% of baseline (p<0.01) and cardiac index to 48+/-6% of baseline (p<0.01) at 8 h after CPB in PLA was attenuated in MP (myocardial function: 72+/-3%, p<0.01 vs PLA; cardiac index: 78+/-6%, p<0.05 vs PLA). Systemic TNFalpha was increased at 8 h in PLA compared to MP (243+/-34 vs 90+/-34 pg/ml, p<0.05). Myocardial TNFalpha was increased at 8 h after CPB compared to baseline and MP (p<0.05). Myocardial TNFalpha immunostaining was more pronounced in PLA than in MP (p<0.05), with TNFalpha-mRNA localization predominantly to cardiomyocytes., Conclusions: Methylprednisolone attenuates both systemic and myocardial TNFalpha increases and progressive myocardial dysfunction induced by cardiac surgery, suggesting a key role for TNFalpha.

Neonatal rat hearts are more tolerant to ischemia compared to adult rat hearts. We hypothesized that opioid receptors and mitochondrial potassium channels are involved in the elevated ischemia tolerance of neonatal rats. Newborn rats were treated by an intraperitoneal injection with sodium chloride (placebo, Pla; n = 7), naloxone (Nal; n = 8), or K+ (ATP) channel blocker 5-hydroxydecanoate (HD; n = 8), or were left untreated (sham; n = 8). Thirty minutes after injection, the rats were sacrificed and hearts were arrested cardioplegically and fixed with aldehyde fixative 90 min after global ischemia at room temperature. For control, newborn rat hearts were fixed immediately after sacrifice. Ventricular tissue blocks were prepared for electron microscopy. Mitochondrial (volume-weighted mean volume of mitochondria) and cardiomyocyte volume (cellular edema index, CEI) were estimated to quantify the ischemic injury. Compared to control myocardium, CEI was increased by 244% +/- 39% in sham, 173% +/- 28% in Nal, 142% +/- 25% in HD, and 101% +/- 24% in Pla (P < 0.05 between groups). Volume-weighted mean volume of mitochondria was increased by 514% +/- 235% in sham, 341% +/- 110% in Nal, 458% +/- 149% in HD, and 175% +/- 70% in Pla. Differences between Pla and other groups were significant (P < 0.01 for all). No significant difference was observed between the other groups. Thus, ischemic injury was smallest with placebo, indicating a mechanism similar to preconditioning induced by the intraperitoneal injection. This response was attenuated by blockade of opioid receptors and mitochondrial potassium channels, suggesting their involvement in the elevated ischemia tolerance of newborn rat hearts.

Surfactant proteins (SP) have an important impact on the function of the pulmonary surfactant. In contrast to humans, rat lungs are immature at birth. Alveolarization starts on postnatal day 4. Little is known about the distribution of SP during postnatal alveolarization. By immunoelectron microscopy, we studied the distribution of SP-A, SP-D, SP-B, and precursors of SP-C in type II pneumocytes before, near the end and after alveolarization and in mature lungs. We determined the subcellular volume fractions and the relative labeling index to obtain information about preferential labeling of compartments and non-randomness of labeling. Independently of alveolarization, the overall cellular distribution of SP was non-random. A preferential labeling for SP-A and SP-D was found in small vesicles and multivesicular bodies (mvb). SP-B and precursors of SP-C were localized in mvb and lamellar bodies (lb). There are no postnatal changes in labeling for all three SP in these compartments. Labeling intensity for SP-B in lb increased in close correlation with a significant increase in the volume fractions of lb during alveolarization. Our results support the concept that postnatal alveolarization in rat lungs is associated with significant increases in the SP-B content in lb and volume fraction of lb in type II pneumocytes. The postnatal compartment-specific distribution of SP-A, precursors of SP-C and SP-D does not change.

Objective: Mild to moderate transient contractile dysfunction is frequently observed after cardiac surgery on cardiopulmonary bypass (CPB) but may also lead to low-cardiac-output (LCO) failure especially in patients with unstable angina, and is often referred to represent myocardial stunning. Whether time course of contractile dysfunction after cardiac surgery is similar to that of myocardial stunning was investigated in pigs., Methods: After baseline measurements of systemic hemodynamics (micromanometry), myocardial contractile function (sonomicrometry), cardiac output and coronary flow (ultrasonic probe), CPB was instituted. Control animals (n = 7) were weaned after 3 h from CPB. In LCO animals (n = 8), global ischemia was induced for 10 min by aortic crossclamping, followed by 1 h of cardioplegic cardiac arrest. After declamping and reperfusion, CPB was terminated after a total of 3 h. Measurements were repeated at 15 min, 4 h and 8 h after CPB. Systemic TNFalpha-plasma concentrations were measured (ELISA) and left ventricular biopsies were analyzed with respect to myocardial TNFalpha (immunohistochemistry) and irreversible cellular damage (light/electron microscopy)., Results: Contractile function decreased in LCO (75 +/- 12%) and control (83 +/-17%) at 15 min compared to baseline (p < 0.05). Thereafter, contractile function remained unchanged in control, but progressively decreased in LCO (52 +/- 12% at 4 h; 36 +/- 5% at 8 h; p < 0.05). Coronary flow remained unchanged in both groups. Cardiac output progressively decreased to 2.8 +/- 0.9 l/min at 8 h in the LCO group compared to baseline (5.9 +/- 1.1 l/min, p < 0.05) and control (5.7 +/- 1.4 l/min, p < 0.05). There was no evidence for myocardial infarction. TNFalpha-plasma concentrations and myocardial TNFalpha-staining were increased at 8 h after CPB in the LCO group compared to baseline and control (p < 0.05)., Conclusions: The progressive pattern of myocardial dysfunction apart from ongoing ischemia after cardiac surgery suggested underlying mechanisms at least partially different from those of myocardial stunning.

The Sox genes define a family of transcription factors that play a key role in the determination of cell fate during development. The preferential expression of the Sox15 in the myogenic precursor cells led us to suggest that the Sox15 is involved in the specification of myogenic cell lineages or in the regulation of the fusion of myoblasts to form myotubes during the development and regeneration of skeletal muscle. To identify the physiological function of Sox15 in mice, we disrupted the Sox15 by homologous recombination in mice. Sox15-deficient mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. Histological analysis revealed the normal ultrastructure of myofibers and the presence of comparable amounts of satellite cells in the skeletal muscles of Sox15(-/-) animals compared to wild-type animals. These results exclude the role of Sox15 in the development of satellite cells. However, cultured Sox15(-/-) myoblasts displayed a marked delay in differentiation potential in vitro. Moreover, skeletal muscle regeneration in Sox15(-/-) mice was attenuated after application of a crush injury. These results suggest a requirement for Sox15 in the myogenic program. Expression analyses of the early myogenic regulated factors MyoD and Myf5 showed the downregulation of the MyoD and upregulation of the Myf5 in Sox15(-/-) myoblasts. These results show an increased proportion of the Myf5-positive cells and suggest a role for Sox15 in determining the early myogenic cell lineages during skeletal muscle development.