Differential Signaling and Hypertrophic Responses in Cyclically Stretched vs Endothelin-1 Stimulated Neonatal Rat Cardiomyocytes.

Numerous neurohumoral factors such as endothelin (ET)-1 and angiotensin (Ang) II as well as the stretch stimulus act concertedly in the in vivo overloaded heart in inducing hypertrophy and failure. The primary culture of rat neonatal cardiomyocytes is the only in vitro model that allows the comparative analysis of growth responses and signaling events in response to different stimuli. In the present study, we examined stretched rat cardiomyocytes grown on flexible bottomed culture plates for hypertrophic growth responses (protein synthesis, protein/DNA ratio, and cell volume), F-actin filaments rearrangement (by confocal laser scanning microscopy), and for signaling events (activation of phospholipase C [PLC]-beta, protein kinase C [PKC], mitogenactivated protein [MAP] kinases) and compared these responses with ET-1 (10-8 M)-stimulated cells. Cyclic stretch for 48 h induced hypertrophic growth in cardiomyocytes indicated by increases in the rate of protein synthesis, cell volume, and diameter, which were less pronounced in comparison to stimulation by ET-1. During cyclic stretch, we observed disoriented F-actin, particularly stress-fibers whereas during ET-1 stimulation, Factins rearranged clearly in alignment with sarcomeres and fibers. The upstream part of signaling by cyclic stretch did not follow the PLCbeta-PKC cascade, which, in contrast, was strongly activated during ET-1 stimulation. Cyclic stretch and, to greater extent, ET-1 stimulated downstream signaling through ERK, p38 MAP kinase, and JNK pathways, but the involvement of tyrosine kinase and PI3 kinase-Akt signaling during cyclic stretch could not be proven. Taken together, our results demonstrate that both cyclic stretch and ET-1 induce hypertrophic responses in cardiomyocytes with different effects on organization of F-actin stress fibers in case of stretch. Furthermore, on the short-term basis, cyclical stretch, unlike ET-1, mediates its hypertrophic response not through activation of PLC-beta and PKC but more likely through integrin-linked pathways, which both lead to downstream activation of the MAP kinase family.

Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.

Presence of SERCA and calcineurin during fetal development of porcine skeletal muscle.

Mechanisms involved in skeletal myofiber differentiation during fetal development of large animals are poorly understood. Studies in small animals suggest that the calcineurin (Cn) pathway is involved in myofiber differentiation. Neural activity is a prerequisite for Cn activity, implying maintenance of sustained low intracellular Ca(2+) concentrations. To study the role of Cn in fetal myofiber differentiation, we monitored the temporal and spatial distribution of Cn subunits, sarcoplasmic reticulum Ca(2+) ATPase (SERCA), phospholamban (PLB), and myosin heavy chain (MyHC) isoforms in relation to ingrowing nerves in porcine semitendinosus muscle (m. semitendinosus) at 55 and 75 days of gestation (dg) and at term. Immunofluorescence analysis revealed the presence of Cn subunits and SERCA isoforms at all analyzed stages. Cn distribution was not fiber-type specific, but expression became more prominent at term. At 75 dg, differential SERCA2 expression was accompanied by perinuclear PLB in primary fibers. SERCA1 was expressed in all fiber types at all stages. No specific MyHC isoform distribution was seen in relation to neuromuscular contacts, although neuromuscular contacts were present. From these results we speculate that in porcine m. semitendinosus differential SERCA2 expression precedes differential Cn expression. The question whether the Cn pathway is involved in prenatal myofiber differentiation needs further studies.

Architecture and mineralization of developing cortical and trabecular bone of the mandible.

Ossification of the presumptive trabecular bone in the mandibular condyle and the presumptive cortical bone in the mandibular corpus of the pig mandible was investigated during development, using micro-computed tomography (microCT). Three-dimensional architecture and mineralization characteristics were assessed from ten pigs of different developmental ages. In the condyle, increases in trabecular thickness and separation and a decrease in the trabecular number, led to an unchanged bone volume fraction. A conversion from rod-like into plate-like trabeculae was observed. Bone volume and trabecular thickness were always higher in the corpus, where an increase in bone volume fraction was caused by an increase in the trabecular thickness and a decrease in separation. A transition from a plate-like structure into a more compact structure took place. The average degree of mineralization in the condyle and the corpus increased with age. In the corpus, the degrees of mineralization were higher than in the condyle. The differences between the condyle and corpus and the changes with age could be explained by differences in the distribution of mineralization within the trabecular elements. Generally, the degrees of mineralization increased from the surface toward the centers of the trabecular elements, indicating growth of the trabecular elements by the surface apposition of new mineral.

Myosin heavy chain isoforms in equine gluteus medius muscle: comparison of mRNA and protein expression profiles.

The major structural protein in skeletal muscle, myosin heavy chain (MyHC), is primarily transcriptionally controlled. We compared the expression of MyHC isoforms on the mRNA and protein level in biopsies from the m. gluteus medius from adult untrained horses. In transverse sections, the majority of fibers showed qualitatively identical mRNA and protein expression patterns. However, coexpression of 2a and 2d/x MyHCs was substantially more common at the protein than at the mRNA level, suggesting a fine-tuning of these two genes in normal muscle not subjected to any training protocol. Because transverse sections give a limited sampling of mRNA expression in the case of uneven distribution of transcripts in a muscle fiber, we also analyzed longitudinal sections. We present, for the first time, evidence that expression of MyHC mRNA and protein was equal along the length of the fiber. Hence, mRNA expression is not regulated by differential expression of isoforms by separate myonuclei. It is concluded that the number of protein hybrid fibers in equine gluteus medius muscle is controlled by alteration of the transcription pattern uniformly along the fiber, rather than by simultaneous transcription of genes. The differences with the results in muscle of small animals and humans are discussed.

Embedding of large specimens in glycol methacrylate: prerequisites for multi-signal detection and high-resolution imaging.

Acrylic resin mixtures are commonly used to study microscopic sections of biological specimens, giving the advantage of good morphological preservation. Existing embedding protocols, however, are suitable for tissue blocks, not exceeding 1 mm in thickness. We have developed a protocol to embed larger specimens (up to 2 cm(3)) in Technovit 8100. This medium allowed us to perform classic histological (trichrome), silver, as well as immunohistochemical staining, needed for multi-signal detection at high-resolution imaging to reconstruct a three-dimensional interpretation of a serially sectioned muscle. The technique was applied to reconstruct the semitendinosus muscle of a fetal pig, 44 days post conception, featuring connective tissue, intramuscular nerves, blood vessels, and muscle fibre types. For the reconstruction, a technique was used that enabled us to insert high-resolution images of histological details into low-resolution images of the entire muscle.

Cyclic stretch and endothelin-1 mediated activation of chloride channels in cultured neonatal rat ventricular myocytes.

To date various types of Cl(-) currents have been recorded in cardiac myocytes from different regions of the heart and from different species. Most of these are silent under basal conditions, but are rapidly activated under the influence of various agonists or physical stress that, in the long term, also lead to development of hypertrophy. Previously, we identified three different Cl(-) channel activities in neonatal rat cardiomyocytes: (i) Ca(2+) regulated, (ii) cAMP regulated (cystic fibrosis transmembrane conductance regulator Cl(-) channels) and (iii) osmoregulated Cl(-) channels. In this study, we examined comparatively the effects of cyclic stretch and endothelin-1 (ET-1) on Cl(-) channel activity in primary cultures of neonatal rat ventricular myocytes using an (125)I-efflux assay. About 4 min after the start of the (125)I-efflux (mean basal rate amounts 6.3% of total (125)I incorporated/min), the addition of 10 nM ET-1 or the application of cyclic stretch rapidly and transiently increased (125)I-efflux by 3.8%/min and 0.8%/min respectively above the basal rate. The stretch induced (125)I-efflux rate could be blocked by 100 microM Gd(3+) but it had no effect on the ET-1 response. After 24 h stimulation by ET-1 or cyclic stretch the myocytes responded by hypertrophy which is detected by increases of (3)H-leucine incorporation into protein and protein/DNA ratio. In conclusion, cyclic stretch as well as ET rapidly and transiently activate Cl(-) channels in rat neonatal cardiomyocytes. The results suggest that the activation of distinct types of Cl(-) channels (co)transduce the stretch- and agonist-induced hypertrophic responses in these myocytes.