Controlled mechanical ventilation (CMV) plays a key role in triggering the impaired diaphragm muscle function and the concomitant delayed weaning from the respirator in critically ill intensive care unit (ICU) patients. To date, experimental and clinical studies have primarily focused on early effects on the diaphragm by CMV, or at specific time points. To improve our understanding of the mechanisms underlying the impaired diaphragm muscle function in response to mechanical ventilation, we have performed time-resolved analyses between 6h and 14days using an experimental rat ICU model allowing detailed studies of the diaphragm in response to long-term CMV. A rapid and early decline in maximum muscle fibre force and preceding muscle fibre atrophy was observed in the diaphragm in response to CMV, resulting in an 85% reduction in residual diaphragm fibre function after 9-14days of CMV. A modest loss of contractile proteins was observed and linked to an early activation of the ubiquitin proteasome pathway, myosin:actin ratios were not affected and the transcriptional regulation of myosin isoforms did not show any dramatic changes during the observation period. Furthermore, small angle X-ray diffraction analyses demonstrate that myosin can bind to actin in an ATP-dependent manner even after 9-14days of exposure to CMV. Thus, quantitative changes in muscle fibre size and contractile proteins are not the dominating factors underlying the dramatic decline in diaphragm muscle function in response to CMV, in contrast to earlier observations in limb muscles. The observed early loss of subsarcolemmal neuronal nitric oxide synthase activity, onset of oxidative stress, intracellular lipid accumulation and post-translational protein modifications strongly argue for significant qualitative changes in contractile proteins causing the severely impaired residual function in diaphragm fibres after long-term mechanical ventilation. For the first time, the present study demonstrates novel changes in the diaphragm structure/function and underlying mechanisms at the gene, protein and cellular levels in response to CMV at a high temporal resolution ranging from 6h to 14days.

The pacemaker current I fof the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those ofI f, suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.

Background-Structural and phenotypic changes of cardiomyocytes characterize atrial fibrillation. We investigated whether changes in the glucose-regulated protein GRP94, which is essential for cell viability, occur in the presence of chronic atrial fibrillation.Methods and Results-Samples of fibrillating atrial myocardium obtained from both goat and human hearts were analyzed for GRP94 expression by an immunologic approach. In goats, atrial fibrillation was induced and maintained for 2, 4, 8, and 16 weeks. After 16 weeks of atrial fibrillation, cardioversion was applied and followed by 8 weeks of sinus rhythm. GRP94 levels doubled in goat atrial myocytes after 4 to 16 weeks of fibrillation with respect to normal atria and returned to control levels in atrial myocardium of cardioverted goats. Immunohistochemical analyses confirm that GRP94 increase occurred within cardiomyocytes. Significantly, increased levels of GRP94 were also observed in samples from human fibrillating atria. In the absence of signs of myocyte irreversible damage, the GRP94 increase in fibrillating atria is comparable to GRP94 levels observed in perinatal goat myocardium. However, calreticulin, another endoplasmic reticulum protein highly expressed in perinatal hearts, does not increase in fibrillating atria, whereas inducible HSP70, a cytoplasm stress protein that is expressed in perinatal goat hearts at levels comparable to those observed in the adult heart, shows a significant increase in chronic fibrillating atria.Conclusions-Our data demonstrate a large, reversible increase in GRP94 in fibrillating atrial myocytes, which may be related to the appearance of a protective phenotype.

We previously showed that skeletal myocytes of the adult rabbit do not accumulate the endoplasmic reticulum glucose-regulated protein GRP94, neither constitutively nor inducibly, at variance with skeletal myocytes during perinatal development (5), Here we show that C2C12 cells upregulate GRP94 during differentiation and, similarly to primary cultures of murine skeletal myocytes, specifically display GRP94 immunoreactivity on the cell surface. Stable transfection of C2C12 cells with grp94 antisense cDNA shows lack of myotube formation in clones displaying >40% reduction in GRP94 amount. The same result is obtained after irt vivo injection of grp94-antisense myoblasts, Conversely, GRP94 overexpression is accompanied by accelerated myotube formation. Analyses of BrdU incorporation, p21 nuclear translocation, and muscle-gene expression show that muscle differentiation is not apparently affected in grp94-antisense clones. In contrast, cell-surface GRP94 is greatly reduced in grp94-antisense clones, as shown by immunocytochemistry and precipitation of cell-surface biotinylated proteins. Thus, cell-surface expression of GRP94 is necessary for maintenance of fusion competence. Furthermore, differentiating C2C12 cells grown in the presence of anti-GRP94 antibody show decreased myotube number suggesting that cell-surface GRP94 is directly involved in myoblast fusion process.

The glucose-regulated protein GRP94 is a stress-inducible glycoprotein that is known to be constitutively and ubiquitously expressed in the endoplasmic reticulum of mammalian cells. From a rabbit heart cDNA library we isolated four overlapping clones coding for the rabbit homologue of GRP94 mRNA. Northern blot analysis shows that a 3200 nt mRNA species corresponding to GRP94 mRNA is detectable in several tissues and it is 5-fold more abundant in the heart than in the skeletal muscle. Hybridization analysis in situ shows that GRP94 mRNA accumulates in cardiac myocytes, whereas in skeletal muscles it is not detectable in myofibres. A monoclonal antibody raised by using a 35 kDa recombinant GRP94 polypeptide as immunogen detects a single reactive polypeptide of 94 kDa in a Western blot of liver and heart homogenates and does not react with skeletal muscle homogenates. Conversely, GRP94 mRNA and protein are detectable in both cardiac and skeletal muscle myocytes of fetal and neonatal rabbits. After 24 h of endotoxin administration to adult rabbits, GRP94 mRNA accumulation increases 3-fold in both heart and skeletal muscle and it is followed by a comparable increase in protein accumulation. However, hybridization and immunohistochemistry in situ do not reveal any change in the expression of GRP94 mRNA and protein in skeletal muscle myocytes after endotoxin treatment. Thus skeletal muscle fibres display a unique regulation of the GRP94 gene, which is up-regulated during perinatal development, whereas in the adult animal it is apparently silent and not responsive to endotoxin treatment.

Intracellular calcium overload of guinea pig cardiomyocytes is accompanied by troponin T cross-linking, which is revealed by changes in immunoreactivity of anti-troponin T antibodies. We presently investigated whether the same process is detectable in the human heart. Immunohistochemistry shows myofibrillar staining with BN-59 anti-troponin T antibody with rare cardiomyocytes in samples obtained at surgery, whereas approximately 50% of myocytes are labeled in heart samples taken at autopsy within 3 hours of death, and every cardiomyocyte is stained after exposure of biopsy sections to 10 mmol/L calcium. Western blot analysis shows reactive polypeptides of approximately 70 and 85 to 90 kd in addition to troponin T in both treated and autopsy heart sections. Neither reactivity in immunohistochemistry nor additional reactive polypeptides in Western blot are detectable when calpain or transglutaminase is inhibited during exposure of sections to high calcium. Troponin T crosslinking occurs also in isolated myofibrils, which show staining with BN-59 at either sarcomeric A or I bands. Labeling with TdT-mediated dUTP nick and labeling (TUNEL) to demonstrate apoptosis reveals DNA fragmentation in BN-59-positive myocytes. Thus, troponin T cross-linking occurs in human cardiac myocytes concomitantly with apoptosis and autopsy autolysis, suggesting that similar cytosolic alterations can be produced by different types of myocyte death.

We investigated the mRNA distribution of three different ryanodine receptors (RyR) and of the intracellular Ca(2+)-release channel/inositol 1,4,5-trisphosphate receptor (IP3R) type 1 in the rat heart during development and aging. In situ hybridization analysis shows that RyR1 mRNA is never expressed in the heart at any of the stages examined: RyR2 mRNA is detectable in cardiomyocytes in the early embryonic stages, whereas RyR3 mRNA accumulates in cardiomyocytes around birth. IP3R mRNA appears at first in the primitive atrium at embryonic day 11 and in subsequent stages it is detectable also in a minor population of ventricular myocytes, which presumably correspond to conduction system precursors. In the adult heart, no apparent difference in hybridization signal intensity is observed between atrial and ventricular working myocytes either with RyR2, RyR3 or IP3R cRNA probes, except for myocytes of the heart conduction system, which differ from working myocytes in the intensity of the hybridization signals for each probe. Additional differences are detected in the senescent heart with the IP3R cRNA probe, which hybridizes with atrial myocytes stronger than with ventricular ones. RNase protection analysis confirms the temporal differences in RyR2 and RyR3 transcript accumulation observed during heart development and reveals a significant increase of IP3R mRNA in the atrial myocardium during aging. Thus, the composition of intracellular Ca(2+)-release channel mRNAs of the rat heart shows temporal and regional variations: such changes might reflect important differences in transcriptional regulation of these genes among myocytes.

We previously demonstrated that conduction-system myocytes of the rabbit heart express cytoskeletal proteins immunologically related to neurofilaments. In order to determine more precisely the nature of these proteins, we screened an expression cDNA library, prepared from the sino-atrial node region of the rabbit heart, using a monoclonal antibody which reacts with the M subunit of neurofilaments. Sequence analysis of the isolated cDNA clones shows high homology with rat and human neurofilament M mRNAs. Northern blot analysis demonstrates hybridization with a transcript expressed in brain, with the size expected for neurofilament M mRNA. An mRNA species of the same size is also detectable in the Northern blot of the sino-atrial node region RNA.In situhybridization documents that in the adult rabbit the transcript accumulates in neurons and is localized in myocytes of the sino-atrial and atrio-ventricular nodes and of the atrio-ventricular bundle and bundle branches, but not in working atrial and ventricular myocytes. Developmental analysis was undertaken in order to determine the distribution of the neurofilament M mRNA in the rabbit embryonic heart.In situhybridization shows that neurofilament M mRNA is detectable in a few ventricular myocytes in proximity to the atrio-ventricular groove after 9.5 days of embryonic development and it is accompanied by the presence of the protein. At subsequent stages of development neurofilament M mRNA is detectable in a number of cardiac myocytes, which are mainly localized at the atrio-ventricular junction and in the ventricular subendocardium and presumably correspond to myocytes of the heart conduction system.

BACKGROUND During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the troponin T subunit is a substrate for calpains, we investigated the effects of irreversible myocyte damage on troponin T immunoreactivity. METHODS AND RESULTS Hearts from adult guinea pigs (n=32) were perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-reperfusion and Ca2+ overloaded hearts show reactive polypeptides of about 240 to 260 kD and 65 to 66 kD in addition to troponin T. A similar pattern of immunoreactivity is observed with an anti-troponin I antibody. Histochemical troponin T immunoreactivity and reactivity on high-molecular-weight polypeptides are detectable in normal heart samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited. CONCLUSIONS The evolution of the ischemic injury is accompanied by changes in troponin T immunoreactivity as a consequence of the calcium-dependent activation of both calpain proteolysis and transglutaminase cross-linking.

Conduction system myocytes are a subpopulation of cardiac myocytes that display unique electrophysiologic properties. Significant differences in cellular components of conduction myocytes have been demonstrated by the application of in situ procedures using both immunologic and molecular probes. Although molecular and cellular biology investigations are still at the beginning, they unequivocally show that conduction myocytes are a highly heterogeneous myocyte population, whose difference from working myocytes might reflect both the degree of functional specialization and the origin from a cell lineage distinct from myocardial cells.

The gene coding for mouse cardiac troponin I (TnI) has been cloned and sequenced. The cardiac TnI gene contains 8 exons and has an exon-intron organization similar to the quail fast skeletal TnI gene except for the region of exons 1-3, which is highly divergent. Comparative analysis suggests that cardiac TnI exon 1 corresponds to fast TnI exons 1 and 2 and that cardiac exon 3, which codes for most of the cardiac-specific amino-terminal extension and has no counterpart in the fast gene, evolved by exon insertion/deletion. The amino acid sequence of cardiac TnI exon 4 shows limited homology (36% identity) with fast TnI exon 4 but is remarkably similar (79% identity) to the corresponding sequence of slow TnI, possibly reflecting an isoform-specific TnC-binding site. The cardiac TnI gene is one of the very few contractile protein genes expressed exclusively in cardiac muscle. To identify the regulatory sequences responsible for the cardiac-specific expression of this gene we transfected cultured cardiac and skeletal muscle cells with fragments up to 4.0 kilobases of the 5'-flanking region linked to a reporter gene. Deletion analysis reveals four major regions in the 5'-flanking sequence, a minimal promoter region, which directs expression at low level in cardiac and skeletal muscle cells, and two upstream cardiac-specific positive regions separated by a negative region.

We have compared the efficiency of direct gene transfer in normal and regenerating rat skeletal muscle. Muscle necrosis and regeneration was induced by intramuscular injection of bupivacaine in the soleus muscle of adult rats. Plasmids containing beta-galactosidase (beta-gal) or chloramphenicol acetyltransferase (CAT) genes driven by viral promoters were injected 3 days after bupivacaine treatment into the regenerating and the contralateral uninjured muscles. Expression of CAT activity was > 80-fold higher in regenerating compared to control muscles at 7 days post-transfection, but decreased at 30 and 60 days. Southern blot analysis showed that the predominant form of CAT DNA was episomal in transfected muscles; however, CAT activity measurements performed on the same transfected muscles showed no precise correlation between enzymatic activity and amount of plasmid DNA. Expression of beta-gal was detected in numerous regenerating fibers of the injured soleus muscles at 7 days post-transfection; in contrast, only rare positive fibers were found in control muscles. Focal infiltrates of mononuclear cells, which surround and invade selectively beta-gal-positive fiber segments, were observed at 30 days post-transfection, suggesting that immune mechanisms are implicated in the progressive loss of transgenes with time. The finding that regenerating muscle fibers display a higher efficiency of transfection may be relevant to gene therapy of Duchenne muscular dystrophy, because regenerating fibers are numerous in the early stages of the disease.

The pattern of expression of the neuropeptide CGRP and its encoding mRNA has been determined by immunohistochemistry and in situ hybridization in the mouse olfactory pathway during development. Specific CGRP transcripts are first detected at E13 followed by the appearance of the peptide at E15. Both peptide and transcript are present until birth; their expression then appears to be down-regulated since postnatally the peptide is only observed in some olfactory receptor neurons. A monoclonal antibody that specifically recognizes the neurofilament subunit NF-M has been used in order to identify olfactory and trigeminal axons. Our results demonstrate that CGRP is expressed in olfactory neurons and their axons during development, thus supporting further its role as a differentiation factor during olfactory bulb ontogenesis.

We have previously established that immunoreactivity for the triplet of polypeptides that comprise the class IV intermediate filament proteins (NFP-triplet) is localized in specific subpopulations of neurons in guinea-pig sensory and autonomic ganglia. Antibodies to novel neurofilament proteins, including a polyclonal antibody to a 57 kDa neuronal intermediate filament polypeptide (NIF57kD) and a monoclonal antibody (CH1) to a 150 kDa intermediate filament, or associated, protein were used in combination with antibodies to the NFP-triplet for double-labelling immunohistochemistry. The results show that different subpopulations of neurons in the guinea-pig dorsal root ganglia, coeliac ganglion and enteric ganglia can be distinguished by their complementary immunoreactivity for these proteins. In dorsal root ganglia, larger neurons are intensely immunoreactive for the NFP-triplet while immunoreactivity with CH1 and NIF57kD antibodies is restricted to the small to medium-sized neurons. In the coeliac ganglion, two regionally defined subpopulations of neurons can be distinguished by their immunoreactivity for either the NFP-triplet or NIF57kD, whereas CH1 labels all neurons with equal intensity. Three classes of morphologically distinct myenteric neuron subpopulations are also distinguished by their immunoreactivity for either the NFP-triplet, NIF57kD or CH1 antibodies. Two classes of submucous neurons are labelled both with CH1 and NIF57kD antibodies but show faint or no immunoreactivity for the NFP-triplet. It is concluded that intermediate filament protein immunoreactivity marks different subpopulations of neurons, which suggests that these proteins may have specific roles in neuronal function.