Time course analysis of mechanical ventilation-induced diaphragm contractile muscle dysfunction in the rat.

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 6 h and 14 days 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 9-14 days 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 9-14 days 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 6 h to 14 days.

Inositol 1,4,5-trisphosphate receptor in heart: evidence for its concentration in Purkinje myocytes of the conduction system.

Inositol 1,4,5-trisphosphate (IP3) is one of the second messengers capable of releasing Ca2+ from sarcoplasmic reticulum/ER subcompartments. The mRNA encoding the intracellular IP3 receptor (Ca2+ channel) has been detected in low amounts in the heart of various species by Northern blot analysis. The myocardium, however, is a heterogeneous tissue composed of working myocytes and conduction system cells, i.e., myocytes specialized for the beat generation and stimulus propagation. In the present study, the cellular distribution of the heart IP3 receptor has been investigated. [3H]IP3 binding experiments, Western blot analysis and immunofluorescence, with anti-peptide antibodies specific for the IP3 receptor, indicated that the majority of Purkinje myocytes (the ventricular conduction system) express much higher IP3 receptor levels than atrial and ventricular myocardium. Heterogeneous distribution of IP3 receptor immunoreactivity was detected both at the cellular and subcellular levels. In situ hybridization to a riboprobe generated from the brain type 1 IP3 receptor cDNA, showed increased accumulation of IP3 receptor mRNA in the heart conduction system. Evidence for IP3-sensitive Ca2+ stores in Purkinje myocytes was obtained by double immunolabeling experiments for IP3 receptor and cardiac calsequestrin, the sarcoplasmic reticulum intralumenal calcium binding protein. The present findings provide a molecular basis for the hypothesis that Ca2+ release from IP3-sensitive Ca2+ stores evoked by alpha 1-adrenergic stimulation is responsible for the increase in automaticity of Purkinje myocytes (del Balzo, U., M. R. Rosen, G. Malfatto, L. M. Kaplan, and S. F. Steinberg. 1990. Circ. Res. 67:1535-1551), and open new perspectives in the hormonal modulation of chronotropism, and generation of arrhythmias.

Myosin types and fiber types in cardiac muscle. II. Atrial myocardium.

Antibodies were produced against myosins isolated from the left atrial myocardium (anti-bAm) and the left ventricular myocardium (anti-bVm) of the bovine heart. Cross-reactive antibodies were removed by cross-absorption. Absorbed anti-bAm and anti-bVm were specific for the myosin heavy chains when tested by enzyme immunoassay combined with SDS gel electrophoresis. Indirect immunofluorescence was used to determine the reactivity of atrial muscle fibers to the two antibodies. Three populations of atrial muscle fibers were distinguished in the bovine heart: (a) fibers reactive with anti-bAm and unreactive with anti-bVm, like most fibers in the left atrium; (b) fibers reactive with both antibodies, especially numerous in the right atrium; (c) fibers reactive with anti-bVm and unreactive with anti-bAm, present only in the interatrial septum and in specific regions of the right atrium, such as the crista terminalis. These findings can be accounted for by postulating the existence of two distinct types of atrial myosin heavy chains, one of which is antigenically related to ventricular myosin. The tendency for fibers labeled by anti-bVm to occur frequently in bundles and their preferential distribution in the crista terminalis, namely along one of the main conduction pathways between the sinus node and the atrioventricular node, and in the interatrial septum, where different internodal tracts are known to converge, suggests that these fibers may be specialized for faster conduction.

Myosin types and fiber types in cardiac muscle. III. Nodal conduction tissue.

The sinoatrial (SA) and atrioventricular (AV) nodes are specialized centers of the heart conduction system and are composed of muscle cells with distinctive morphological and electrophysiological properties. We report here results of immunofluorescence and immunoperoxidase studies on the bovine heart showing that a large number of SA and AV nodal cells share a distinct type of myosin heavy chain (MHC) which is not found in other myocardial cells and can thus be used as a cell-type-specific marker. The antibody used in this study was raised against fetal skeletal myosin and reacted with fetal skeletal but not with adult skeletal MHCs. Both atrial and ventricular fibers, as well as fibers of the ventricular conduction tissue were unlabeled by this antibody. Specific reactivity was exclusively seen in most cells in the central portions of the SA and AV nodes and rare cells in perinodal areas. However, a number of nodal cells, particularly those located in the peripheral nodal regions, were unreactive with this antibody. The myosin composition of nodal tissues was also explored using two antibodies reacting specifically with alpha-MHC, the predominant atrial isoform, and beta-MHC, the predominant ventricular isoform. Most nodal cells were reactive for alpha-MHC and a number of them also for beta-MHC. Variation in reactivity with the two antibodies was also observed in perinodal areas: at these sites a population of large fibers reacted exclusively for beta-MHC. These findings point to the existence of muscle cell heterogeneity with respect to myosin composition both in nodal and perinodal tissues.

VAMP/synaptobrevin isoforms 1 and 2 are widely and differentially expressed in nonneuronal tissues.

VAMP/synaptobrevin is part of the synaptic vesicle docking and fusion complex and plays a central role in neuroexocytosis. Two VAMP (vesicle-associated membrane protein) isoforms are expressed in the nervous system and are differently distributed among the specialized parts of the tissue. Here, VAMP-1 and -2 are shown to be present in all rat tissues tested, including kidney, adrenal gland, liver, pancreas, thyroid, heart, and smooth muscle. The two isoforms are differentially expressed in various tissues and their level may depend on differentiation. VAMP-1 is restricted to exocrine pancreas and to kidney tubular cells, whereas VAMP-2 is the predominant isoform present in Langerhans islets and in glomerular cells. Both isoforms show a patchy vesicular intracellular distribution in confocal microscopy. The present results provide evidence for the importance of neuronal VAMP proteins in the physiology of all cells.

Type 2X-myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene.

We have previously reported the identification of a distinct myosin heavy chain (MyHC) isoform in a major subpopulation of rat skeletal muscle fibers, referred to as 2X fibers (Schiaffino, S., L. Gorza, S. Sartore, L. Saggin, M. Vianello, K. Gundersen, and T. Lømo. 1989. J. Muscle Res. Cell Motil. 10:197-205). However, it was not known whether 2X-MyHC is the product of posttranslational modification of other MyHCs or is coded by a distinct mRNA. We report here the isolation and characterization of cDNAs coding a MyHC isoform that is expressed in type 2X skeletal muscle fibers. 2X-MyHC transcripts differ from other MyHC transcripts in their restriction map and 3' end sequence and are thus derived from a distinct gene. In situ hybridization analyses show that 2X-MyHC transcripts are expressed at high levels in the diaphragm and fast hindlimb muscles and can be coexpressed either with 2B- or 2A-MyHC transcripts in a number of fibers. At the single fiber level the distribution of each MyHC mRNA closely matches that of the corresponding protein, determined by specific antibodies on serial sections. In hindlimb muscles 2X-, 2A-, and 2B-MyHC transcripts are first detected by postnatal day 2-5 and display from the earliest stages a distinct pattern of distribution in different muscles and different fibers. The emergence of type 2 MyHC isoforms thus defines a distinct neonatal phase of fiber type differentiation during muscle development. The functional significance of MyHC isoforms is discussed with particular reference to the velocity of shortening of skeletal muscle fibers.

Myosin types and fiber types in cardiac muscle. I. Ventricular myocardium.

Antisera against bovine atrial myosin were raised in rabbits, purified by affinity chromatography, and absorbed with insolubilized ventricular myosin. Specific anti-bovine atrial myosin (anti-bAm) antibodies reacted selectively with atrial myosin heavy chains, as determined by enzyme immunoassay combined with SDS-gel electrophoresis. In direct and indirect immunofluorescence assay, anti-bAm was found to stain all atrial muscle fibers and a minor proportion of ventricular muscle fibers in the right ventricle of the bovine heart. In contrast, almost all muscle fibers in the left ventricle were unreactive. Purkinje fibers showed variable reactivity. In the rabbit heart, all atrial muscle fibers were stained by anti-bAm, whereas ventricular fibers showed a variable response in both the right and left ventricle, with a tendency for reactive fibers to be more numerous in the right ventricle and in subepicardial regions. Diversification of fiber types with respect to anti-bAm reactivity was found to occur during late stages of postnatal development in the rabbit heart and to be influenced by thyroid hormone. All ventricular muscle fibers became strongly reactive after thyroxine treatment, whereas they became unreactive or poorly reactive after propylthiouracil treatment. These findings are consistent with the existence of different ventricular isomyosins whose relative proportions can vary according to the thyroid state. Variations in ventricular isomyosin composition can account for the changes in myosin Ca2+-activated ATPase activity previously observed in cardiac muscle from hyper- and hypothyroid animals and may be responsible for the changes in the velocity of contraction of ventricular myocardium that occur under these conditions. The differential distribution of ventricular isomyosins in the normal heart suggests that fiber types with different contractile properties may coexist in the ventricular myocardium.

The human gene coding for HCN2, a pacemaker channel of the heart.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, underlying 'pacemaker' currents (I(f)/Ih), are involved in pacemaker activity of cardiac sinoatrial node myocytes and central neurons. Several cDNAs deriving from four different genes were recently identified which code for channels characterized by six transmembrane domains and a cyclic nucleotide binding domain. We report here the identification of the human HCN2 gene and show that its functional expression in a human kidney cell line generates a current with properties similar to the native pacemaker f-channel of the heart. The hHCN2 gene maps to the telomeric region of chromosome 19, band p13.3. This is the first identification of a genetic locus coding for an HCN channel.

Increased myocardial GRP94 amounts during sustained atrial fibrillation: a protective response?

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.

Troponin isoform switching in the developing heart and its functional consequences.

The subunits of the troponin complex-troponin C, troponin T, and troponin I-are responsible for the Ca(2+)-dependent regulation of contractile activity in heart and skeletal muscle. Distinct troponin T and I isoforms, generated by different genes or by alternative splicing from the same gene, are expressed during cardiac development. Troponin switching affects the Ca(2+) sensitivity of the contractile system and may account for the greater resistance to hypoxia and acidosis, and the impaired responsiveness to adrenergic stimulation of the fetal and neonatal heart.

Molecular and cellular diversity of heart conduction system myocytes.

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.

Immunocytochemistry of rhabdomyosarcoma. The use of four different markers.

Fast myosin and slow myosin are specific markers of skeletal muscle, in addition to myoglobin. This study of 15 specimens of rhabdomyosarcomas from 13 patients using specific antisera for the three markers as well as for desmin led to positive findings in all cases with at least one antiserum. Desmin was present in all cases; fast myosin and myoglobin were present in 10 cases each. Slow myosin was present in six cases. It appears that the combination of several markers is helpful in differentiating rhabdomyosarcomas from other tumors. The markers considered were generally more abundant in neoplastic elements with large amounts of cytoplasm. This finding suggests that the larger cells of rhabdomyosarcomas are more differentiated than smaller rhabdomyoblasts, which were often negative with some of the antisera used.

Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies.

A novel type of myosin heavy chain (MHC), called 2X, has been recently identified in type 2 fibers of rat skeletal muscles using an immunochemical approach. In the present study, the same panel of anti-MHC monoclonal antibodies was used in immunohistochemistry combined with enzyme histochemistry to identify and compare type 2X fibers in hindlimb skeletal muscles of rat, mouse, and guinea pig. Immunohistochemistry shows that 2X MHC is localized in a large subset of type 2 fibers and is co-expressed with 2A or 2B MHC in a small number of fibers. Enzyme histochemistry shows that type 2X fibers display low myosin ATPase activity after pre-incubation at pH 4.3 and high activity after paraformaldehyde pre-incubation at pH 10.4. After pre-incubation at pH 4.6, myosin ATPase shows intermediate and high activity in rat and mouse 2X fibers, respectively, whereas it is low in guinea pig 2X fibers. Succinate dehydrogenase displays moderate to high activity in 2X fibers of all species. Taken together, these staining patterns allow this novel fiber population to be distinguished from the other type 2 fibers using only enzyme histochemistry. Nevertheless, the combined use of immuno- and enzyme histochemistry prevents incorrect fiber typing due to the interspecies variability of myosin ATPase activity among the correspondent fiber types, and completely modifies the presently used classification of mouse type 2 fibers.

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.

Myosin isoform expression in rat rhabdomyosarcoma induced by Moloney murine sarcoma virus.

Myosin isoform expression was analyzed in experimental rhabdomyosarcoma (RMS) using monoclonal antibodies (mAbs) and immunofluorescence techniques. Tumors induced by inoculating newborn rats with Moloney murine sarcoma virus (Mo-MSV) were examined 30-90 days after birth. Nine tumors and two lymph node metastases were studied by direct, indirect, and double immunofluorescence assays using a panel of five anti-myosin mAbs. The mAb BF-45 was specifically reactive with embryonic myosin heavy chain (MHC), mAb BF-34 was specific for a neonatal MHC epitope, mAb BF-B6 was directed against an epitope present in both embryonic and neonatal MHC, and mAbs BF-F3 and BF-32 detected epitopes present in adult MHC isoforms. Anti-desmin antibodies were also used for comparison. The results of this study show that: (1) the majority of neoplastic cells stained for desmin while only a minority of neoplastic cells were labeled by anti-myosin antibodies; (2) myosin positive tumor cells contained predominantly embryonic and neonatal MHC types but rare RMS cells reacted exclusively with anti-adult myosin antibodies; and (3) adult and embryonic MHC phenotypes were occasionally detected within the same tumor cell especially in RMS with the longest latencies. Together these results would suggest that the mechanism(s) regulating MHC gene expression in skeletal muscle cells can be altered by the transforming activity of Mo-MSV.

Heart conduction system: a neural crest derivative?

Using the anti-neurofilament monoclonal antibody iC8 we report here that muscle fibers of the conduction system of the adult and developing rabbit heart express a cytoskeletal protein antigenically and electrophoretically similar to the middle subunit of neurofilaments (NF-M). In the 11-day embryo a number of cardiac muscle cells also express a neural crest surface marker recognized by the monoclonal antibody HNK-1. Both markers are found in many cells of the 3rd and 4th branchial arches, which are populated by cells of neural crest origin. In the 11-day embryo cells of the 4th branchial arch are in close proximity to and intermingled with the atrial myocardium: cells co-expressing sarcomeric myosin heavy chain with iC8 and HNK-1 immunoreactivity are seen at these sites. The findings suggest that conduction tissue cells of the rabbit heart originate from a population of neural crest-derived cells migrating from the branchial arches into the developing heart.

[Cardiac myosins and myocardial contraction]. / Miosine cardiache e contrattilità del miocardio.

The contractile properties of cardiac muscle cells are determined by the molecular composition of the contractile apparatus and in particular by the structure of myosin. Three isoforms of myosin heavy chains have been recently identified in the mammalian heart: alpha and beta myosin heavy chains, present in atrial and ventricular myocardium, and nodal myosin heavy chain, present in sino-atrial and atrio-ventricular nodes. The alpha and beta isoforms are coded by two distinct genes whose expression is tissue and developmental stage-specific, and can be regulated by hormonal and mechanical factors. The relative concentration of the two isoforms is correlated with the maximal velocity of shortening and with the energy cost of force generation. In hyperthyroid myocardium the predominant isoform is the alpha, high ATPase myosin heavy chain and the contraction is fast but less economical; in hypothyroid and in mechanically overloaded myocardium the beta, low ATPase isoform is predominant and the contraction is slower and more economical.

Reduced amount of the glucose-regulated protein GRP94 in skeletal myoblasts results in loss of fusion competence.

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 up-regulate 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 in 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.

Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody.

Using an immunological approach, we demonstrated previously that a neurofilament-like protein is expressed in rabbit heart conduction tissue myocytes, and we proposed that these specialized cardiac muscle cells are of neuroectodermal origin. In the present study, we used the expression of the neurofilament-like protein as a marker for identifying conduction tissue cells and studying their distribution in the developing heart. In 11-day-old rabbit embryos, myocytes expressing the neurofilament-like protein were localized at the atrioventricular and the sinoatrial junctions and had a ring-like distribution. At embryonic day 12, reactive myocytes were found also in the subendocardial layer of the dorsal ventricular wall, in continuity with labeled myocytes at the atrioventricular junction. Examination of older embryos and of neonatal and adult hearts revealed that the expression of the neurofilament-like protein was not restricted to myocytes of conduction tissue regions, but it was also detectable in myocytes of the sinoatrial ring bundle, in scattered myocytes localized in the left sinal horn wall, and in the right atrium in proximity to atrioventricular sulcus tissue. Thus, using an intracellular marker, we show that precursors of adult atrial conduction tissue are distributed at the sinoatrial and atrioventricular junctions; at variance, ventricular conduction tissue precursors do not have a ring-like distribution but are localized in the subendocardial layer, in continuity with the atrioventricular junctional myocytes.