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J C Perriard

Publications and source records attributed to J C Perriard.

At least 19 recordsLinked to original sources
PubMed2002

Progressive cardiac hypertrophy and dysfunction in atrial natriuretic peptide receptor (GC-A) deficient mice.

OBJECTIVE: To investigate how permanent inhibition of guanylyl cyclase A receptor (GC-A) affects cardiac function. METHODS: Hearts of GC-A-/- and corresponding wild type mice (GC-A+/+) were characterised by histological, western blotting, and northern blotting analyses. Cardiac function was evaluated in isolated, working heart preparations. RESULTS: At 4 months of age, GC-A-/- mice had global cardiac hypertrophy (about a 40% increase in cardiac weight) without interstitial fibrosis. Examination of heart function found a significant delay in the time of relaxation; all other parameters of cardiac contractility were similar to those in wild type mice. At 12 months, the hypertrophic changes were much more severe (about a 61% increase in cardiac weight), together with a shift in cardiac gene expression (enhanced concentrations of atrial natriuretic peptide (3.8-fold), B type natriuretic peptide (2-fold), beta myosin heavy chain (1.6-fold) and alpha skeletal actin (1.7-fold) mRNA), increased expression of cytoskeletal tubulin and desmin (by 29.6% and 25.6%, respectively), and pronounced interstitial fibrosis. These changes were associated with significantly impaired cardiac contractility (+dP/dt decreased by about 10%) and relaxation (-dP/dt decreased by 21%), as well as depressed contractile responses to pressure load (all p < 0.05). CONCLUSIONS: Chronic hypertension in GC-A-/- mice is associated with progressive cardiac changes--namely, initially compensated cardiomyocyte hypertrophy, which is complicated by interstitial fibrosis and impaired cardiac contractility at later stages.

Animals↗
PubMed2001

Prosomes form sarcomere-like banding patterns in skeletal, cardiac, and smooth muscle cells.

Prosomes (20S proteasomes) constitute the catalytic core of the 26S proteasomes, but were first observed as factors associated with unstranslated mRNA. Recently, their RNase activity was discovered together with the fact that their proteolytic function is dispensable in adapted human cells. By indirect immunofluorescence using monoclonal antibodies, we demonstrate as a general phenomenon, regular intercalation of specific types of prosomes into the sarcomeric structure of all types of striated muscle. Surprisingly, in cultured smooth muscle cells without sarcomeric organization, some prosomes also form regular striations in extended projections of cytoplasmic regions. The significance of their sarcomeric distribution is not understood as yet, but the pattern we observe is very similar to that shown by others for muscle-specific mRNAs, identified by in situ hybridization, and that of the cognate proteins. A role of prosomes in the cotranslational assembly of the myofibrillar proteins is suggested, since prosomes organize into pseudo-sarcomeric patterns prior to formation de novo of the actin-myosin arrangement.

Animals↗
PubMed2001

Alterations at the intercalated disk associated with the absence of muscle LIM protein.

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain- containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.

Animals↗
PubMed2001

Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes.

Expression of epitope-tagged sarcomeric proteins in cardiomyocytes is a powerful approach for the characterization of interacting domains. Here, we report a new strategy for the study of the targeting of contractile proteins in cardiomyocytes by Sindbis virus (SIN)-mediated gene transfer. Two recombinant SIN were generated, one encoding the myosin-light chain MLC3f-eGFP fusion protein (SINrep5/MLC3f-eGFP), and the other encoding the alpha-actinin-DsRed fusion protein (SINrep5/alpha-actinin-DsRed). After infection of long-term cultured neonatal and adult rat cardiomyocytes with SINrep5/MLC3f-eGFP, the exogenous MLC3f-eGFP fusion protein localized to the sarcomeres. Freshly isolated rod-shaped ventricular cardiomyocytes infected with SINrep5/alpha-actinin-DsRed exhibited a correct incorporation of the newly synthesized alpha-actinin-DsRed fusion protein at the Z-band of the sarcomere. This allows the assumption that the exogenous protein is assembled into myofibrils in living cardiomyocy-tes using the same molecular interactions equally to the endogenous counterpart. It has been thus demonstrated that the SIN expression system makes possible the straightforward analysis of the localization of sarcomeric proteins in cultured cardiomyocytes and may offer new possibilities for the characterization of mutant proteins involved in hypertrophic cardiomyopathies.

Actinin↗
PubMed2001

Characterisation of postnatal growth of the murine heart.

Using a new technique to isolate rod-shaped cardiomyocytes from small tissue pieces we were able to analyse the developmental profile of postnatal cardiomyocyte growth in the mouse. During the first 4 postnatal days the volume of the cardiomyocytes remains relatively constant despite a concomitant increase in heart weight, indicating growth due to cell division of the cardiomyocytes, also called hyperplasia. After postnatal day 5 the volume of the cardiomyocytes increases dramatically until postnatal day 14, when the increment of the volume curve decreases again. The cardiomyocytes reach their adult volume at around 3 months of age. These measurements present the first detailed analysis of the phase of so-called developmental hypertrophy, i.e. normal cardiomyocyte growth in the mouse, and provide an essential base-line for the analysis of growth parameters in mouse models for cardiomyopathies. We used this method to characterise the growth characteristics of cardiomyocytes from MLP (muscle LIM protein) knockout mice, a mouse model for dilated cardiomyopathy. During the first 2 postnatal weeks there is no significant difference in the growth parameters between MLP knockout and wildtype mice. However, in the adult animals cardiomyocytes from MLP knockout mice are not only characterised by a more irregular shape, but also by a high variability in size compared to cardiomyocytes from wildtype animals. This suggests that the alterations in ventricular morphology in the MLP heart are not due to a general elongation of the cardiomyocytes but to myocyte disarray and ventricular wall thinning caused by the heterogeneous volume of the cardiomyocyte population.

Aging↗
PubMed2001

Mass production of embryoid bodies in microbeads.

Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell-derived, differentiated cells are of increasing importance as a potential source for non-proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula-like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.

Animals↗
PubMed2000

A novel marker for vertebrate embryonic heart, the EH-myomesin isoform.

Myomesin is a structural component of the M-band that is expressed in all types of striated muscle. Its primary function may be the maintenance of the thick filament lattice and its anchoring to the elastic filament system composed of titin. Different myomesin isoforms have been described in chicken and mice, but no particular function has been assigned to them. Here we investigate the spatio-temporal expression pattern of myomesin isoforms by means of reverse transcriptase-polymerase chain reaction and isoform-specific antibodies. We find that two alternative splicing events give rise to four myomesin isoforms in chicken contrary to only one splicing event with two possible isoforms in mice. A splicing event at the C terminus results in two splice variants termed H-myomesin and S-myomesin, which represent the major myomesin species in heart and skeletal muscle of avian species, respectively. In contrast, in mammalian heart and skeletal muscle only S-myomesin is expressed. In embryonic heart of birds and mammals, alternative splicing in the central part of the molecule gives rise to the isoform that we termed EH-myomesin. It represents the major myomesin isoform at early embryonic stages of heart but is rapidly down-regulated around birth. Thus, the strict developmental regulation of the EH-myomesin makes it an ideally suited marker for embryonic heart.

Alternative Splicing↗
PubMed2000

Cardiomyocyte cytoskeleton and myofibrillogenesis in healthy and diseased heart.

The unique cytoarchitecture of cardiomyocytes arises by complex interactions of different filamentous structures of the cytoskeleton. Intermediate filaments of the non-sarcomeric cytoskeleton are not essential for development but important for maintenance of myofibrils. Myofibrils consist of contractile proteins involved in force generation and the muscle cytoskeleton framework. The latter is essential for proper assembly and maintenance as well as for interaction with other cardiomyocytes or the extracellular matrix, thus being involved in force transmission. The information for sarcomere assembly is encoded in the proteins and some domains essential for faithful incorporation have been identified by epitope tagging experiments. Many KO mutations result in embryonic lethal phenotypes and new techniques e.g. using cardiomyocytes derived from ES cell-lines will have to be developed that allow to study such mutations in cardiomyocytes rather than whole organisms. Alterations in the expression levels of several proteins of the muscle cytoskeleton or impairment of their function by point mutations can result in increased mechanical stress in the cardiomyocytes which finally leads to cellular responses such as the development of dilated cardiomyopathy (DCM). MLP (muscle-LIM-protein) deficient mice develop DCM and changes in the mechanical coupling of cardiomyocytes result in alterations at the intercalated disks and enhanced accumulation of adherens junction proteins. Therefore, controlled interactions between proteins of the muscle cytoskeleton and contractile proteins are essential to ensure proper cardiac function and a more detailed insight in these processes might provide new tools to improve the contractile efficiency of the cardiomyocytes and thus working output in cardiomyopathies.

Animals↗
PubMed1999

Thick filament assembly occurs after the formation of a cytoskeletal scaffold.

The development of myofibrils involves the formation of contractile filaments and their assembly into the strikingly regular structure of the sarcomere. We analysed this assembly process in cultured human skeletal muscle cells and in rat neonatal cardiomyocytes by immunofluorescence microscopy using antibodies directed against cytoskeletal and contractile proteins. In particular, the question in which temporal order the respective proteins are integrated into developing sarcomeres was addressed. Although sarcomeric myosin heavy chain is expressed as one of the first myofibrillar proteins, its characteristic A band arrangement is reached at a very late stage. In contrast, titin, then myomesin and finally C-protein (MyBP-C) gradually form a regularly arranged scaffold on stress fiber-like structures (SFLS), on non-striated myofibrils (NSMF) and on nascent striated myofibrils (naSMF). Immediately subsequent to the completion of sarcomere cytoskeleton formation, the labeling pattern of myosin changes from the continuous staining of SFLS to the periodic staining characteristic for mature myofibrils. This series of events can be seen most clearly in the skeletal muscle cell cultures and--probably due to a faster developmental progression less well in cardiomyocytes. We therefore conclude that the correct assembly of a cytoskeletal scaffold is a prerequisite for correct thick filament assembly and for the integration of the contractile apparatus into the myofibril.

Actin Cytoskeleton↗
PubMed1999

Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting.

Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.

Animals↗
PubMed1999

Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments.

Myofibrillogenesis in situ was investigated by confocal microscopy of immunofluorescently labelled whole mount preparations of early embryonic chicken heart rudiments. The time-course of incorporation of several components into myofibrils was compared in triple-stained specimens, taken around the time when beating starts. All sarcomeric proteins investigated so far were already expressed before the first contractions and myofibril assembly happened within a few hours. No typical stress fibre-like structures or premyofibrils, structures observed in cultured cardiomyocytes, could be detected during myofibrillogenesis in the heart. Sarcomeric proteins like (&agr;)-actinin, titin and actin were found in a defined localisation pattern even in cardiomyocytes that did not yet contain myofibrils, making up dense body-like structures. As soon as the heart started to beat, all myofibrillar proteins were already located at their exact position in the sarcomere. The maturation of the sarcomeres was characterised by a short delay in the establishment of the pattern for M-line epitopes of titin with respect to Z-disk epitopes and the incorporation of the M-line component myomesin, which preceded that of myosin binding protein-C. Thus dense body-like structures, made up of titin, (&agr;)-actinin and actin filaments serve as the first organised complexes also during myofibrillogenesis in situ and titin functions as a ruler for sarcomere assembly as soon as its C termini have become localised. We suggest that assembly of thin and thick filament occurs independently during myofibrillogenesis in situ and that myomesin might be important for integrating thick filaments with the M-line end of titin.

Animals↗
PubMed1999

Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes.

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (&agr;)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle (&agr;)-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.

Actins↗
PubMed1999

Developmental expression of creatine kinase isoenzymes in chicken growth cartilage.

We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.

Animals↗
PubMed1998

Different behaviour of the non-sarcomeric cytoskeleton in neonatal and adult rat cardiomyocytes.

Neonatal and adult rat cardiomyocytes display differences when isolated and cultured in vitro. Whereas cells obtained from juvenile hearts adapt quite rapidly as judged by their beating, cells from adult animals undergo a complex degeneration-regeneration process of their myofibrillar apparatus. These differences are also reflected by a distinct sensitivity to drugs that affect the non-sarcomeric cytoskeleton. After long-term treatment with nocodazole, which disassembles microtubules, neonatal rat cardiomyocytes (NRC) remain relatively unaffected, whereas adult rat cardiomyocytes (ARC) are unable to spread on the substrate or to undergo the remodelling process of their myofibrils. If microfilaments are destroyed by cytochalasin D, neither NRC nor ARC spread, and they lose the capacity to assemble new myofibrils. The effects of drug treatment with both cytochalasin and nocodazole, respectively, were reversible, since normal myofibrillogenesis took place after the cells had been washed and cultivated in standard medium for 4 days. This study demonstrates that microfilaments are essential for assembly of new sarcomeres in vitro, and underlines intrinsic differences between NRC and ARC in their requirement for intact microtubules. Adult cardiomyocytes have lost a certain degree of flexibility due to their longer adaptation to the specific situation in the heart, whereas cardiomyocytes isolated from neonatal animals can maintain and assemble myofibrils in vitro even after their microtubules were destroyed.

Actin Cytoskeleton↗
PubMed1998

Permeation and pathways of human calcitonin (hCT) across excised bovine nasal mucosa.

In vitro permeation of human calcitonin (hCT), salmon calcitonin (sCT), and the somatostatin analog octreotide (SMS) through excised bovine nasal mucosa was studied applying donor/receiver experiments and confocal laser scanning microscopy. Permeabilities of gonadorelin, buserelin, Hoe013, and of thymopoietin fragments TP5 and TP4 were also included. Apparent permeability coefficients (Peff) ranged between 4 x 10(-5) (SMS) and 1.7 x 10(-5) cm s(-1) (TP4). Such Peff are typical for leaky-type airway epithelia. The order of permeabilities was: SMS >> hCT, sCT > buserelin, Hoe013 >> TP5 > TP4, LHRH. The relatively high permeability of hCT and sCT contrasted to their high molecular weight. At 37 degrees C, the permeability of hCT from mucosal to serosal (m-to-s) was found two-fold higher (p < 0.05) than from serosal to mucosal (s-to-m). Controls using 3H-mannitol showed equal permeabilities in both directions. At 4 degrees C, permeation of hCT was reduced but equal in both directions (m-to-s and s-to-m). As evaluated by confocal laser scanning microscopy, uptake studies with FITC-18-hCT revealed intracellular fluorescence in the epithelial cells, at 10 min/10 microM exposure in the form of fluorescent vesicles. By combination of these findings, an endocytotic pathway is suggested to contribute to the transport of hCT through nasal epithelium.

Animals↗
PubMed1997

Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

Actins↗
PubMed1997

MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure.

MLP is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at actin-based structures involved in cytoarchitecture organization. To assess its role in muscle differentiation, we disrupted the MLP gene in mice. MLP (-/-) mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Ultrastructural analysis revealed dramatic disruption of cardiomyocyte cytoarchitecture. At birth, these hearts were not hypertrophic, but already abnormally soft, with cell-autonomous and MLP-sensitive alterations in cytoarchitecture. Thus, MLP promotes proper cardiomyocyte cytoarchitecture, whose perturbation can lead to dilated cardiomyopathy. In vivo analysis revealed that MLP-deficient mice reproduce the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans, providing the first model for this condition in a genetically manipulatable organism.

Animals↗