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Human Molecular Genetics Advance Access originally published online on November 3, 2004
Human Molecular Genetics 2005 14(1):7-17; doi:10.1093/hmg/ddi002
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Human Molecular Genetics, Vol. 14, No. 1 © Oxford University Press 2005; all rights reserved

Expression of cardiac myosin-binding protein-C (cMyBP-C) in Drosophila as a model for the study of human cardiomyopathies

Thien Phong Vu Manh1,{dagger}, Mustapha Mokrane1,{dagger}, Emmanuelle Georgenthum1, Jeanne Flavigny2, Lucie Carrier2,3, Michel Sémériva1, Michel Piovant1 and Laurence Röder1,*

1CNRS, UMR6545, LGPD-IBDM, Université de la Méditerranée, F-13288, Marseille, France, 2INSERM, U582, Paris, F-75013, France and 3Institute of Experimental and Clinical Pharmacology, University Hospital Eppendorf, Hamburg, Germany

* To whom correspondence should be addressed at: LGPD-IBDM, Campus de Luminy Case 907, 13288 Marseille Cedex 09, France. Tel: +33 491269612; Fax: +33 491820682; Email: mailto:roder{at}ibdm.univ-mrs.fr

Received July 20, 2004; Revised September 26, 2004; Accepted October 21, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the MYBPC3 gene encoding human cardiac myosin-binding protein-C (cMyBP-C) are associated with familial hypertrophic cardiomyopathy (FHC), but the molecular mechanisms involved are not fully understood. In addition, development of FHC is sensitive to genetic background, and the search for candidate modifier genes is crucial with a view to proposing diagnosis and exploring new therapies. We used Drosophila as the model to investigate the in vivo consequences of human cMyBP-C mutations. We first produced transgenic flies that specifically express human wild-type or two C-terminal truncated cMyBP-Cs in indirect flight muscles (IFM), a tissue particularly amenable to genetic and molecular analyses. First, incorporation of human cMyBP-C into the IFM led to sarcomeric structural abnormalities and to a flightless phenotype aggravated by age and human gene dosage. Second, transcriptome analysis of transgenic IFM using nylon microarrays showed the remodelling of a transcriptional program involving 97 out of 3570 Drosophila genes. Among them, the Calmodulin gene encoding a key component of muscle contraction, found up-regulated in transgenic IFM, was evaluated as a potential modifier gene. Calmodulin mutant alleles rescued the flightless phenotype, and therefore behave as dominant suppressors of the flightless phenotype suggesting that Calmodulin might be a modifier gene in the context of human FHC. In conclusion, we suggest that the combination of heterologous transgenesis and transcriptome analysis in Drosophila could be of great value as a way to glean insights into the molecular mechanisms underlying FHC and to propose potential candidate modifier genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ability to discover new susceptibility genes for human disorders depends on the description of the genetic pathways that are disturbed by mutations in disease-associated genes. With the achievement of genome sequencing programs and the development of functional genomics strategies, it is now possible to switch from the single gene approach to a genome-wide analysis and to obtain a description of biological pathways on the basis of a more extensive survey. Furthermore, analyses of similar molecular pathways in experimental model systems that are amenable to interdisciplinary functional studies are helpful in depicting the molecular processes involved in the disease and can provide insights into the disease gene functions. Interestingly, genetic pathways are highly conserved between human and fly. Drosophila melanogaster shows 67% of similarity at the proteome level and approximately three-quarters of human disease loci have their counterparts in the fruit fly (1Go). Drosophila is actually a verified advantageous model for the study of complex human pathologies such as neurodegenerative diseases, diabetes, ageing or glaucoma (reviewed in 2Go–5Go, respectively).

Concerning cardiomyopathies, Ferrus et al. (6Go) have already shown that Drosophila is a suitable experimental system for genetic screening of muscle protein interactions leading to suppressor identification that may provide new insights into heterogeneous diseases. The structural and functional molecular bases are highly conserved in all muscles of the phylum. Sarcomeres are well-organized structures showing a high degree of order and composed mostly of highly conserved proteins. Likewise, the physiological and molecular bases of the coupling between contraction and excitation are also conserved from invertebrates to vertebrates. The aim of this study was to assess the potential of Drosophila as a model organism to help unravel some aspects of the molecular mechanisms involved in familial hypertrophic cardiomyopathy (FHC) development and provide a basis for proposing candidate modifiers genes.

FHC is a monogene disorder inherited in an autosomal-dominant fashion, for which mutations in at least 13 distinct genes, most of them encoding sarcomeric proteins, have been identified (7Go,8Go). More than 40% of the genotyped families carry a mutation in the MYBPC3 gene encoding cardiac myosin-binding protein-C (cMyBP-C) (9Go) and most mutations identified in the MYBPC3 gene are predicted to produce C-terminal truncated proteins (10Go). In addition, the phenotypic expression of FHC may be modulated by gene modifiers (reviewed in 7Go). The molecular bases of the hypertrophic response have not been fully elucidated, partly because of the difficulty of accessing human cardiac tissue. However, in vitro and in vivo studies have demonstrated that disruption of sarcomere structure and/or function may be a stimulus for hypertrophy (11Go). Other studies have implicated Ca2+ as a primary signal for cardiac hypertrophy (12Go). Recently, altered cardiac energetics has been proposed as a general mechanism underlying the phenotypic expression of FHC in both humans and mice (13Go,14Go). Furthermore, cardiac hypertrophy is accompanied by modifications of gene expression such as reactivation of fetal gene programs, up-regulation of several growth factors and down-regulation of adult forms of several cardiac genes including the cardiac ß-myosin heavy chain gene (15Go). However, the cascade of molecular events leading to this switch has yet to be determined.

In the present study, we have used a strategy involving three steps. First, a trans-heterologous approach to induce the expression of different forms of the human cMyBP-C ina specific Drosophila tissue. Second, the molecular consequences analysis of the cMyBP-C misexpression in a large-scale perspective using the cDNA nylon microarray technology. Third, the genetic validation of a candidate modifier gene, Calmodulin. The originality of our approach consists in using transcriptome analysis in order to select potential modifier genes, which accelerate the discovery of genetic interactions necessary to resolve multigenic pathologies.

We used the in vivo UAS/GAL4 system (16Go), to express wild-type or two truncated forms of cMyBP-C in Drosophila indirect flight muscles (IFM). IFM rather than Drosophila heart were chosen as the expression site mainly because of their experimental advantages (reviewed in 6Go). In particular, they fill a large part of the fly thorax and, therefore, constitute a material amenable to rapid and relatively easy structural and molecular analyses. Components of the contractile apparatus of striated muscle fibres are highly conserved in human; moreover, IFM and cardiac muscles share common physiological properties (17Go). IFM are asynchronous muscles whose contraction frequency is independent of nervous stimulation and they display stretch activation properties. IFM are also very sensitive to environmental and genetic backgrounds and slight disturbances of their structure and/or organization lead to ‘flightless’ flies, which are nonetheless viable and fertile. In the present study, we show that human cMyBP-C was readily incorporated into the IFM sarcomeres leading to structural and morphological alterations of the myofibrils and to flightless flies, a phenotype aggravated by the age and human gene dosage. Second, the transcription of 97 out of 3570 Drosophila genes was remodelled in transgenic IFM. Most of the deregulated genes have a vertebrate homologue, suggesting that elements of gene networks are conserved between flies and humans. Third, we tested the effect of mutants for Calmodulin gene which was recovered up-regulated in the transgenic IFM, on the flightless phenotype. We show that Calmodulin behaves as a modifier of the transgenic phenotype and that transcriptome analysis might thus provide the basis for a rapid screen to point out candidate modifier genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human cMyBP-C was targeted in Drosophila IFM
The human cMyBP-C protein is arranged in eight immunoglobulin-like C2-type (IgC2-like) domains and three fibronectin type-III (FN3) domains (Fig. 1) and shares the specific molecular architecture of the Myosin-binding proteins family (18Go,19Go). No cMyBP-C homologue has been identified in the Drosophila genome (1Go). However, by a BLAST search of the entire bulk of Drosophila predicted proteins with cMyBP-C protein sequence, a fairly good homology score (P=4.6e–79) was obtained with Projectin protein, a member of the invertebrate titin-like family (20Go). The best sequence alignment corresponds to 455 amino acid of the human protein arranged in an Ig–FN3–FN3–Ig–FN3 repeated motif. Projectin contains a long repeated sequence composed of this motif, but only one occurrence, located at the N-terminal end of the Drosophila protein, was found to be homologue of the cMyBP-C protein (Fig. 1A). A reciprocal BLAST search of the collection of human reference proteins generated by the NCBI (21Go) with the 455 amino acid Projectin domain produced best homology scores with four human Titin isoforms (E=1e–73) and with cMyBP-C (E=1e–55).



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  		Figure 1. The multidomain human cMyBP-C and Drosophila Projectin proteins. (A) Diagram of WT human cMyBP-C (1274 amino acids, 150 kDa, Swis-Prot/TrEMBL Protein Knowledgebase accession number: Q14896 [GenBank] ) and Drosophila Projectin (9120 amino acids, 1 MDa, Swis-Prot/TrEMBL Protein Knowledgebase accession number: O76281) proteins alignment. cMyBP-C and Projectin are composed of the specific arrangement of 8 Ig/3 FN3 and 39 Ig/39 FN3 modules, respectively. cMyBP-C phosphorylation sites and projectin kinase domain are indicated. The 455 amino acids sequence homologous between cMyBP-C and Projectin is boxed. (B) Diagram of WTt, M0t and M6t cMyBP-C transgenic proteins showing Actin, Myosin and Titin binding sites and phosphorylation sites. Ig and FN3 modules are identified as C0–C10, N- to C-terminal. C-terminal truncated cMyBP-C M6t lacks part of C10 domain, responsible for binding Myosin, and contains 19 additional amino acids (black rectangle). M0t lacks the C5–C10 domains, responsible for Myosin and Titin binding, and contains two additional amino acid residues (black rectangle).

 
The absence of a cMyBP-C counterpart in the Drosophila genome ruled out considering the loss of function mutation approach, and consequently, a human cMyBP-C misexpression study was carried out. Using the UAS–GAL4 system (see Materials and Methods), transgenic flies were produced in which a full-length wild-type (WTt) or two C-terminal truncated forms of human cMyBP-C proteins (M6t and M0t, Fig. 1B) were expressed in the IFM. The SG29.1-GAL4 driver used here induces expression during metamorphosis in the developing IFM, which is de novo built-up during this stage (22Go) and during the adult life. The human protein expressed with the aid of the UAS–GAL4 system was expected to be targeted to the sarcomere through unwarranted interactions with at least one specific endogenous sarcomeric protein. The conserved Ig-like and type-III fibronectin motifs are known to mediate protein–protein interactions. This could lead to a disturbance of the Drosophila protein network functions, similar to the way dominant-negative variants can alter protein–protein interactions, and thus induce measurable muscle dysfunction.

Human cMyBP-C protein expression in IFM led to flightless flies
The correct size and antigenicity of the expressed proteins were checked by western blot using an anti-Myc or an anti-cMyBP-C antibody. Within IFM of 7-day-old flies, the three human cMyBP-Cs appeared to be stably expressed and undegraded (data not shown).

Whatever the cMyBP-C forms expressed in IFM, transgenic flies exhibited a flightless phenotype, which was associated with an abnormal wing position when compared with control lines (data not shown). These phenotypes are age- and dose-dependent. Among the different transgenic lines, which all produced similar effects, six have been analysed in detail (Fig. 2; see Materials and Methods). On the one hand, considering an equal copy number of a specific transgene, the proportion of flightless flies increases with age. For example, 50% of flies were unable to fly at day 8 when bearing two M6t copies compared with 100% at day 16 (Fig. 2B). On the other hand, at a given day of adult life, the percentage of flightless flies increased with the copy number of the transgene (Fig. 2A–C). For example, 50% of flies were unable to fly at day 6 with four copies of any of the human transgene when compared with day 8–9 with two copies (compare Fig. 2A–C). Moreover, a significant difference was observed between the different transgenic lines carrying only one copy of the transgenes. At day 13, 16 and 18, 50% of flightless flies expressing one M0t, M6t or WTt transgene copy were obtained, respectively (Fig. 2A–C). Interestingly, this result indicates that the expression of the shortest M0t transgene induces a more drastic IFM dysfunction than M6t or WTt proteins, an effect measurable at a threshold obtained with one copy. However, we cannot rule out that the origin of this graded effect was due to a position effect related to the transgene genomic insertion site.



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  		Figure 2. cMyBP-C expression in the IFM induces an age- and dose-dependent flightless phenotype. Percentage of flightless transgenic flies expressing four, two or one copy of WTt (A), M6t (B) or M0t (C) cMyBP-C transgenes under the control of SG29.1 driver as a function of age. The percentage of flightless flies increases with age and with transgene copy number. The effect of transgene copy number is highlighted with vertical and horizontal dashed lines; for example, expression of four, two or one copy of the WTt transgene leads to 73, 50 or 15% of flightless flies, respectively, at day 9 (vertical), and to 50% flightless flies at day 6, 9 or 18 (horizontal). Whatever the WTt (A), M6t (B) or M0t (C) transgene form, extremely significant differences were observed in function of the copy number (**P=0 for 4, 2 and 1 copies). Considering a given transgene copy number: 4, 2 or 1, the differences induced by WTt, M6t and M0t transgenes expression are, respectively, very significant (*P=0.019), not significant (P=0.69) and extremely significant (**P=8.29e–09) (log-rank test and flight test control lines, see Materials and Methods).

 
Human cMyBP-C proteins were incorporated into the fly sarcomere
Immunofluorescent stainings showed that each transgenic human protein is incorporated into the same location of IFM sarcomeres, i.e. the Z–I region. In double labelling experiments, the transgenic human proteins co-localized with Kettin at the Z-band of IFM sarcomere (Fig. 3A–C). At physiological sarcomere length, I-band of the Drosophila IFM sarcomere is very short (23Go). In addition, ultrastructural studies reported further suggest that after 10 days most of the IFM sarcomeres in transgenic flies were recovered in contracted state, even in relaxing medium. In contracted muscles, because the I-band is virtually absent or very sharp and can be squeezed with the Z-band, the localization of the human transgenic proteins was examined in mechanically stretched IFM fibres. Transgenic proteins were located within the I-band and excluded from the Z-band (Fig. 3C–E). Interestingly, these results indicate that human cMyBP-C can specifically bind at least one Drosophila sarcomeric protein, localized within the I-band. Moreover, this interaction must involve cMyBP-C protein domains comprised within the shortest transgenic form (Fig. 1B).



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  		Figure 3. Transgenic cMyBP-C localizes in the Z–I region of the IFM sarcomere. Adult myofibril double stained with anti-Myc which reveals the human WTt cMyBP-C protein (green, A) and anti-Kettin (red, B) used as a marker of Z-band border (54Go,55Go). (C) Shows the merged image (yellow in overlapping regions). All cMyBP-C forms were incorporated in the same region of the IFM sarcomere and co-localized with Kettin. (D) A mechanically stretched myofibril: phase contrast observation shows a photon dense Z-line (arrow) and anti-Myc staining reveals the expressed cMyBP-C protein (red, E). cMyBP-C was located in the I-band and excluded from the Z-line (arrow) as shown in the merge (F). IFM were prepared from 7-day-old-transgenic flies expressing two copies of cMyBP-C transgenes.

 
IFM myofibrillar structure was affected by expression of transgenic cMyBP-C
The ultrastructure of transgenic IFM fibres of 15-day-old flightless animals expressing two copies of the human gene was examined by electron microscopy. Longitudinal sections showed disturbed myofibrillar organization mainly involving distortion and streaming of the Z-band across the sarcomere (Fig. 4). Occasionally, M-line deformation was also observed. Furthermore, transgenic sarcomere lengths were found to be ~10% shorter on average than in SG29.1-GAL4 control flies (Fig. 5). In addition, our results suggest that M0t expression induced a more severe sarcomere length reduction when compared with WTt and M6t forms. Shorter sarcomeres observed under relaxing conditions might indicate a contracted state. The number of structural defects recovered within the transgenic sarcomeres increasing with age suggests a causal correlation between structural defects and inability to fly. The affected muscles would appear not to be able to execute any longer the contraction–relaxation cycles required for wing movement.



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  		Figure 4. Electron micrographs of adult IFM longitudinal sections. (A) Control, (B) WTt (C) M6t and (D) M0t transgenic IFM. In B–D, the Z-line was distorted (asterisk; compare with control) and M-line distortions were also sometimes observed (black arrow); the I-band appears stretched and disorganized (white arrows). The IFM were prepared from 15-day-old (SG29.1-GAL4) control flies and transgenic flies expressing two copies of cMyBP-C transgenes. Scale bar represents 0.5 µm.

 


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  		Figure 5. Expression of cMyBP-C protein in IFM induces sarcomere length reduction. Bar graph showing the sarcomere length (SL) of 15-day-old IFM expressing two copies of M0t, M6t or WT transgenes (mean±SEM, n=22, 22 and 18, respectively) compared with control line (C: SG29.1-GAL4, mean±SEM, n=21). The distance from a Z-line to the adjacent Z-line (sarcomere length) was measured by electronic microscopy. The length of adult control sarcomere (average: 3.13 µm) is consistent with the length of wild-type IFM sarcomere (56Go). § indicates the statistical significance between M0t or M6t or WTt and control (**P<0.001). {dagger} indicates the statistical significance between M0t and WTt (*P=0.04). {ddagger} and ¶ indicate the statistical significance between M6t and M0t (ns: P=0.053) and WTt and M6t (ns: P=0.89), respectively (Student's t-test, see Materials and Methods).

 
Transcriptional regulation in IFM was globally affected by transgenic human cMyBP-C expression
We next investigated whether the disarray of IFM myofibrillar structure and/or function induced by expression of human cMyBP-C correlatively results in remodelling of gene expression pattern using a preliminary large-scale trancriptome analysis. We manufactured nylon microarrays using 3570 non-redundant cDNA clones. We compared 8-day-old transgenic IFM expressing two copies of human M0t truncated protein with non-transgenic IFM. We made this choice because M0t could have a stronger effect when compared with the other cMyBP-C transgenes, as suggested by the results reported earlier.

The human protein expression triggered a thorough alteration of the Drosophila IFM transcriptional pattern. Up to 97 genes were deregulated, among which 33 were up-regulated and 64 were down-regulated. In total, 73% of the deregulated Drosophila known genes have vertebrate homologues. They mostly belong to protein metabolism and catabolism, carbohydrate metabolism, energy metabolism, response to stress, signal transduction, transcription, transport, calcium signalling and muscle contraction functional groups (Table 1). This latter group is composed of six genes encoding muscle sarcomere proteins: troponin I (wings up A) and troponin T (upheld) and tropomodulin (sanpodo), as well as flightin (flightin) and paramyosin (paramyosin), two specific components of invertebrate sarcomere (24Go,25Go) and projectin (bent). Surprisingly, overexpression of a human exogenous myofilament protein capable of being incorporated into the IFM fibre is therefore accompanied by the coordinate down-regulation of the Drosophila genes encoding interrelated proteins.


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  		Table 1. Example of genes differentially expressed in 8-day-old M0t transgenic IFM
 
Calmodulin is a modifier of the flightless phenotype
Differences in gene transcription might be related to the phenotype of transgenic flies (26Go), and deregulated genes could be potential modifiers. Among the deregulated genes, we found two genes encoding proteins involved in calcium signalling (Table 1). In particular, the Calmodulin (Cam) gene was up-regulated in 8-day-old M0t transgenic flies. There is only one Cam gene in Drosophila encoding a single Ca2+-binding protein (27Go). Calmodulin is the major transducer of calcium signal mediating the muscle contraction. This intracellular sensor of Ca2+ activates specific downstream signalling pathways in response to local changes in Ca2+ concentration. In transgenic flies, the increase of Calmodulin in response to human protein expression might concomitantly induce an increase in Ca2+/Calmodulin-dependent signalling and/or influence the Ca2+ storage, which in turn might, in part, be responsible for the pathological flightless phenotype. We hypothesized that the Drosophila Cam gene might behave as a suppressor of the flightless phenotype and therefore that reduction of Cam concentration and consequently lowering of Ca2+/Calmodulin signalization could induce a phenotypic rescue.

To test this, transgenic IFM expressing two copies of cMyBP-C transgenes and bearing heterozygous loss of function mutations, Cam03909/+ or Camk04213/+, were generated. Heterozygous flies for these alleles do not display any mutant phenotype on their own. Inactivation of one dose of Cam in transgenic flies strongly reduced the percentage of flightless flies when compared with control transgenic flies having two wild-type alleles of Cam (Fig. 6). For example, 26, 12 and 10% of, respectively, WTt, M6t and M0t cMyBP-C transgenic flies heterozygous for Cam03909 were flightless at day 8 versus 44, 46 and 44% of the corresponding control lines. Likewise, Camk04213 heterozygosity led also to partial rescue of the flightless phenotype induced by cMyBP-C expression in the IFM (Fig. 6). For some unexplained reason, a slightly stronger rescue was observed with Cam03909 in the case of M0t when compared with the WTt and M6t (Fig. 6C). Unfortunately, the extent of phenotype rescue by Cam mutant homozygote could not be assessed because of the lethality of the recessive Cam mutations. These results indicate that Cam behaves as a modifier gene of the flightless phenotype.



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  		Figure 6. Lowering Calmodulin gene dosage in transgenic flies expressing cMyBP-Cs induces an age dependent rescue of the flightless phenotype. The percentage of flightless transgenic flies expressing two copies of WTt (A), M6t (B) or M0t (C) cMyBP-C homozygous for wild-type Cam allele (+/+, control line) or heterozygous for a Cam mutant alleles (Cam/+) as a function of age. Percentage of flightless transgenic flies heterozygous for mutant Cam allele was greatly reduced when compared with control. Whatever the WTt (A), M6t (B) or M0t (C) transgene form, differences obtained between Cam heterozygous and homozygous flies were extremely significant (**P<0.001) (log-rank test, see Materials and Methods).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this research, we propose to use Drosophila as an in vivo system to investigate cardiomyopathies. Our aim was to assess the potential of this invertebrate model system by evaluating the structural, functional and molecular impacts of the expression of human cMyBP-Cs proteins on insect IFM. IFM is an ideal system for the expression of human protein. In particular, the IFM structure and function are quite similar to that of the vertebrate striated muscles and most protein constituents have counterparts in both types of organisms. Moreover, the high degree of order in the IFM makes it a sensitive indicator of alterations within the structure and the function of myofibrillar proteins.

Wild-type and two C-terminal truncated cMyBP-C proteins have been driven in the IFM by means of the UAS–GAL4 system (16Go). We show that in the fly, the human cMyBP-Cs were incorporated into the sarcomere even though a human cMyBP-C counterpart was not identified in Drosophila. This result reveals that the human protein retained the ability to interact with the endogeneous Drosophila sarcomeric protein(s) due to the highly conserved Ig-like and type-III fibronectin domains. In the IFM, cMyBP-C incorporates the I-band. In vertebrates, cMyBP-C is located in the A-band (28Go). This difference probably reflects the vertebrates versus invertebrates' sarcomere specificities; for instance, Titin-like proteins are found in the invertebrate striated muscles instead of a unique Titin molecule in vertebrates (20Go). The precise targeting of the human protein to the IFM sarcomere indicates its interaction with at least one of the components of the I-band. As Drosophila Projectin and human cMyBP-C show structural homology, an attractive hypothesis would be that cMyBP-C partially displaces and competes with projectin for binding to an I-band protein. In support of this hypothesis, Projectin has been shown to localize in the IFM I-band (29Go). Furthermore, in synchronous flight muscles, both Projectin (30Go) and cMyBP-C are found in the A-band (data not shown). The competition of cMyBP-C and Projectin for binding Actin could explain the ability of the human proteins to insert within the I-band of the Drosophila sarcomere. It has been reported that Projectin interacts with the thin filament (31Go) and that the Projectin N-terminal end may weakly attach to the actin filament in the I-band (32Go). Furthermore, cMyBP-C has been reported to bind Actin (33Go) and the Actin-binding site is localized in the N-terminal region (34Go,35Go), which is present in all cMyBP-C forms used in this study.

The integration of human cMyBP-Cs in the Drosophila sarcomeres was correlated with the appearance of dose- and age-dependent phenotypes. These progressive phenotypes were reminiscent of the late-onset pathology induced by cMyBP-C mutations in humans (36Go,37Go). The ultrastructural analysis of adult transgenic IFM showed myofibrillar defects characterized by shorter transgenic sarcomeres and the presence of misaligned Z-band sometimes accompanied by M-line distortions. These structural alterations may be responsible for the flightless phenotype. New born transgenic flies are able to fly and exhibit normal muscle morphology. Furthermore, cMyBP-C is detected from the first day of the transgenic adult life and immunodetection analysis does not reveal progressive human protein incorporation in function of animal age (data not shown). These observations rule out an abnormal muscle developmental program and tend rather to indicate that the continuous use of a dysfunctional muscle during the adult life is at the origin of the progressive phenotypes.

The incorporation of cMyBP-C proteins in the I-band could, directly or indirectly, weaken the Z-band whose collapse might account for the functional muscle defect. This hypothesis has been proposed in the case of MLP(–/–) mice which showed Z-band defects without cardiac hypertrophy at birth (38Go). The IFM functional defects might also be correlated with shorter sarcomeres observed in relaxing conditions, which could indicate either muscle hypercontraction or permanent contraction. Hypercontraction phenotype is unlikely to occur here. For example, neither double overlap of thin filaments at the M-line level nor separation and accumulation of muscle fibres material to one or both attachment sites were observed (39Go). We therefore favoured abnormal maintenance in a contracted state and the inability of the transgenic IFM to relax. This hypothesis is supported by the fact that actin immunofluorescence stainings in relaxed transgenic IFM myofibrils did not show the characteristic doublet, which is the signature of relaxed fibres, but rather systematically a single bright band at the H-zone (data not shown). More thorough structural and physiological analyses, including contraction forces and contractility measurements, would obviously be required to really understand the basis of the dysfunction triggered by cMyBP-C expression.

In order to gain insights into the molecular mechanisms that underlie the human protein integration within the sarcomere architecture, a genomic approach has been adopted using cDNA microarray technology. We show that the expression of the human M0t cMyBP-C truncated form in the IFM triggered an overall transcriptional change. Likewise, several studies have shown that FHC are accompanied by a dramatic change in the global pattern of cardiac genes expression (15Go). Among the 3570 Drosophila genes analysed using home-made nylon cDNA microarrays, 97 were deregulated. This transcriptome study is currently being carried out with pan genome microarrays in order to evaluate the gene expression remodelling in function of age. However, the preliminary results obtained with the analysis of 25% of the predicted Drosophila genes have already provided noteworthy information. In particular, we show that the overexpression of cMyBP-C, which leads to flightless phenotype, is associated with the transcriptional down-regulation of six sarcomeric protein encoding genes.

The flightless phenotype could be due, at least in part, to the imbalance of the sarcomere proteins stoichiometry that is critical for muscle structure and/or function. Our analysis suggests that this disturbance is first transmitted at the transcriptional level and induces a coordinated down-regulation of genes encoding interrelated sarcomeric proteins. This type of cross-talk has already been mentioned in zebrafish mutant heart lacking troponin T in which failure of sarcomere assembly was associated with down-regulation of at least troponin I and tropomyosin (40Go). Likewise, mouse models of ablation and overexpression of myofilament genes in the heart suggest that transcriptional control may be one of the regulatory mechanisms involved in the regulation of myofilament protein stoichiometry (41Go,42Go).

The negative co-regulation of the six Drosophila genes could have significant consequences and, therefore, might explain, at least in part, the flightless phenotype. Loss of function mutations in wings up A and upheld genes encoding, respectively, Troponin I and Troponin T and flightin gene encoding a specific IFM expressed protein have been shown to induce reduction of the sarcomere length in the adult IFM associated, in the case of flightin, with alterations of Z-band alignment; these structural abnormalities have been correlated with flightless phenotypes (39Go). In addition, the reduction of sanpodo (Tropomodulin), bent (Projectin) and paramyosin (Paramyosin) gene expression might be correlated with the shortening of transgenic sarcomere length as they encode proteins that play a critical role in the regulation of the length and organization of thin (43Go) and thick (25Go) filaments. It was also of particular interest to note that a large proportion of the fluctuating genes were involved in energetic metabolism. Three genes encoding enzymes in the glycolytic pathway were found to be down-regulated. The expression level of five genes encoding enzymatic constituents of the tricarboxylic acid cycle and oxidative phosphorylation was also reduced. This observation could be particularly relevant with regard to the recent proposal of the energy depletion hypothesis to explain the pathophysiology encountered in FHC (44Go).

The penetrance of the pathology linked to FHC promoting mutations has been shown to be extremely sensitive to genetic background (15Go). The search for candidate modifier genes is therefore of crucial importance with a view to proposing diagnosis and investigating new therapies. Any gene, whose expression level is modified during the pathology, can theoretically be considered as a potential modifier gene. In Drosophila, a mutation in a candidate gene identified with cDNA microarrays can be easily tested using classical genetic crosses, for its ability to modify the phenotype. The relevance of this strategy was checked with the Cam gene, encoding the calcium-binding protein, Calmodulin, which was up-regulated in M0t transgenic flies. We show that the flightless phenotype is strongly reduced in transgenic flies heterozygotes for Cam loss of function allele. Therefore, Cam mutation behaves as a dominant suppressor of the flightless phenotype produced by cMyBP-C expression. The data cannot on its own provide any explanation for the observed effect of Cam dosage on the flightless phenotype. They illustrate, however, the potential of coupling microarray data with the search for modifiers. The interest of this result is highlighted by the recent discovery of an association between polymorphism in the promoter of a human Calmodulin gene and the modulation of FHC development associated with mutations in MYBPC3 and MYH7 genes (L. Carrier, unpublished data). The approach proposed here could therefore be of value for systematically identifying potential candidate modifier genes. Moreover, a comparison of both suppressed and unsuppressed transcriptome signatures could be a great value for deciphering suppression mechanisms.

Our study shows that some characteristics of the phenotype induced by misexpression of cMyBP-C in the Drosophila IFM are similar to those of FHC pathology: the progressive development of muscle dysfunction, the correlated alterations in the organization and structure of the muscle sarcomeres and the change in the overall pattern of gene expression. One might argue that any mutant or WT sarcomeric protein incorporation into this tissue would lead systematically to a flightless phenotype. In order to settle this question, a similar process was started with the cardiac troponin T protein (cTnT). We observed an IFM progressive dysfunction (flightless phenotype) that is sensitive to Calmodulin gene dosage (data not shown). This result raises the question of the specificity of the suppression effect exerted by Calmodulin. It will be important to determine whether the Calmodulin gene expression level is modulated in cTnT transgenic IFM. In addition, quantitative differences observed in normal and rescued kinetics between cMyBP-C and cTnT transgenic IFM regarding Calmodulin background could mean that the mechanisms involved in the two cases are slightly different. These mechanisms could be described at molecular level by comparing again the suppressed and unsuppressed transcriptome signatures.

Flight muscle dysfunction in flies and cardiac hypertrophy in humans are obviously very distant pathologies, and the main concern will be to extract, from the data obtained in flies, those that might be relevant in human. The high conservation of the molecules constituting a muscle, as well as the physiological basis of muscle function, might give grounds for confidence in the final result. Eleven other sarcomeric genes have been implicated in FHC, of which nine have orthologues in fly. A systematic and more thorough analysis of the transcriptional changes triggered by these genes should lead to the identification of relevant molecular signatures associated with their expression in Drosophila. This approach might lead to the identification of common as well as specific molecular features shared by Drosophila muscles whose function is altered by incorporation of defective sarcomeric proteins. Conserved elements in human might be considered as relevant modifier candidate genes that have to be checked in higher vertebrates for validation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
cDNAs and vectors
Experiments were carried out with pMT myc-tagged vector carrying different cMyBP-Cs (WT, M6t and M0t) as described previously (45Go,46Go) and cloned in the pUAST transformation vectors (16Go).

Drosophila stocks
Germline transformation of y, w individuals was performed as previously described (47Go). Among the 14 independent responder lines obtained for WT, M6t and M0t cMyBP-C transgenes, six were used in this study: UAS-WTt(6P5), UAS-M0t(7), UAS-M6t(13) (insertions on chromosome II) and UAS-WTt (6P3), UAS-M0t(2), UAS-M6t(9) (insertions on chromosome III). All responder lines are homozygous viable.

UAS-cMyBP-C responder flies were crossed with SG29.1-GAL4 driver flies (16Go). This specific driver induces the IFM expression of the cMyBP-C cDNA (48Go). As SG29.1-GAL4 is on the X, crosses between responder (or Cam mutant line) and driver lines were designed to obtain the same expression level of SG29.1-GAL4 in male and female offsprings in order to eliminate dosage compensation effect. Cam03909 (FBal0008045) and Camk04213 (FBal0064739) recessive alleles were obtained from Fly Stock Center. The offspring was reared at 29°C. Genotype of the transgenic lines: [yw, SG29.1-GAL4; UAS-WT(6P5)/+]; [yw, SG29.1-GAL4; +/+; UAS-WT(6P5)/UAS-WT(6P5)]; [yw, SG29.1-GAL4; UAS-WT(3P5)/UAS-WT(3P5; UAS-WT(6P5)/UAS-WT(6P5)]; [yw, SG29.1-GAL4; +/+; UAS-M6(9)/+]; [yw, SG29.1-GAL4; +/+; UAS-M6t(9)/UAS-M6t(9)]; [yw, SG29.1-GAL4; UAS-M6t(13)/UAS-M6t(13); UAS-M6t(9)/UAS-M6t(9)]; [yw, SG29.1-GAL4; +/+; UAS-M0t(2)/+]; [yw, SG29.1-GAL4; +/+; UAS-M0t(2)/UAS-M0t(2)]; [yw, SG29.1-GAL4; UAS-M0t(7)/UAS-M0t(7)/+; UAS-M0t(2)/UAS-M0t(2)]; [yw, SG29.1-GAL4; Camk04213/+; UAS-WT(6P5)/UAS-WT(6P5)]; [yw, SG29.1-GAL4; Camk04213/+; UAS-M6t(9)/UAS-M6t(9)]; [yw, SG29.1-GAL4; Camk04213/+; UAS-M0t(2)/UAS-M0t(2)]; [yw, SG29.1-GAL4; Cam03909/+; UAS-WT(6P5)/UAS-WT(6P5)]; [yw, SG29.1-GAL4; Cam03909/+; UAS-M6t(9)/UAS-M6t(9)]; [yw, SG29.1-GAL4; Cam03909/+; UAS-M0t(2)/UAS-M0t(2)].

Immunodetection
Flies were anaesthetized using CO2 and then quickly beheaded. Thoraces were separated from the abdomen and carefully opened on ice (4°C) in relaxing buffer containing protease inhibitors (50% glycerol, 20 mM phosphate buffer, pH. 7.0, 2 mM MgCl2, 1 mM EGTA, 8 mM DTT, 1 mM PMSF, 0.2 mM leupeptin, 5 mM ATP) to avoid proteolysis and mechanical damage of the fibres that, in particular, might shorten them. Glycerol was eliminated by several washes with relaxing buffer (PBS, 5 mM MgCl2, 1 mM EGTA and 5 mM ATP) without glycerol, and all steps of the immunolabelling were performed in relaxing buffer.

IFM fibres were labelled using a monoclonal anti-myc antibody (9E10; Tebu-Bio). Monoclonal IgG anti-kettin Ig16 (MAC 155) (49Go) was diluted (1 : 50) in relaxing buffer. FITC and TRITC conjugated IgG secondary antibodies were purchased from Jackson Immuno Research Laboratories. Muscle fibres were mounted onto glass slides using a Vectashield mounting buffer (Vector Laboratories) and observed under an Axiophot ZEISS or a 410 ZEISS confocal microscope.

Electron microscopy
Hemi thoraces ultrathin sections were prepared according to Yamaguchi et al. (50Go) and observed under a transmission LEO 912 electron microscope. Images were treated with digital scanner AGFA DUOSCAN.

Flight test
Drosophila stocks were tested for flight ability in a cubic transparent Plexiglas box (100x100x100 cm3) with a light source on the top part to encourage flies to fly. The flies were slightly anaesthetized using CO2 and laid out in a beaker (20 cm height and 10 cm in diameter). The internal vertical surfaces were covered with aminopropyltriethoxysilane to prevent the flies from climbing. The container was stirred every 5 min. Flies that were not able to leave the container within 20 min were considered as flightless.

Three lines were used as control for the flight tests: the SG29.1-GAL4 driver line reared at 29°C, the responder line bearing two copies of transgene reared at 29°C and the transgenic flies expressing two copies of M0t under the control of SG29.1 driver reared at 18°C. On average, a maximum of 2% of flightless flies were observed at day 18 for these control lines.

Statistical analysis
Flight test data are presented as average of two independent experiments and the difference between groups of data was assessed using log-rank test. Sarcomere length data are presented as mean±SEM and analysed using Student's t-test. All results were considered as not significant (ns) when P>0.05, very significant when 0.01<P<0.05 (*) and extremely significant when P<0.001 (**).

Total RNA extraction
Thoraces were prepared as described earlier and dissected immediately. IFM were detached from the inner side of cuticles of 50 thoraces and were homogenized in 1 ml TRIzol reagent (Life Technologies). After extraction (TRIzol protocol), total RNA quality was assessed by electrophoresis on 1% agarose gel. According to Figure 2 the whole population of transgenic flies used for dissection included 50% of flightless flies.

Microarray production
In total, 3570 amplification PCR products of unique full-length cDNA clones were spotted onto nylon filters using a GMS 427 microarrayer (Genetics MicroSystems, Affymetrix). The size of the amplicons ranges from ~0.2 to 3 kb (UG15-26, UG27-38, UG42-44, UG46-48, UG61-63 and UG65-71). The clones, obtained from the Berkeley Drosophila Genome Project, represent a subset of the Drosophila Gene collection release version 1.0, which corresponds to full-length cDNA ranging from ~0.2 to 8 kb (1Go). This resource was prepared within the Marseille-Nice Genopole facilities.

Hybridizations were carried out with complex targets prepared from 1.0 µg of total RNA and labelled with 33P during an oligo-dT primed reverse transcription (51Go,52Go). Three different hybridizations (three replicates) were performed for transgenic and control tissues to evaluate the reproducibility. Image acquisition was performed using a high-resolution imaging plate device (Fuji BAS 5000). The image was treated with the quantification software ArrayGauge (53Go). For each clone, the intensity obtained with the complex probe was submitted to a standard correction and was then divided by the intensity obtained previously with the oligonucleotide vector probe. This step corrects for possible differences in DNA content. The resulting normalized intensity was then divided by the median of the normalized intensities of all complex target values. This step corrects for experimental differences between the complex probe hybridizations and allows an approximate calculation of mRNA abundance values. Every normalized value that is below the mean of the background value plus one standard deviation was estimated as transcriptionally inactive. After this treatment, 450 genes were considered to be expressed in the non-transgenic IFM and 463 in the M0 transgenic IFM. The ratios non-transgenic/transgenic and transgenic/non-transgenic were calculated. Ninety-seven genes showed a ratio >2 and were taken into account.


   ACKNOWLEDGEMENTS
 
We thank David Martin and Bernard Jacq (Laboratoire de Génétique et Physiologie du développement, Institut de Biologie du Développement de Marseille) for their advice concerning microarray functional analysis using Gene Ontology terms and Badih Ghattas (Institut de Mathématiques de Marseille, Université de la Méditerranée) for his assistance in the flight test statistical analysis. We thank Nathalie Lalevée for reading the manuscript and for helpful comments. We would like to warmly thank Ketty Schwartz for her interest in and enthusiasm for using Drosophila animal model to study FHC. Nylon microarrays were manufactured on the Marseille-Nice Genopole platform and supported by CNRS and Research Ministry dedicated programs. This work was supported by the Association Française contre les Myopathies (AFM). T.P.V.M. was supported by an AFM doctoral fellowship.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


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Sarcomere Mutations in Cardiomyopathy With Left Ventricular Hypertrabeculation
Circ Cardiovasc Genet, October 1, 2009; 2(5): 442 - 449.
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 Eur Heart JHome page
F. W. Friedrich, P. Bausero, Y. Sun, A. Treszl, E. Kramer, D. Juhr, P. Richard, K. Wegscheider, K. Schwartz, D. Brito, et al.
A new polymorphism in human calmodulin III gene promoter is a potential modifier gene for familial hypertrophic cardiomyopathy
Eur. Heart J., July 1, 2009; 30(13): 1648 - 1655.
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 J Am Coll CardiolHome page
P. Richard, E. Villard, P. Charron, and R. Isnard
The Genetic Bases of Cardiomyopathies
J. Am. Coll. Cardiol., October 27, 2006; 48(9_Suppl_A): A79 - A89.
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