Periodic variation in R-R intervals and cardiovascular autonomic regulation in young adult Syrian hamsters.

Several hamster strains are commonly used as models for cardiomyopathic phenotypes evolving toward heart failure. However, little is known about heart rate variability (HRV) in this species. Prolonged surface ECG recording, a prerequisite to HRV studies, can be obtained either by telemetry or by restraints. Here, we performed long time ECG recording using telemetry on young adult Syrian hamsters and we analyzed time series of interbeat intervals. Standard statistics showed that the mean of normal R-R intervals slightly increased with age, with standard deviation of normal R-R intervals remaining stable over time. However, time domain analysis using Poincaré plots revealed dynamic changes in the HRV. Analysis of frequency domains revealed that the ratio of spectral components (low frequency/high frequency) exhibited a maturation pattern. Thus refined analysis of HRV revealed a more complex pattern than common statistical analysis would translate. Unlike other rodents, hamsters display a great spontaneous variability of their heart rate. As the complexity canvas of HRV might be the consequence of extracardiac regulation factors, we assessed the sympathovagal balance in both time and frequency domain of heart rate. Pharmacological tests revealed that both sympathetic and vagal tones contribute to HRV in Syrian hamsters. Thus Syrian hamsters have a broad intrinsic HRV with large influences of the neurovegetative system. However, the influence of the previous beat seems to prevail over the autonomic oscillators. These animals present a high sensitivity to artificially altered cardiac regulation and might be great models for the diagnosis of early alterations in the HRV related to pathology. Therefore, Syrian hamsters represent a unique model for HRV studies.

Myoblast xenotransplantation as a tool to evaluate the appropriateness of nanoparticular versus cellular trackers.

Myoblast transplantation is being considered as a potential strategy to improve muscle function in myopathies; hence, it is important to identify the transplanted cells and to have available efficient reagents to track these cells. We first validated a human to mouse xenotransplantation model warranting the complete and rapid rejection of the cells. We then used this model to assess the appropriateness of a nanoparticle reagent to track the transplanted cells. Human myoblasts were loaded with ferrite nanoparticles and injected into the tibialis muscle of immunocompetent mice. Upon collection and histological analysis of muscle sections at different time points, we observed the total disappearance of the human cells within 6 days while ferrite particles remained detectable and colocalized with mouse infiltrating and neighboring cells at the injection site. These results suggest that the use of exogenous markers such as ferrite nanoparticles may lead to false-positive results and misinterpretation of cell fate.

Non-invasive restrained ECG recording in conscious small rodents: a new tool for cardiac electrical activity investigation.

In vivo electrophysiology remains a suitable method to monitor cardiac activity; however, surface electrocardiogram (ECG) monitoring remains complicated in the case of small animals. Sedation has helped to maintain the animal still; however, it is known that anesthetic drugs impair the regulation of the cardiac electrical activity. To circumvent this problem, ECG monitoring using telemetry or restraints has been developed. This study reports a new methodology, based on a restraining system without further sedation, for recording ECGs on small animal models. We investigated its efficacy in Syrian hamsters and in several strains of mice, and we compared these data to those obtained with telemetry devices. We show that this new system can easily be used in animals of different sizes ranging from adult hamsters to newborn mice. When compared to telemetry, this restrained ECG monitoring method shows a very good yield, as 65% of total beats can be used for further analysis. When recorded in the same animals, RR intervals distributions are identical for both techniques. In conclusion, this restrained ECG monitoring technique is a well-suited tool for exploring various aspects of cardiac electrophysiology in a wide variety of small animals including very young mice.

Intrapericardial administration of novel DNA formulations based on thermosensitive Poloxamer 407 gel.

Inherited cardiopathies are leading to life-threatening conditions such as heart failure. Moreover, treatments currently available fail in altering the cardiac phenotype. Thus, gene therapy appears as an attracting alternative to conventional treatments. However, gene delivery remains a major hurdle in achieving this goal. To obtain regional delivery of plasmid DNA, intrapericardial administration seems to be an interesting approach. In order to improve retention time at the site of injection, formulations based on a thermosensitive gel of Poloxamer 407 were assessed. Protection and condensation of plasmid DNA was initially performed through complexation with polyethyleneimine (PEI), a widely used polymer. Characterization of the size and zeta potential of the complexes suggested interactions between the polyplexes and the Poloxamer gel through significant increase of the size of the polyplexes and shielding of the surface charges. In vivo evaluation has highlighted the toxicity of PEI/DNA polyplexes toward the myocardium. However, feasibility of intrapericardial injection of Poloxamer based formulations as well as their very low toxicity has been established.

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies.

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.

Can exogenous stem cells be used in transplantation?

Today's most urgent problem in transplantation is the lack of suitable donor organs and tissues and as the population ages, demands for organs and tissue therapies will only increase. One alternative to organ transplantation is cell therapy whose aim is to replace, repair or enhance the biological function of damaged tissue or diseased organs. One goal of cellular transplantation thus has been to find a renewable source of cells that could be used in humans. Embryonic stem (ES) cells have the potential to proliferate in vitro in an undifferentiated and pluripotent state. Theoretically, ES cells are capable of unlimited proliferation in vitro. ES cells spontaneously differentiate into derivatives of all three primary germ layers: endoderm, ectoderm and mesoderm, hence providing cells in vitro which can theoretically be isolated and used for transplantation. Furthermore, these pluripotent stem cells can potentially be used to produce large numbers of cells that can be genetically modified in vitro. Once available, this source of cells may obviate some of the critical needs for organ transplantation. Murine ES cells have been extensively studied and all available evidence indicates that all aforementioned expectations are indeed fulfilled by ES cells. ES cells as well as embryonic germ cells have recently been isolated and maintained in culture. The recent descriptions of human ES cells portend the eventual use of allogeneic in vitro differentiated cells for human therapy. This goal, however, is fraught with obstacles. Our aim is first to review the recent advances made with murine ES cells and then to point out potentials and difficulties associated with the use of human ES cells for transplantation.

Regulated splicing of an alternative exon of beta-tropomyosin pre-mRNAs in myogenic cells depends on the strength of pyrimidine-rich intronic enhancer elements.

Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.

Gene delivery to the myocardium by intrapericardial injection.

Several studies have demonstrated the feasibility of gene transfer into the heart muscle. However, all the available data also indicate that the extent of transfection remains limited. As an alternative method to intravascular administration, we have developed a novel strategy which uses the pericardial sac. When a replication-deficient adenovirus containing the cDNA encoding a bacterial beta-galactosidase is injected into the pericardial sac of adult Wistar rats the staining is exclusively restricted to the pericardial cell layers. However, injecting a mixture of collagenase and hyaluronidase together with the virus, leads to a large diffusion of the transgene activity, reaching up to 40% of the myocardium. Transgene expression is predominant in the left ventricle and the interventricular septum but limited in the right ventricle. In vivo echocardiographic measurements of the left ventricular diameters at end diastolic and end systolic times show no difference between virus- and sham-injected animals, thus indicating a good clinical tolerance to this strategy of virus delivery. The same protocol has been used with the same efficiency in mice, which leads us to propose injection into the pericardial sac as an effective and harmless method for gene transfer into the heart muscle.

Cardiac myosin binding protein C gene is specifically expressed in heart during murine and human development.

Cardiac myosin binding protein C (MyBP-C) is a substantial component of the sarcomere, with both structural and regulatory roles. The gene encoding cardiac MyBP-C in humans is located on chromosome 11p11.2, and mutations that are most predicted to produce truncated proteins have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). To understand better the pathophysiology of FHC and with a view to the development of animal models for this disease, we have investigated by in situ hybridization the pattern of expression of the cardiac MyBP-C gene during human and mouse development using species-specific oligonucleotide probes. From 4 weeks of human development, a strong labeling of cardiac MyBP-C mRNAs was unambiguously detected in all heart compartments, and no signal could be visualized in somites. In murine embryos, from embryonic day 9.5 until birth, a strong signal was detected exclusively in the heart. Our results showed that during both human and murine development, in contrast to chicken development, the cardiac MyBP-C gene is abundantly and specifically expressed in the heart.

The transcriptional activity of a muscle-specific promoter depends critically on the structure of the TATA element and its binding protein.

We have previously characterized the proximal promoter of the mouse IIB myosin heavy chain (MyHC) gene, which is expressed only in fast-contracting glycolytic skeletal muscle fibers. We show here that the substitution into this promoter of a non-canonical TATA sequence from the IgH gene results in inactivity in muscle cells, even though TATA-binding protein (TBP) can bind strongly to this mutated promoter. Chemical foot-printing data show, however, that TBP makes different DNA contacts on this heterologous TATA sequence. The inactivity of such a non-canonical TATA motif in the IIB promoter context appears to be caused by a non-functional conformation of the bound TBP-DNA complex that is incapable of sustaining transcription. The conclusions imply that the precise sequence of the promoter TATA motif needs to be matched with the specific functional class of upstream activator proteins present in a given cell type in order for the gene to be transcriptionally active.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase gene is regulated at the transcriptional level during compensated left ventricular hypertrophy in the rat.

In mammalian myocardium, relaxation is mainly triggered by the reuptake of calcium from the cytosol to the lumen of the sarcoplasmic reticulum (SR) through the cardiac isoform of the sarco(endo)plasmic reticulum calcium ATPase, SERCA2a. Relaxation abnormalities related to deficient SR Ca(2+)-uptake have been identified in human heart failure and in animal models of cardiac hypertrophy and failure. These alterations have been associated with a reduction in SERCA2a activity and in steady-state SERCA2a protein and mRNA levels. As a first step in the analysis of the mechanisms responsible for this reduction, we have studied a possible down-regulation of the SERCA2 gene transcription during left ventricular hypertrophy (LVH) induced by constriction of the ascending aorta in the rat. Quantifications of the mRNA levels demonstrated no alteration, compared to sham-operated rats, at 5 d after imposition of the pressure overload, whereas a significant decrease was observed at 11 d. Transcription in-vitro experiments (cardiac nuclear run-on assays) performed in isolated cardiomyocytes nuclei showed no changes at 5 d and a 37% reduction of the SERCA2 gene transcription at 11 d. These results strongly suggest that SERCA2 gene expression down-regulation during cardiac hypertrophy occurs, at least in part, at the level of the transcription.

Molecular cloning and analysis of the human cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) gene promoter.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in regulating Ca2+ movements in myocardium. In cardiac hypertrophy and human heart failure, the decrease in mRNA and protein levels of SERCA2 might account for the reduced diastolic Ca2+ re-uptake seen in these conditions. To investigate the regulation of human SERCA2 gene expression, an 18.6-kb human genomic clone that contains exons 1,2 and 3 of the SERCA2 gene has been isolated, and 13 kb of 5' upstream flanking sequence of which the proximal 2.5 kb of the promoter have been sequenced. Similar to the rabbit gene, the human SERCA2 promoter possesses a TATA-like box (-25 bp), a CAAT-box (-78 bp) and a number of consensus cis-regulatory elements including three Sp1 sites, a CACCC-box, and an OTF-1 binding sequence. No CArG box (present in the rabbit SERCA2 promoter) was identified in the human proximal promoter. Two putative thyroid response elements (TRE) are also present, suggesting that the human SERCA2 gene is also regulated by thyroid hormone as are the rat and rabbit genes. To study transcriptional activity of the human SERCA2 promoter in vitro, luciferase reporter plasmids containing a series of 5' deleted promoter constructs from -2577 bp to +170 bp were transfected into neonatal rat cardiomyocytes and C2C12 myotubes. The results suggest that: (a) the sequences from the transcription start site to -263 bp are necessary to obtain maximal transcriptional activity; (b) sequences from the transcription start site to -125 bp are essential for basal transcriptional activity; (c) at least one positive regulatory element is located between -263 bp and -125 bp; and (d) at least one negative regulatory element is present between -1741 bp and -412 bp.

Sequence divergence associated with species-specific splicing of the nonmuscle beta-tropomyosin alternative exon.

Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.

Myotube-specific activity of the human aldolase A M-promoter requires an overlapping binding site for NF1 and MEF2 factors in addition to a binding site (M1) for unknown proteins.

The human aldolase A gene is expressed in several tissues through the use of three alternative promoters. The activity of one of the promoters, pM, is restricted to skeletal muscle. We reported previously that a proximal 280 bp pM fragment confers tissue-specific expression to a CAT reporter gene in transgenic mice. This small regulatory region directs expression to muscle composed mainly of fast-twitch fibers. Here we show that a minimal promoter fragment from base-pairs -164 to +45 is sufficient to highly active pM during myoblast differentiation in cell culture and demonstrate that two DNA elements play a major role in this activation. These elements consist of a binding site (M1) for unknown ubiquitous proteins and an overlapping binding site for MEF2 and NF1 families of transcription factors. The NF1 factor constitute the main binding activity on the MEF2/NF1 site and, interestingly, some of the DNA-protein complexes that form with muscle nuclear extracts on the NF1 element differ from those that form with non-muscular extracts.

Splicing of the alternative exons of the chicken, rat, and Xenopus beta tropomyosin transcripts requires class-specific elements.

The diversity of protein isoforms is often generated from single genes by alternative splicing of the primary transcript. Using transfection of beta tropomyosin minigene constructs into homologous and heterologous cell systems, we show that there are differences, among higher vertebrates, in the components of the splicing machinery which control the conserved regulated splicing pattern of two mutually exclusive exons (6A and 6B) present in this gene. These experiments demonstrate that genes which give rise to alternative transcripts may require an appropriate combination of splicing factors which are species-specific, or at least restricted to the same taxonomic subgroup (class). An important practical implication is that the splicing of these genes may be deregulated in heterologous systems in vitro and in vivo, i.e. in transgenic animals.

Clones of human satellite cells can express in vitro both fast and slow myosin heavy chains.

In order to test the diversification among satellite cells in man, satellite cells were isolated from human quadriceps and masseter muscles. The growth kinetics and morphological features of these cells were determined in vitro and the expression of the different myosin heavy (embryonic, fetal, fast, and slow) and light chain isoforms was analyzed. In all satellite cell cultures, only the four fast-type light chains (MLC1emb, MLC1F, MLC2F, and MLC3F) were synthesized and no slow myosin light chains were ever detected. However, we found that fused cultures of human satellite cells express both adult fast and slow myosin heavy chains (MHCs), in addition to embryonic and fetal isoforms. In order to determine if distinct fast and slow cell lineages could be detected among the satellite cells, a clonal analysis was carried out on both cell populations. This analysis was first carried out on clonal populations and was confirmed by the analysis of isolated clones. Double-labeling experiments confirmed that all myogenic clones which expressed fast MHC also coexpressed slow MHC. Therefore, we found no evidence for the existence of different fast and slow satellite cell lineages in human postnatal skeletal muscle.

Promoter elements and transcriptional control of the chicken tropomyosin gene [corrected].

The chicken beta tropomyosin (beta TM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the beta TM gene, we have analyzed the 5' regions associated with each CAP site. Truncated regions 5' to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (-168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5' to the beta TMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterized an enhancer element (-201/-68 nt) which contains an E box (-177), a variant CArG box (-104) and a stretch of 7Cs (-147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner.

[Analysis of the diversity of tropomyosin isoforms]. / Analyse de la diversité des isoformes de tropomyosine.

Tropomyosins are a family of actin filament binding proteins. Like many structural proteins, tropomyosin isoform expression involves the use of multiple genes, but diversity is to a large extent generated by alternative processing of RNA. The tropomyosin family consists of 15 to 20 different protein isoforms which are coded by four genes. Each of these genes code for multiple proteins ranging from two up to as many as nine different isoforms. These genes have been named alpha CTM, alpha FTM, alpha STM and beta TM after to the striated muscle specific subunit of tropomyosin which they code. Their multiple coding potential is based upon the existence of multiple exons associated with initiation of transcription, multiple exons associated with polyadenylation signals and multiple mutually exclusive internal exons which are alternatively spliced. The regulation of this process of alternative splicing have been extensively studied both in the case of exons 2a/2b of the alpha FTM gene and in the case of exons 6a/6b of the beta TM gene. In both cases, one exon is specifically used in one type of muscle tissue, exon 2a is smooth muscle specific and exon 6b is skeletal muscle specific. In both cases, alternative splicing involves a combination of negative regulation, on exon 2b in smooth muscle and on exon 6b in non muscle tissues, and of competition in the alternative situation.

The muscle specific promoter of chick beta tropomyosin gene requires helix-loop-helix myogenic regulatory factors and ubiquitous transcription factors.

The chick beta tropomyosin (TM) gene has two alternative transcription initiation start sites which are used in muscle or non muscle tissue. A recombinant plasmid containing 805 nucleotides (nt) of the sequence upstream to the muscle CAP site driving the bacterial chloramphenicol acetyltransferase gene is sufficient for muscle specific expression. Of the two E boxes present in this construct, only the E box proximal to the CAP site is functional since deletion or mutation of this E box causes a decrease of CAT activity (about 40%). Separate mutation of Sp1 motifs also reduces the transcription driven by the 805nt fragment. Double mutation of E box and Sp1 motifs show that helix-loop-helix muscle regulatory factors and ubiquitous Sp1 transcription factor are required in the initiation of the transcription of the chick beta TM gene in muscle tissue. Our results also suggest that other factors may participate to this process.

Pre-mRNA secondary structure and the regulation of splicing.

Nuclear pre-mRNAs must be precisely processed to give rise to mature cytoplasmic mRNAs. This maturation process, known as splicing, involves excision of intron sequences and ligation of the exon sequences. One of the major problems in understanding this process is how splice sites, the sequences which form the boundaries between introns and exons, can be accurately selected. A number of studies have defined conserved sequences within introns which were later shown to interact with small nuclear ribonucleoproteins (snRNPs). However, due to the simplicity of these conserved sequences it has become clear that other elements must be involved and a number of studies have indicated the importance of secondary structures within pre-mRNAs. Using various examples, we shall show that such structures can help to specify splice sites by modifying physical distances within introns or by being involved in the definition of exons and lastly, that they can be part of the regulation of alternative splicing.