For example, researchers are able to specifically target a gene to remove it, add a new gene or to edit its genetic sequence by inserting a mutation observed in familial ALS. This technology not only provides greater precision but significantly reduces the time taken to develop the model systems. Applications requesting funding to test compounds of interest in rodent models with many characteristics of the human disease must be well designed and should adhere to the guidelines developed by the community.
The naturally occurring dog model that carries a spontaneous mutation for SOD1 is extensively used for therapy and biomarker development. The Association supports a mouse model repository at Jackson Laboratories to enable detailed characterization and global distribution of these model systems. Increased identification of gene mutations associated with ALS through big data initiatives enables the generation of a wide variety of new model systems.
Motor neurons, astrocytes and co-cultured systems of multiple cell types, including muscle in a dish, are useful for studying the response of individual cells to toxins or potential therapies. Motor neurons carrying the mutant SOD1 gene have been used to study the process of protein aggregation, which researchers believe is harmful to motor neurons. Several studies have demonstrated that astrocytes secrete toxic factors that damage motor neurons and significant research has been dedicated to isolating these potentially toxic factors.
Exciting new opportunities have opened up with the development of induced pluripotent stem cells iPSCs. These cells can be derived from the skin of an ALS patient, genetically modified and used to generate motor neurons in a dish. This should allow researchers to gain a deeper understanding of the disease process as it differs among different patients. See more information on the stem cell focus area page. Its use has many advantages. If the heart is seriously compromised, the fetus rarely survives past ED Thus, homozygous animals carrying the null mutation at both alleles would not come to term but rather would die during embryogenesis and be resorbed Before one goes to the trouble of making a targeted animal it is also prudent to have the analytical infrastructure in place for the necessary analyses and these have been largely lacking for an assessment of cardiac function in utero.
Our abilities to discern cardiac contractile function in the intact, mid-gestation embryo, as reflected by both stroke volume and heart rate have been quite limited but are developing rapidly A second concern is that gene targeting is very time consuming, and it is not unusual for an experiment to take years, versus months if a transgenic approach is used.
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This is particularly true if one is trying to make a mutation in the gene, and not simply ablate it, as those experiments require two separate targeting events Nevertheless, gene targeting remains the more elegant and intellectually satisfying of the two approaches as it is more precise.. Familial Hypertrophic Cardiomyopathy. The familial hypertrophic cardiomyopathies FHC include a group of primary cardiac muscle disorders characterized by a high incidence of morbidity and mortality. Their classification is based on their pathophysiology and etiology.
Cardiomyopathy was originally defined as a non-coronary disease of heart muscle In , the World Health Organization redefined the term to mean heart disease of undefined etiology. These cardiomyopathies are inherited, rather than acquired as a secondary consequence to altered cardiac load 22 , are autosomal dominant, and cause hypertrophic cardiomyopathy, as measured by an increase in mass. FHC is characterized by unexplained cardiac hypertrophy without increased cardiac load, or in the absence of other systemic abnormalities.
Histologically, there is well-characterized disarray at both the myocyte and myofiber levels. Bizarre nuclear morphology and karyomegaly are also often present although these characteristics can be age dependent and may not present in children The histopathology also often includes extensive fibrosis that can be either focal or interstitial in nature As a group of diseases, the familial hypertrophic cardiomyopathies are somewhat common, with independent studies yielding estimates that individuals are affected 26, However, FHC is a major cause of sudden death in otherwise healthy appearing young adults A recent study estimated that, in the sudden cardiac death of 35 young adults, 12 were due to FHC Even within a family in which the disease is due to a single genetic defect, the severity, onset and penetrance of the pathology is highly variable presumably due to the existence of modifier loci, although this has not yet been formally demonstrated.
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Penetrance is age-related, with onset usually occurring in adolescence 25,30, Beginning with the seminal paper of Geisterfer-Lowrance et al 32 , a genetic basis for the primary etiology of the disease began to be established. Using gene linkage techniques coupled with molecular biological approaches, and taking advantage of a large pedigree, the Seidman's established that FHC mapped to chromosome 14q The gene encodes the major motor protein responsible for pumping in the adult ventricle.
The mutation converted a highly conserved arginine residue Arg to a glutamine ArgGln. The large number of different mutations, and their various locations in the MyHC molecule helps, in part to explain the disease's widely varying severity: different mutations have different effects on MyHC's ability to carry out its function.
For example, the ArgGln mutation is highly penetrant and has severe effects on morbidity and mortality of the affected patient population. The residue is located near the nucleotide-binding pocket, and is at a site in the myosin' s head that participates in actin-myosin interactions. Thus, the mutation lies at and near critical sites for the motor' s functions These disorders, like FHC, may present heterogeneous clinical features and morbidity and other modifier genes undoubtedly influence mortality..
We now know that FHC is an autosomal dominant genetic disorder resulting from mutations in different genes encoding sarcomeric proteins that make up the thick, thin and titin filaments.
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Clinically, FHC typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. Diagnosis is usually made based. Although the molecular genetics of understanding the disease's etiology are quite advanced, the pathogenic processes that actually are responsible for morbidity and mortality have remained obscure. This is because their description must necessarily take place over prolonged periods of time, and in a population whose behavior and environment is uncontrolled.
To mitigate these difficulties, a substantial amount of effort has been devoted towards generating relevant animal models in which disease progression can be studied closely in a controlled manner. Development of animal models has shed significant light into the pathogenesis of FHC, linking the genetic mutations to particular functional deficits. In theory , a mutant sarcomeric protein that is produced and presumably causes disease development can alter sarcomere function via at least two different mechanisms:.
Below, we will briefly consider relevant examples that illustrate the power of animal models in uncovering the primary disease deficits.. Underlying the different motor functions of the different muscle types, the myosin heavy chains exist as a gene family 37 and, in the heart, two isoforms, termed alpha and beta are expressed.
MYH7 Is composed of 40 exons, and encodes a protein of amino acid residues Figure 1. V 1 and V3 display different intrinsic A TPase activities; V 1 being the most active by about three to four-fold. Thus, the human ventricle consists mostly of V3 38,39 while the adult mouse ventricle, which has a rate of beats per minute, contains V A The human mutations previously described are indicated, the red color referring to the more malignant ones. The intron-exon organization of the gene is represented and the affected exons are shown in blue.
The mutations that have been engineered in animal models are shown below the intron-exon diagram. C Principle characteristics of animal models reproducing some of the human mutations, comparison with the human disease and disease pathogenesis.
Missense mutations are represented by the letter encoding the amino acid aa residue that is mutated followed by the aa position number, followed by the letter of the new aa. To date, more than 63 distinct mutations have been defined. Missense and nonsense mutations and deletions have all been reported, and this diversity is responsible, at least in part, for the widely varying clinical picture of FHC. Thus, the clinician sees tremendous differences in terms of the degree of hypertrophy, onset and evolution of the symptoms of the disease, occurrence of sudden cardiac death SCD , and the patient' s prognosis classified as benign or malignant.
The effects of the different mutations on the different functions of the myosin heavy chain lead to different prognoses. The principle clinical mutations associated with a malignant prognosis include ArgGln 32, , ArgCys 33,43,44 , GlyArg 33,45,46 , ArgTrp 45,47,48 , and, depending on the ethnic origin of the patients, ValMet 42,44, In order to study the consequences of this mutation, the missense mutation, ArgGln RQ was generated in the mouse using gene targeting This mouse has subsequently been analyzed extensively over its lifetime and has yielded valuable information concerning the natural history of the disease.
In the human, the mutation only presents in heterozygous form and, consistent with this observation, the homozygote mice do not live for more than a few days after birth Sedentary heterozygotes survived, demonstrating that the presence of ArgGln myosin in the sarcomere leads to abnormal cardiac function but is compatible with life.
The heterozygotes exhibited the same cardiac histopathology and pathophysiology observed in human FHC. Cardiac dysfunction preceded histopathologic changes, and myocyte disarray, hypertrophy, and fozygotes showed a more severe phenotype than females, as assessed by gender-specific electrophysiologic abnormalities 5easons for these gender differences remain obscure. Exercise capacity is compromised in the heterozygotes 53, In the future, this mouse model for FHC will continue to allow us to study the impact of background genotype, diet and physical activity on phenotype..
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The functional consequences of the ArgGln mutation at the molecular level 61 have been assessed: actin-activated cycling is accelerated, and this could account for the faster rate of rise in pressure observed in vivo 55 , as well as the faster rate of force development in muscle strips Such a gain of function is consistent with the hemodynamic phenotype observed in humans, and is likely to stimulate the development of a compensatory hypertrophy that is not due to reduced power-generating capacity but instead is related to chronically increased energetic demands on the myocardium.
In this model, compensatory hypertrophy would increase tissue mass to reduce wall stress and energy utilization per unit volume of myocardium. How valid is it to extrapolate the mouse data to the human? Interestingly, the degree of enzymatic impairment of the mutant myosins correlated with the clinical phenotype of patients carrying the corresponding mutation. These data suggest that, despite specie differences, the animal models may well be predictive for the human. Another approach to answering this question is to generate larger animals carrying the disease locus.
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There was a high incidence of premature death, and the observed phenotype was very similar to that of the human. The mutant protein exerted a dominant-negative effect, consistent with the genetics of the human population. Thus, the transgenic rabbit expressing the ArgGln mutant protein is a desirable model to study the pathogenesis and search for therapeutic targets for human HCM.
The rabbit is also more suitable for noninvasive functional studies using echocardiography or electrophysiology. Although these techniques have been developed for the mouse 64 , they remain restricted to a small number of specialized academic centers, and the rabbit model will certainly be more accessible to the general cardiology community for detailed study.
In particular, extensive electrophysiological studies have already been performed in rabbit 65 , providing useful data on the arrhythmogenic dysfunction that is common in FHC, especially the occurrence of sudden cardiac death.. These small proteins appear to have both structural and regulatory roles in myosin function, stabilizing the long alpha helical neck of the myosin head and affecting its rigidity or stiffness They belong to the superfamily of EF-hand proteins, which includes calmodulin and troponin C.
Cardiac compartment-specific isoforms are expressed in most vertebrate species, including man. A The human mutations previously described are indicated. The mutati ons that have been engineered in animal models are shown below the intron-exon diagram. The concept that mutations in the myosin light chains might be causative for FHC was a logical outgrowth of the accumulating data that alterations in other sarcomeric proteins could lead to the disease. Additionally, a large cluster of MyHC mutations mapped to the domains that bound the light chains, implicating this region as being critically important to the normal function of the motor.
Structure-function studies show that there is an increase in actin translocation velocity in an in vitro motility assay 67, Seven missense mutations plus one truncation mutation in RLC have also be en documented 67, More than half of these Ala13Thr, Phe18Leu, Glu22Lys, Pro94Arg occurred at highly conserved amino-acid residues, and modeling indicated that disruption of the phosphorylation site might result. This, in turn, could affect flexibility of the myosin neck.
Both light chain mutations are associated with a rare and striking cardiac phenotype, which involves massive hypertrophy of the cardiac papillary muscles and adjacent ventricular tissue, causing a mid cavity obstruction MLC mutations may interfere with the stretch-activation response of papillary muscle and adjacent ventricular tissue, a property only found in portions of the heart that increase power output These changes might translate into impairment in the elasti city of the neck region of myosin, leading to a reduction in the oscillatory power of the papillary muscles, which is normally augmented by a strong str etch-activation response.
The authors hypothesized that over time, a compensatory hypertrophic response takes place to increase power, but eventually obstructs the ventricular cavity.. The phenotype was recapitulated This model provided the opportunity to study the stretch-activation response before the hearts were distorted by the hypertrophic process and confirmed that the stretch-activation response might playa role in the mammalian heart, providing a new way to modulate human cardiac function.
Moreover, a novel transgenic mouse in which the phosphorylatable site was ablated showed the importance of this post-translational modification Indeed, pharmaceutical modulation of cardiac RLC phosphorylation may provide a new target to therapeutic intervention in heart failure.. As noted above, the ELC transgenic mice were made with an intact human genomic fragment. Thus, although the phenotype was accurately recapitulated, the experiment was subject to some ambiguity in terms of the MetVal mutation being causative, as there were many other sequences present.
In order to unambiguously establish a causal relationship for the regulatory and essential light chain mutations in cardiac hypertrophy, multiple lines of mice that expressed either the wild type or mutated forms, utilizing cDNA clones encompassing only the gene loci's coding regions were made. Because of the discordance of these data with the data obtained in the transgenic mice containing the human genomic fragment, the current view, that these point mutations by themselves cause significant cardiac hypertrophy, should be reassessed.
The possibility remains that these point mutations may simply be closely linked to the real, yet undiscovered lesion in the human locus.. These data show the usefulness of transgenesis and animal models for establishing the unique structure-function relationships in the mutated proteins. Genetic remodeling allows the scientific community to test the causality of particular mutations. Such experiments have significant implications for clinical practice as, before wide spread screenings are contemplated, let alone implemented, establishing a direct cause-an-effect relationship for the particular mutation is imperative..
MyBP-C, a major component of the thick filament, binds to both the myosin thick and titin filament systems. Discovered over twenty-seven years ago 73 , interest in the protein' s role s intensified after multiple mutations in the polypeptide were linked to familial hypertrophic cardiomyopathy MyBP-C is localized in the C region of the A band, and has an unique organization, with axial bands in each half-sarcomere. Like other myosin binding proteins and titin, MyBP-C belongs to the intracellular immunoglobulin Ig superfamily and is composed of repeated Ig and fibronectin domains In vitro modeling and reconstitution experiments, as well as experiments carried out using cell transfections, indicate that the protein probably plays an important role in assembling and maintaining the overall architecture of the sarcomere 75, The cardiac isoform, consisting of 35 exons Figure 3 , is encoded by the human MYBPC3 gene, which is localized on chromosome 11p The truncations most frequently occur at the COOH-terminus, which contains a myosin-binding site, with the titin-binding site sometimes still present, sometimes not.
Interestingly, presentation of the disease is often delayed until the fifth-sixth decades, and is characterized by late-onset HCM, incomplete penetrance, and a relatively favorable clinical profile 81 , contrasting with other, more malignant sarcomeric gene mutations. Because of these characteristics, longitudinal pathological changes linked to the disease state in the MYBP-C patients are hard to obtain..
The transgenic approach should provide a powerful tool for studying the longitudinal pathological processes involved in the disease's onset and progression late in life.