Catrin S. Rutland

Cardiomyopathy: A Systematic Review of Disease-Causing Mutations in Myosin Heavy Chain 7 and Their Phenotypic Manifestations

Background:Mutations in myosin heavy chain 7 (MYH7) commonly cause cardiomyopathy. However, the relationship between mutation location, cardiomyopathy type, change in amino acid composition and disease severity is poorly understood. This systematic review aims to provide, on a large scale, important insights into the role mutations in MYH7 play in cardiomyopathy. Methods: The literature was searched from 1966 to March 2009. The mutation location, type of mutation and disease type and severity were documented. When the severity of disease was known, the change in charge and hydropathy of the mutation was determined. Where appropriate, either a χ2 test was used or a relative risk ratio was calculated in order to evaluate the data. Results:The data presented in this study demonstrate that there are proportionately more mutations in the head and neck regions of this gene than in the tail. Importantly, mutations in the head of the gene, those that cause large changes in the hydropathy of the amino acid and non-conservative mutations are more likely to lead to a severe phenotype. Conclusions: This study suggests that mutation location in the MYH7 gene and changes in amino acid composition can have a negative impact on the disease outcome in individuals with cardiomyopathy.

Induction of Intrauterine Growth Restriction by Reducing Placental Vascular Growth with the Angioinhibin TNP-470

Abstract The placenta is a specialized vascular interface between the maternal and fetal circulations that increases in size to accommodate the nutritional and metabolic demands of the growing fetus. Vascular proliferation and expansion are critical components of placental development and, consequently, interference with vascular growth has the potential to severely restrict concurrent development of both the placenta and fetus. In this study, we describe the effects of an antiangiogenic agent, TNP-470, on placental vascular development and the induction of a form of intrauterine growth restriction (IUGR) in mice. Administration of TNP-470 to dams in the second half of pregnancy resulted in a smaller maternal weight gain accompanied by decreased placental and fetal sizes in comparison with control animals. Total numbers of fetuses per litter were not affected significantly. Stereological analysis of placentas revealed no changes in the combined lengths of vessels. However, the mean cross-sectional areas of maternal and fetal vessels in the labyrinth of TNP-470-treated mice were reduced at Embryonic Day 13.5 (E13.5) but not at E18.5. Further analysis showed reduced placental endothelial proliferation at E13.5 and E18.5 in TNP-470-treated animals. No other structural or morphometric differences in placentas were detected between TNP-470-treated and control mice at E18.5. This study provides conclusive evidence that administration of TNP-470 interferes with placental vascular proliferation and vessel caliber and results in a reproducible model of IUGR.

Unique vascular phenotypes following over-expression of individual VEGFA isoforms from the developing lens

Formation of a correctly organised vasculature and subsequently embryonic survival is critically dependent on the dosage and site-specific expression of VEGF. Murine VEGF exists in three common isoforms (viz. 120, 164 and 188 amino acids) having different organ specific distribution levels. Gene knock-in studies show that expression of any of the individual isoforms of VEGF extends survival until birth, although each is associated with distinct organ-specific abnormalities. Comparison of the effects of VEGF isoform expression is complicated by the general lethality of mis-expression, in addition to cumulative effects of adjacent tissues from the inappropriately patterned vasculature. Here we investigate the effects of over-expression of individual VEGFA isoforms from the lens-specific αA-Crystallin promoter and characterise their effects on the vessel morphology of the hyaloid and developing retinal vasculature. Since the hyaloid vasculature is an anatomically distinct, transient vasculature of the eye, comprising 3 cell types (endothelium, pericytes and macrophages) it is possible to more readily interpret the role of individual VEGF-A isoforms in vascular pattern formation in this model. The severity of the vascular phenotype, characterised by a hyperplastic hyaloid at E13.5 and subsequently retinal vascular patterning and ocular defects, is most severe in transgenics over-expressing the more diffusible forms of VEGFA (120 and 164), whereas in VEGFA188 transgenics the hyaloid vascular defects partially resolve post-natally. The results of this study indicate that individual isoforms of VEGFA induce distinct vascular phenotypes in the eye during embryonic development and that their relative doses provide instructive cues for vascular patterning.

Knockdown of alpha myosin heavy chain disrupts the cytoskeleton and leads to multiple defects during chick cardiogenesis

Atrial septal defects are a common congenital heart defect in humans. Although mutations in different genes are now frequently being described, little is known about the processes and mechanisms behind the early stages of atrial septal development. By utilizing morpholino‐induced knockdown in the chick we have analysed the role of alpha myosin heavy chain during early cardiogenesis in a temporal manner. Upon knockdown of alpha myosin heavy chain, three different phenotypes of the atrial septum were observed: (1) the atrial septum failed to initiate, (2) the septum was initiated but was growth restricted, or (3) incorrect specification occurred resulting in multiple septa forming. In addition, at a lower frequency, decreased alpha myosin heavy chain was found to give rise to an abnormally looped heart or an enlarged heart. Staining of the actin cytoskeleton indicated that many of the myofibrils in the knockdown hearts were not as mature as those observed in the controls, suggesting a mechanism for the defects seen. Therefore, these data suggest a role for alpha myosin heavy chain in modelling of the early heart and the range of defects to the atrial septum suggest roles in its initiation, specification and growth during development.

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.

A predictive model for canine dilated cardiomyopathy—a meta-analysis of Doberman Pinscher data

Dilated cardiomyopathy is a prevalent and often fatal disease in humans and dogs. Indeed dilated cardiomyopathy is the third most common form of cardiac disease in humans, reported to affect approximately 36 individuals per 100,000 individuals. In dogs, dilated cardiomyopathy is the second most common cardiac disease and is most prevalent in the Irish Wolfhound, Doberman Pinscher and Newfoundland breeds. Dilated cardiomyopathy is characterised by ventricular chamber enlargement and systolic dysfunction which often leads to congestive heart failure. Although multiple human loci have been implicated in the pathogenesis of dilated cardiomyopathy, the identified variants are typically associated with rare monogenic forms of dilated cardiomyopathy. The potential for multigenic interactions contributing to human dilated cardiomyopathy remains poorly understood. Consistent with this, several known human dilated cardiomyopathy loci have been excluded as common causes of canine dilated cardiomyopathy, although canine dilated cardiomyopathy resembles the human disease functionally. This suggests additional genetic factors contribute to the dilated cardiomyopathy phenotype.This study represents a meta-analysis of available canine dilated cardiomyopathy genetic datasets with the goal of determining potential multigenic interactions relating the sex chromosome genotype (XX vs. XY) with known dilated cardiomyopathy associated loci on chromosome 5 and the PDK4 gene in the incidence and progression of dilated cardiomyopathy. The results show an interaction between known canine dilated cardiomyopathy loci and an unknown X-linked locus. Our study is the first to test a multigenic contribution to dilated cardiomyopathy and suggest a genetic basis for the known sex-disparity in dilated cardiomyopathy outcomes.

Multiple Genetic Associations with Irish Wolfhound Dilated Cardiomyopathy

Cardiac disease is a leading cause of morbidity and mortality in dogs and humans, with dilated cardiomyopathy being a large contributor to this. The Irish Wolfhound (IWH) is one of the most commonly affected breeds and one of the few breeds with genetic loci associated with the disease. Mutations in more than 50 genes are associated with human dilated cardiomyopathy (DCM), yet very few are also associated with canine DCM. Furthermore, none of the identified canine loci explain many cases of the disease and previous work has indicated that genotypes at multiple loci may act together to influence disease development. In this study, loci previously associated with DCM in IWH were tested for associations in a new cohort both individually and in combination. We have identified loci significantly associated with the disease individually, but no genotypes individually or in pairs conferred a significantly greater risk of developing DCM than the population risk. However combining three loci together did result in the identification of a genotype which conferred a greater risk of disease than the overall population risk. This study suggests multiple rather than individual genetic factors, cooperating to influence DCM risk in IWH.

Genetics of Human and Canine Dilated Cardiomyopathy

Cardiovascular disease is a leading cause of death in both humans and dogs. Dilated cardiomyopathy (DCM) accounts for a large number of these cases, reported to be the third most common form of cardiac disease in humans and the second most common in dogs. In human studies of DCM there are more than 50 genetic loci associated with the disease. Despite canine DCM having similar disease progression to human DCM studies into the genetic basis of canine DCM lag far behind those of human DCM. In this review the aetiology, epidemiology, and clinical characteristics of canine DCM are examined, along with highlighting possible different subtypes of canine DCM and their potential relevance to human DCM. Finally the current position of genetic research into canine and human DCM, including the genetic loci, is identified and the reasons many studies may have failed to find a genetic association with canine DCM are reviewed.

Tropomyosin 1: Multiple roles in the developing heart and in the formation of congenital heart defects

Tropomyosin 1 (TPM1) is an essential sarcomeric component, stabilising the thin filament and facilitating actins interaction with myosin. A number of sarcomeric proteins, such as alpha myosin heavy chain, play crucial roles in cardiac development. Mutations in these genes have been linked to congenital heart defects (CHDs), occurring in approximately 1 in 145 live births. To date, TPM1 has not been associated with isolated CHDs. Analysis of 380 CHD cases revealed three novel mutations in the TPM1 gene; IVS1 + 2T > C, I130V, S229F and a polyadenylation signal site variant GATAAA/AATAAA. Analysis of IVS1 + 2T > C revealed aberrant pre-mRNA splicing. In addition, abnormal structural properties were found in hearts transfected with TPM1 carrying I130V and S229F mutations. Phenotypic analysis of TPM1 morpholino-treated embryos revealed roles for TPM1 in cardiac looping, atrial septation and ventricular trabeculae formation and increased apoptosis was seen within the heart. In addition, sarcomere assembly was affected and altered action potentials were exhibited. This study demonstrated that sarcomeric TPM1 plays vital roles in cardiogenesis and is a suitable candidate gene for screening individuals with isolated CHDs.

Gene Therapy Using Plasmid DNA Encoding Vascular Endothelial Growth Factor 164 and Fibroblast Growth Factor 2 Genes for the Treatment of Horse Tendinitis and Desmitis: Case Reports

In this clinical study, for the first time we used the direct gene therapy to restore severe injuries of the suspensory ligament branch and superficial digital flexor tendon in horses (Equus caballus). We injected the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor 164 and fibroblast growth factor 2 at the site of injury in the suspensory ligament branch and tendon. Treatment effects were evaluated with the use of clinical observation and ultrasound imaging during a period of a few months. We showed that gene therapy used within a period of 2–3 months after the injury resulted in the complete recovery of functions and full restoration of the severely damaged suspensory ligament and superficial digital flexor tendon.