Cardiomyopathies are the most common form of genetic disorders featuring primary abnormalities in the structure and function of the heart. Over the past few decades, tremendous progress has been made in elucidating the genetic basis of cardiac disorders. However, the development of specific and effective therapies remains largely limited due to the lack of suitable therapeutic targets. Nucleoli are polyfunctional subnuclear domains that are heavily involved in ribosomal RNA production. Recent studies have identified nucleolar structure perturbations and functional defects associated with different types of cardiomyopathies. Additionally, several mutations have been identified in several ribosomal genes that are linked to cardiomyopathy in human patients. We previously identified a nucleolar DEAD-box RNA helicase, DDX27, as a critical regulator of myogenesis. This study aimed to investigate the role of ddx27 deficiency in cardiac muscle and expand the understanding of DDX27 mediated pathways that are involved in myopathies. In this study, we used zebrafish models to investigate ddx27 deficiency in cardiac muscle. Phenotype characterization, cardiac function testing, transmission electron microscopy and histological analysis of ddx27 mutants revealed corresponding dilated cardiomyopathy and skeletal muscle hypotrophy. Furthermore, knockdown of DDX27 ortholog, Rs1, in cardiac muscle was fatal for Drosophila larvae. However, other tissues (i.e., neural or gastrointestinal) were unaffected suggesting that abnormalities caused by Ddx27 deficiency are specific to cardiac and skeletal muscle. Immunofluorescence, northern blotting and polysomal profiling of ddx27 zebrafish myofibers revealed that DDX27 is necessary for preserving nucleolar architecture and ribosome biogenesis. Here we have shown that DDX27 is essential for normal function of cardiac and skeletal myogenic processes due to its critical role in ribosomal regulation. Additionally, we provide novel evidence for DdX27 deficiency contributing to dilated cardiomyopathy. Overall, the findings of this study provide further evidence for the role of RNA helicases, specifically DDX27, in cardiac and skeletal muscle pathogenesis as well as provide novel insight into the molecular pathways of therapeutic benefit for afflicted patients of these diseases. / 2022-06-04T00:00:00Z

Objectives: We sought to identify a novel gene for dilated cardiomyopathy (DCM). Background: DCM is a heritable, genetically heterogeneous disorder that remains idiopathic in the majority of patients. Familial cases provide an opportunity to discover unsuspected molecular bases of DCM, enabling pre-clinical risk detection. Methods: Two large families with autosomal-dominant DCM were studied. Genome-wide linkage analysis was used to identify a disease locus, followed by fine mapping and positional candidate gene sequencing. Mutation scanning was then performed in 278 unrelated subjects with idiopathic DCM, prospectively identified at the Mayo Clinic. Results: Overlapping loci for DCM were independently mapped to chromosome 10q25-q26. Deoxyribonucleic acid sequencing of affected individuals in each family revealed distinct heterozygous missense mutations in exon 9 of RBM20, encoding ribonucleic acid (RNA) binding motif protein 20. Comprehensive coding sequence analyses identified missense mutations clustered within this same exon in 6 additional DCM families. Mutations segregated with DCM (peak composite logarithm of the odds score >11.49), were absent in 480 control samples, and altered residues within a highly conserved arginine/serine (RS)-rich region. Expression of RBM20 messenger RNA was confirmed in human heart tissue. Conclusions: Our findings establish RBM20 as a DCM gene and reveal a mutation hotspot in the RS domain. RBM20 is preferentially expressed in the heart and encodes motifs prototypical of spliceosome proteins that regulate alternative pre-messenger RNA splicing, thus implicating a functionally distinct gene in human cardiomyopathy. RBM20 mutations are associated with young age at diagnosis, end-stage heart failure, and high mortality.

Heart disease has been the leading cause of death in the United States for more than 90 years. The leading cause of death in individuals aged 65 and older has remained diseases of the heart from 1950 to the current time. According to the CDC, once diagnosed with heart disease, individuals have an approximately 50% chance of dying within 5 years, regardless of race. Mortality related to heart disease increased dramatically from the start of the 1900s to 1921, but subsequently experienced a steady decline from the mid-1960’s to 2000. However, when the decrease in heart disease is examined at the level of race it is clear that the decrease is not equally shared. While the leading cause of death among both Caucasian American men and women and African American men and women remains heart disease, the decrease in incidence of coronary heart disease among African American men was only half of the decrease in incidence among Caucasian American men. Genetic variants in BAG3 (Bcl-2 associated athanogene 3), a highly evolutionarily conserved gene that has recently emerged as a major dilated cardiomyopathy locus, are prevalent in isolated populations. This led us to hypothesize that variants in BAG3 might contribute to the increased prevalence of IDC in individuals of African ancestry. Expressed predominantly in the heart, the skeletal muscle and in many cancers, BAG3 has pleotropic effects in the heart. It inhibits apoptosis by binding to Bcl-2, facilitates protein quality control by binding to both large and small heat shock proteins, mediates adrenergic responsiveness by coupling the β-adrenergic receptor and the L-type Ca2+ channel, and maintains the integrity of the sarcomere by anchoring actin filaments to the Z disc. However, a paucity of subjects of African ancestry have been included in cohorts of probands with familial dilated cardiomyopathy whose exomes or genomes have been sequenced. Based on our previous observations and reports from other groups we postulated: 1) that mice with haplo-insufficiency of BAG3 will re-capitulate disease seen in humans and serve as a model for studying the pathogenesis of BAG3. 2) The prevalence or identification of specific BAG3 variants will differ by race and/or ethnicity. 3) SNVs of BAG3 may contribute to disease progression and thereby be pathogenic. Our study points out that we cannot understand population-based differences without enhancing the diversity of populations included in genomic studies. Similarly, in the era of big data, efforts must be undertaken to assess the genetic profile of both probands and their family members as without the ability to measure segregation, penetrance and plasticity we can only ascribe associations to functional genetic variants. / Biomedical Sciences

Dilated cardiomyopathy (DCM) or round heart disease is a muscle disease of the heart characterized by left ventricular dilatation and abnormal systolic and diastolic ventricular function. In animals, including turkeys and humans, DCM is a major cause of morbidity and mortality that results in heart failure. In the turkey, DCM can be idiopathic or induced. Since idiopathic or spontaneous DCM occurs in about 2-4 % of normal turkeys, it is of significant concern to the poultry industry. This dissertation was designed to increase our understanding of the pathophysiology of DCM in commercial turkeys. Specific objectives included: evaluating the influence of dietary selenium (Se) and vitamin E on poults fed toxic levels of furazolidone (Fz). Evaluating differences among reciprocal crosses of turkey varieties in susceptibility to a toxic level of Fz that induces DCM were used to assess the role of genetics in DCM. Using glutathione (GSH), glutathione peroxidase (GPx), malondialdehyde (MDA), and plasma uric acid (PUA) as biomarkers, oxidative stress (OS) levels were evaluated. Oxidative stress was also evaluated in poults fed diets containing varying concentration and combinations of vitamin E (0, 50 and 100 IU/kg) and Se (0.0, 0.3 and 0.5 mg/kg). Results from echocardiography measurements at four weeks of age, for poults fed toxic levels of Fz, showed the Narragansett x Bourbon Red reciprocal cross had the lowest internal-diastolic (LVIDd) and systolic dimensions (LVISd), while the Bourbon Red x Narragansett reciprocal cross had the largest LVIDd and LVISd. Left ventricular internal-diastolic and systolic dimension were lower for cross bred than parental poults. In treatment poults, heterosis for ventricular dilation was most significant for Bourbon Red x Narragansett cross. The data suggest that reciprocal crosses respond differently to toxin that induces DCM and genetics may influence a turkey's response to toxic levels of Fz that causes DCM. Results from OS measurements in poults fed normal and those fed normal diets with Fz at two weeks of age, showed no significant differences in MDA and GPx levels. PUA and GSH levels were however significantly increased for poults fed Fz-containing diets. At four weeks of age, no differences were observed for MDA and GPx measurements between poults fed normal and Fz-containing diets. PUA levels increased for poults fed normal diets with Fz, while GSH levels increased only for those fed normal diets. Differences between poults fed normal and Fz-containing diets were significant for GPx measurements. Results of this study showed that, feeding diets with Fz does not increase OS. Measure of the influence of feeding diets supplemented with different concentrations and combinations of Se and vitamin E to poults fed either normal or normal diets with Fz at two and four wks of age, showed higher MDA levels for poults fed Fz-containing diets supplemented with 0.3 mg/kg Se and 100 IU/kg vitamin E. For antioxidant biomarkers, GPx activity were increased for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se and those fed 100 IU/kg vitamin E. Poults fed normal diets supplemented with 100 IU/kg vitamin E had the highest GPx. PUA levels were higher for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se at two wks of age. At four wks of age, PUA concentrations were higher for poults fed Fz-containing diets supplemented with 100 IU/kg vitamin E. Increased PUA were also observed for poults fed diets supplemented with 0.5 mg/kg Se and 50 IU/kg vitamin E and 0.5 mg/kg and 100 IU/kg vitamin E. Poults fed diets supplemented with 50 and 100 IU/kg vitamin E had the highest GSH at two wks of age. Measurements taken at 2 wks of age, for poults fed normal diets supplemented with different concentrations and combinations of Se and vitamin E had increased GSH levels when compared with those fed diets with Fz at four wks of age. In this study, we showed that supplementation of poults fed normal diets with Fz with different concentrations and combinations of Se and vitamin E did not reduce DCM at 2 wks of age. However, at 4 wks of age, though DCM was not decreased by feeding diets supplemented with different concentrations and combinations of Se and vitamin E, reduced oxidant and antioxidant biomarkers were observed. / Ph. D.

Dilated cardiomyopathy (DCM), a disease of the myocardium, causes morbidity and premature death in humans and other domestic animals including turkeys. Though DCM results from many different factors including those that are unknown or idiopathic, genetic factor is a major cause of idiopathic DCM. In this study, I assessed the molecular basis of toxin-induced DCM in turkeys by evaluating the association and effect of mutations in candidate genes in the nucleus and mitochondria on the incidence and severity of this disease. Echocardiographic measurements at 3 weeks of age showed that birds on furazolidone-containing diet exhibited a significant DCM phenotype (increased left ventricular end diastolic dimension and left ventricular end systolic dimension) with a marked decrease in the left ventricular shortening fraction. Pathological phenotype confirmed the dilated heart with extended cell necrosis. Two mutations, both in NADH dehydrogenase genes, were found to be associated with DCM. Real-time RT-PCR quantification indicated that mRNA expression of alpha cardiac actin gene (ACTC) were significantly different between control and treatment birds. While ACTC expression increased, though moderately, in control birds from week 1 to 3, it decreased significantly in treatment birds. These findings suggest that the mitochondrial DNA variation and ACTC expression may be associated with the turkey's response to toxin. Therefore, further research is needed to investigate the molecular mechanism of toxin-induced DCM in the turkey. / Master of Science

Dilated cardiomyopathy (DCM), a heart disease, affects many vertebrates including humans and poultry. The disease can be either idiopathic (IDCM) or toxin-induced. Idiopathic DCM often occurs without a consensus cause. Though genetic and other studies of IDCM are extensive, the specific etiology of toxin-induced is still unknown. Here, our objective was to compare the level of mRNA expression of two candidate genes including troponin T (cTnT) and phospholamban (PLN) using quantitative reverse transcription polymerase chain reaction (RT-PCR) in toxin-induced DCM affected and unaffected turkeys. Cardiac TnT and PLN were chosen because their spontaneous expression has been reported to be associated with IDCM. We also scanned these genes for single nucleotide polymorphisms (SNPs) that could be useful in evaluating their functions in the incidence and severity of toxin-induced DCM in turkeys. There were no significant differences between affected and unaffected birds in the expression of both cTnT and PLN. A total of 12 SNPs were detected in cTnT and PLN DNA sequences. One of the seven haplotypes detected in cTnT was the most frequent. Linkage analysis showed that cTnT gene was unlinked on the current turkey genetic map. Resources developed here, including SNPs, haplotypes, cDNA sequences, and the PCR-RFLP genotype procedure will be used for future investigations involving cTnT and PLN and toxin-induced DCM. / Master of Science

No description available.

Les Entérovirus, et notamment les coxsackievirus B (CV-B), sont une cause commune de myocardite. Cette maladie peut évoluer vers une myocardite chronique jusqu’au stade de cardiomyopathie dilatée (CMD). Le mécanisme moléculaire permettant le passage de l’infection aigue à l’infection persistante dans le tissu cardiaque humain reste méconnu. Pour étudier les activités de réplication des CV-B persistant, un réplicon a été généré à partir d’une souche cardiotrope. Il a permis de caractériser les activités de réplication de CV-B persistant dans des cellules cardiaques humaines. Une analyse par séquençage à moyen débit a permis de mettre en évidence la sélection de variants tronqués dans le cœur pouvant expliquer la progression de la myocardite virale vers la CMD. De plus, l’existence de populations majoritaires tronquées de 19 à 50 nucléotides associées à des formes virales minoritaires complètes a été mise en évidence chez 8 patients atteints de CMD. La proportion de populations tronquées s’est révélée négativement corrélée au ratio ARN+/ARN- et à la charge virale (R2=0,748; P=0,016; R2= 0,36, P=0,038, respectivement). Des études immuno-histologiques et par hybridation in situ des tissus cardiaques ont montrées que le clivage de la dystrophine était uniquement retrouvé dans les cardiomyocytes infectés par les CV-B. La transfection d’ARN de synthèse complets et tronqués dans des cultures de cardiomyocytes humains primaires a mis en évidence une trans-complémentation entre les 2 formes virales induisant de faibles activités de réplication. Nos résultats démontre l’existence d’un nouveau mécanisme moléculaire de coopération entre des populations persistantes d’entérovirus B tronquées et complètes qui contribue à la physiopathologie de la CMD. / Enteroviruses, especially group B coxsackieviruses (CV-B) are considered to be a common cause of acute human myocarditis, a disease that is a precursor of chronic myocarditis cases as well as dilated cardiomyopathy (DCM). The molecular mechanisms related to the switch from the acute to the CV-B chronic persistent infection in human cardiac tissues are still unknown. To study the replication activities of CV-B a replicon from a cardiotropic prototype strain was generated and was used to study persistent CV-B replication activities into human cardiac cells. Using NGS analyses, our results evidenced that the molecular selection of TD viral forms in heart could explain pathophysiological progression from acute viral myocarditis to DCM. Moreover, we demonstrated in 8 end-stage DCM patients the existence of CV-B major populations characterized by 5’NTR deletions ranging from 19 to 50 nucleotides that were associated with minor full-length viral forms. The amounts of persistent deleted populations appeared to be negatively correlated to RNA(+)/RNA(-) minus ratio and viral load values (R2=0.748; P=0.016; R2= 0.36, P=0.038, respectively). In situ hybridization and immunohistological assays in cardiac tissues demonstrated that the dystrophin disruption was only present in EV-B infected cardiomyocytes. Transfection of deleted and full-length RNA-populations in cultured primary human cardiomyocytes evidenced a trans-acting genomic complementation system between the two viral forms resulting in low viral replication activities. Our findings suggest a new molecular mechanism through which persistent low replicative EV-B deleted and full-length collaborative populations contribute to the pathogenesis of idiopathic DCM cases.

Dilated Cardiomyopathy (DCM) with conduction disease and Atrial Fibrillation (AF) are the two cardiac-specific diseases associated with lamin A/C gene (LMNA) mutations. Protein Kinase C Alpha, (PKCα) functions as a nodal integrator of cardiac contractility by “sensing” intracellular calcium and signal transduction. PKCα has been implicated in heart failure and cardiac hypertrophy. Moreover, abnormal PKCα function results in irregular atrial potassium channel activity associated with chronic AF PKCα is a lamin A/C binding partner. Thus, the deregulation of PKCα signaling can contribute to the development of DCM and AF. Our hypothesis is that the AF (Thr528Met), DCM-associated (Arg541Cys) and (Arg541Gly) and DCM/AF-associated (Tyr481Stop) LMNA variants will disrupt the cellular distribution of PKCα therefore resulting in impaired PKCα function. The first objective was to phenotypically characterise Arg541Cys LMNA variant in murine skeletal myoblasts cell line (C2C12) in comparison to cellular phenotypes induced by LMNA variants associated with AF, DCM and DCM with AF. Arg541Cys lamin A and C variants formed circular and sickle-shaped lamin A/C in the nucleus of C2C12 cells. The second objective was to determine the effect of these lamin variants on cellular distribution of PKCα in C2C12 cells. PKCα mislocalized into the nucleus of C2C12 cells transfected with AF and DCM-associated variants (Thr528Met and Arg541Cys). Colocalization analysis showed significant increase in PKCα in the nucleus of AF (Thr528Met) and DCM (Arg541Cys) variants when lamin A and C, were co-transfected compared to wild-type, DCM (Arg541Gly) and DCM/AF (Tyr481Stop) variants. Densitometry analysis showed statistically significant increase in phosphorylated PKCα, the active form of PKCα, in nuclear and cytoplasmic extracts of C2C12 cells expressing Arg541Cys variant. Densitometry analysis also showed statistically significant increase in non-phosphorylated PKCα in the nuclear extract of Thr528Met variant expressing cells. The third objective was to determine the effect of AF and DCM-associated variants on the activity of PKCα. PKCα activity is quantified by measuring the phosphorylation of a known phosphorylated PKCα substrate. Alpha-6-tubulin phospho (Ser165) is phosphorylated by PKCα. Hence, this was used to quantify PKCα activity. No statistical significance was observed in the level of phosphorylated alpha-6-tubulin at (Ser165) in the C2C12 cells that were transfected with lamin A and C variants compared to wild type. Furthermore, PKCα phosphorylation state is cyclic in nature and this could have had an impact on the phosphorylation state of the chosen substrate in this study. The functional consequence of nuclear translocation of PKCα with respect to laminopathies is unknown. Abnormal activation of the Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2) which are branches of the mitogen-activated protein kinase (MAPK) signalling cascade in hearts of mice, and humans prior to the onset of cardiomyopathy. These findings have been associated to cardiac disease-causing lamin A/C alteration to signal transduction pathways implicated in heart function and cardiomyopathy. Human LMNA cardiomyopathy, could lead to abnormal activation of MAPK signalling pathways via abnormal PKCα activation in cardiomyocytes.

No description available.