Rationale/Background: The adult mammalian heart has a very limited potential for regeneration due to cardiomyocyte cell cycle withdrawal which occurs shortly after birth. One potential avenue to repair the heart following stress/injury is to reprogram pre-existing cardiomyocytes to re-enter the cell cycle. The E2F family is a group of transcription factors which ubiquitously regulate the cell cycle, but it has previously been difficult to fully appreciate their role in the post-natal myocardium due to either redundancy or embryonic lethality of genetic models. Thus we generated a dominant negative model of the E2F/Rb pathway via expression of the unique transcriptional repressor E2F6 in postnatal myocardium. E2F6 transgenic (Tg) mice developed dose dependent Dilated Cardiomyopathy (DCM) and sudden death without hypertrophy or apoptosis. This was accompanied by the partial loss of E2F3 (critical for cardiac development) and connexin-43 important for metabolic and electrical coupling. Methods/Results: In this thesis E2F6-Tg mice were examined for markers of cardiac differentiation/ function and exposed to stressors to evaluate the capacity for the E2F pathway to regulate cardiomyocyte growth (isoproterenol) and death (doxorubicin and cobalt chloride). E2F6-Tg mice were twice as sensitive to isoproterenol as their Wt counterparts due to the observed activation of a β2-adrenergic survival pathway. Cardiac hypertrophy in E2F6-Tg mice was accompanied by the rescue of E2F3 expression. Treatment of neonatal cardiomyocytes isolated from Wt and E2F6-Tg pups with cobalt chloride revealed a protective effect for E2F6 against apoptosis. Doxorubicin exposure led to the loss of E2F6 protein and abolished its protective effect. Examination of neonatal hearts and cardiomyocytes isolated from them demonstrated a shift in the cell cycle and metabolic profiles of E2F6-Tg myocardium. Tg cardiomyocytes show decreased glycolysis and a dramatic increase in the regulator of ketolysis, β-hydroxybutyrate dehydrogenase (BDH1), prior to DCM. The substrate of BDH1 (β-hydroxybutyrate) was demonstrated to influence the levels of CX-43 in cardiomyocytes. E2F6 also deregulated expression of T-cap which has been linked to human DCM. Conclusions: I provide evidence that the E2F pathway can regulate growth, death, and differentiation through a variety of mechanisms which link the cell cycle and metabolism to growth and survival to critically govern post-natal cardiac function. Furthermore, I reveal a new biomarker (BDH1) for early DCM which may be useful in diagnosis/ treatment of idiopathic cases of disease.

Dilated cardiomyopathy (DCM) or round heart disease is a muscle disease of the heart which is characterized by ventricular dilatation and abnormal systolic and diastolic left ventricular function. In animals, including turkeys and humans, DCM is the major cause of morbidity and mortality which results from heart failure. In the turkey, DCM can be idiopathic or induced. Since idiopathic or spontaneous DCM occurs in about 1-4% of normal turkeys, it is of significant concern to the poultry industry. In this study, it was proposed that the incidence and severity of DCM in the turkey may have a genetic basis. To test this hypothesis, I investigated differences in the incidence and severity of DCM in five domesticated turkey varieties including Blue Slate (BS), Bourbon Red (BR), Narragansett (N), Royal Palm (RP) and Spanish Black (SB). Preliminary investigations tested the reliability of echocardiography (ECHO) as a non-invasive and non-destructive technique for diagnosing DCM in a large number of birds from hatch to four weeks-of-age. One-day-old poults for both the preliminary and hypothesis testing investigations were obtained from Privett Hatcheries (Portales, New Mexico). The birds were raised under standard management conditions. In the preliminary investigation and to test my hypothesis, DCM was induced by feeding birds ad libitum standard diets containing 700 parts per million furazolidone. Results of the preliminary investigations showed that left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) were the most consistent ECHO indicators of DCM from hatch to 4 weeks-of-age. Variety differences in response to furazolidone were evaluated using these parameters as well as percent mortality. At 9 days-of-age, differences between control and treatment birds for percent mortality and LVESD were significant in the RP variety only but significant for LVEDD in RP and SB. At 29 and 33 days-of-age, all the pair-wise comparisons between control and treatment birds were significant for both LVEDD and LVESD. On average, the BR variety had the smallest dilatation of the heart and lowest mortality at 33 days-of-age when compared to other varieties. The results described in this thesis show, for the first time, variety differences in the turkey's response to diets containing furazolidone. They provide strong evidence that, like previous reports for idiopathic DCM, an animal's response to Fz-induced DCM has a strong genetic component. / Master of Science

Objectives: The purpose of this study was to investigate a family of Great Danes with known dilated cardiomyopathy (DCM) using serial echocardiographic evaluation. Animals, Materials, and Methods: Twenty-two dogs were included in this study. They were split into two groups, clinically normal and those with DCM. The dogs were scanned using 2D and M-mode echocardiography every thirty to sixty days beginning at approximately14-20 days of age. Data were collected and analyzed using generalized additive mixed regression, linear regression, and non-linear regression. Results: All dogs demonstrated progressive echocardiographic changes. The Great Danes with DCM showed several echocardiographic differences when compared to the normal dogs. They included differences in left ventricular diameter, left atrial diameter, interventricular septal thickness, ejection fraction, and fractional shortening. Conclusions: The present study shows that progressive echocardiographic changes occur in both clinically normal Great Danes and those with DCM as they mature. Additionally, the two groups differed with regards to left ventricular diameter, left atrial diameter, interventricular septal thickness, ejection fraction, and fractional shortening.

Evolution of viral myocarditis to dilated cardiomyopathy (DCM)represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. RESULTS: IRF9-/- mice showed dramatically increased mortality compared to the wildtype littermates (0% WT vs 72% IRF-9-/- on day 14, P<0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9-/- mice compared to WT controls (p<0.05). Onn day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p<0.05). CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.

The E2F family of transcription factors regulate cellular growth, death and differentiation, but their role in cardiac biology remains to be fully explored. We hypothesized that the balance of the E2F pathway would determine cardiac development and function. We provide evidence for this via modulation of the E2F6 repressor, in a transgenic (Tg) mouse model. Targeted expression of E2F6 in the heart led to dilated cardiomyopathy (DCM) and death. Microarray analysis revealed that E2F responsive pathways were activated in Tg mice. Furthermore, we found that E2F6 and YY1 (E2F-co-factor) were translocated to the nucleus in Tg mice, providing a potential mechanism for the observed transcriptional activation. We also observed a marked decrease of Connexin43 protein in the myocardium, and reduced atrial conductivity in Tg mice which may lead to reduced cardiac function. The data demonstrates a novel role for E2F pathway outside of cell cycle control in the heart.

The domestic dog, Canis familiaris, with over 450 naturally-occurring hereditary diseases, serves as a valuable model organism for study of the genetics underlying many human hereditary diseases. Approximately half of the diseases that afflict the dog are clinically very similar to various human hereditary diseases. Several cardiac diseases are in this category. Our laboratory is interested in cardiac diseases because they are common causes of death in the human and are also a widespread problem in many breeds of dog. The specific focus of my work is on understanding the genetics of dilated cardiomyopathy (DCM). DCM is a disease characterized by enlargement of the left ventricle leading to an inability of the heart to pump sufficient blood to the body. It is one of the most common cardiac diseases in the dog and has a high mortality. The Great Dane is the second most commonly affected breed. It is seen in many families of Great Danes, and this suggests that DCM has a genetic component. The mode of inheritance of DCM in the Great Dane is currently unknown, although studies have reported both autosomal recessive and autosomal dominant transmission. Many different genes cause DCM, indicating the complexity of the disease. These typically produce proteins that are involved in the sarcomere or cytoskeletal components, leading to problems with contraction or cardiac cell integrity. In order to identify causative or susceptibility genes for DCM in the Great Dane, a whole-genome linkage screen was conducted in a family of Great Danes. One candidate gene, gamma-sarcoglycan (SGCG), was identified through linkage and sequenced in affected and unaffected dogs. Sequencing data revealed no mutations in the coding regions of SGCG, most likely excluding it as a candidate gene for DCM. Continued evaluation of this gene and others, both in sequence content and additional properties such as epigenetic effects, protein structure, and interaction with other genes will increase understanding of DCM in both the dog and the human.

The E2F family of transcription factors regulate cellular growth, death and differentiation, but their role in cardiac biology remains to be fully explored. We hypothesized that the balance of the E2F pathway would determine cardiac development and function. We provide evidence for this via modulation of the E2F6 repressor, in a transgenic (Tg) mouse model. Targeted expression of E2F6 in the heart led to dilated cardiomyopathy (DCM) and death. Microarray analysis revealed that E2F responsive pathways were activated in Tg mice. Furthermore, we found that E2F6 and YY1 (E2F-co-factor) were translocated to the nucleus in Tg mice, providing a potential mechanism for the observed transcriptional activation. We also observed a marked decrease of Connexin43 protein in the myocardium, and reduced atrial conductivity in Tg mice which may lead to reduced cardiac function. The data demonstrates a novel role for E2F pathway outside of cell cycle control in the heart.

Evolution of viral myocarditis to dilated cardiomyopathy (DCM)represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. RESULTS: IRF9-/- mice showed dramatically increased mortality compared to the wildtype littermates (0% WT vs 72% IRF-9-/- on day 14, P<0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9-/- mice compared to WT controls (p<0.05). Onn day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p<0.05). CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.

Abstract Hereditary hemochromatosis (HH) is a common autosomal recessive disorder estimated to affect one out of every 250–400 Caucasian individuals. It is a disorder of iron metabolism, in which excessive iron accumulation in the body may induce serious clinical manifestations (e.g. liver cirrhosis, hepatocellular carcinoma, diabetes, and cardiomyopathy). HH is caused by mutations in the HFE gene, and HFE genotyping thus enables early diagnosis of the disease and detection of the individuals at risk for HH. HFE mutations have also been proposed to predispose to certain other diseases, such as various hematological malignancies and cardiomyopathy. The present evaluation of the clinical utility and outcome of HFE genotyping in search for HH was based on data obtained from 137 subjects referred for HFE mutation analysis during the years 1999–2001. The C282Y and H63D mutations were determined for each subject. HFE genotyping was also used to examine the association between HFE mutations with various hematological disorders and idiopathic dilated cardiomyopathy (IDCM). The C282Y and H63D mutations were determined from 232 patients with various hematological disorders and the C282Y, H63D, and S65C mutations from 91 patients with IDCM and 102 control subjects. High frequencies of C282Y homozygotes (16.8%) and C282Y/H63D compound heterozygotes (5.1%) were found among the subjects referred for HFE genotyping, and the rate of positive findings for HH increased steadily over the years 1999–2001. The frequencies of HFE mutations did not differ significantly in patients with various hematological disorders and IDCM compared to controls. At the end of the follow-up period, left ventricular end-diastolic diameter (LVEDD) was significantly higher in IDCM patients carrying the C282Y mutation than in those without this mutation (p = 0.037). The present study supports active testing for the HFE gene mutations C282Y and H63D in public health care. Serum transferrin saturation is considered the most useful test for selecting subjects for such analysis. Although increasing numbers of HH cases are recognized by physicians, it may still be an underdiagnosed disease. HFE mutations do not seem to significantly increase the risk for various hematological disorders or IDCM. The C282Y mutation may, nevertheless, mediate the progression of IDCM by modifying LV dilation and remodeling.

The E2F family of transcription factors regulate cellular growth, death and differentiation, but their role in cardiac biology remains to be fully explored. We hypothesized that the balance of the E2F pathway would determine cardiac development and function. We provide evidence for this via modulation of the E2F6 repressor, in a transgenic (Tg) mouse model. Targeted expression of E2F6 in the heart led to dilated cardiomyopathy (DCM) and death. Microarray analysis revealed that E2F responsive pathways were activated in Tg mice. Furthermore, we found that E2F6 and YY1 (E2F-co-factor) were translocated to the nucleus in Tg mice, providing a potential mechanism for the observed transcriptional activation. We also observed a marked decrease of Connexin43 protein in the myocardium, and reduced atrial conductivity in Tg mice which may lead to reduced cardiac function. The data demonstrates a novel role for E2F pathway outside of cell cycle control in the heart.