Background: Different types of cardiomyopathies are associated with variable hypertrophic response. A number of growth factors are thought to play a role in pathologic cardiac remodeling. Aims: We compared the modulation of the TGF-ƓÒ superfamily and IGF-1 signaling pathways and their target genes, the cell cycle regulatory proteins in tachycardia-induced dilated cardiomyopathy, a model with no detectable hypertrophy and in ischemic cardiomyopathy, a model with a marked hypertrophic reaction. Methods: In the first study, endomyocardial biopsies were obtained weekly in 15 dogs, during the development of tachycardiomyopaty. Genes involved in the myostatin-TGF-ƓÒ-Activin-A/Smad signaling pathway, p21 and cyclin D were quantified and correlated to echocardiographic measures of hypertrophy. In the second study, myocardial tissue samples were obtained in 8 dogs with a healed myocardial infarction, in 8 dogs with heart failure induced by overpacing and in 7 healthy dogs. We measured gene expression of IGF-1, its receptor (IGF-1R) and cyclins A, B, D1, D2, D3 and E and correlated them to the level of hypertrophy. Results: Tachycardiomyopathy was characterized by chambers dilation with no identifiable hypertrophy. Ischemic cardiomyopathy was characterized by eccentric hypertrophy. In tachycardiomyopathy, Activin-A mRNA was 4-fold higher than at baseline. Smad7 was overexpressed in severe heart failure; p21, a direct target gene of the Smad pathway was upregulated 8-fold and cyclin D1 was down-regulated. In that model, IGF-1 was overexpressed but neither IGF-1R nor any of the cyclins studied. In ischemic cardiomyopathy, IGF-1, IGF-R, and cyclins B, D1, D3 and E gene expression were upregulated. In tachycardiomyopathy, Activin-A and p21 were inversely correlated to the thickness of the interventricular septum. In normal dogs and in the both models of cardiomyopathy, IGF-1R was correlated to the thickness of the interventricular septum and to cyclins. Conclusions: Taken together, these results agree with the notion that Activin-A, IGF and cyclins are involved in the modulation of hypertrophic response observed in cardiomyopathies.

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.

Vascular complications, in microvessels resulting in nephropathy, retinopathy and neuropathy and in macrovessels resulting in atherosclerosis caused by hyperglycemia contribute greatly to the morbidity and mortality in diabetes mellitus. In the vasculature, the endothelial cells (ECs) are first to be damaged by hyperglycemia due to their unique location as the inner lining of all vessels. There are several mechanisms involved in endothelial injury or dysfunction, however, the degradation of heparan sulfate proteoglycan (HSPG) on the cell surface and in the extra cellular matrix (ECM) is considered to be of importance. Heparanase is believed to degrade heparan sulfate (HS). Our objectives were to determine if heparanase is responsible for endothelial injury and dysfunction in diabetes. <p>To determine if hyperglycemia and heparanase cause endothelial injury, high concentrations of glucose (30mM), mimicking hyperglycemia and optimal doses of heparinase I were used to treat cultured porcine aortic endothelial cells (PAECs). Cell injury was measured by determining live cell number and lactate dehydrogenase (LDH) release. To determine if heparanase is expressed in high glucose treated PAECs, reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify heparanase mRNA. In addition, heparanase activity was measured by incubating cell lysates with 35S-labelled ECM from cultured bovine corneal ECs, where released radioactive HS was analyzed by Sepharose gel filtration followed by â-scintillation counting. To help understand the mechanism of high glucose injury, heparanase mRNA and activity were also measured in PAECs treated with H2O2 or mannitol to determine if free radical injury or osmolarity caused effects similar to high glucose treatment. As well, high glucose or heparinase I treated PAECs were also treated with heparin (0.5 ìg/ml) and/or insulin (1 U/ml) and/or basic fibroblast growth factor (bFGF, 1 ng/ml) to determine if these compounds protected ECs from injury or inhibited heparanase expression induced by high glucose. p* PAECs injured by high glucose or heparinase I (0.3 U/ml in serum free medium) showed a significantly decreased live cell number and increased LDH release compared to control cells. High glucose or heparinase I treated ECs showed an increase in live cell number and decrease in LDH release when treated with heparin and/or insulin and bFGF. Heparanase mRNA and activity was expressed in PAECs treated with high glucose or H2O2. Heparin and/or insulin, but not bFGF prevented heparanase mRNA expression and activity in high glucose treated PAECs. Mannitol did not induce the upregulation of heparanase mRNA and activity. bFGF showed variable protection in cells treated with high glucose or heparinase I when combined with insulin or heparin. <p> From these results we conclude that hyperglycemia is a main cause of endothelial injury. Heparanase production induced by hyperglycemia is responsible for EC injury and vascular dysfunction likely through the degradation of HS, resulting in increased vascular permeability and detachment of cells from the basement membrane. The mechanism of heparanase upregulation may be related to the formation of reactive oxygen species, but not due to changes in osmolarity. Heparin and/or insulin and bFGF protect cells from injury caused by high glucose or heparinase I. Heparin and/or insulin but not bFGF inhibit heparanase mRNA upregulation induced by high glucose. This study provides new insight into the causes of vascular injury associated with diabetes and suggests possible treatments to reduce endothelial injury.

Vascular complications, in microvessels resulting in nephropathy, retinopathy and neuropathy and in macrovessels resulting in atherosclerosis caused by hyperglycemia contribute greatly to the morbidity and mortality in diabetes mellitus. In the vasculature, the endothelial cells (ECs) are first to be damaged by hyperglycemia due to their unique location as the inner lining of all vessels. There are several mechanisms involved in endothelial injury or dysfunction, however, the degradation of heparan sulfate proteoglycan (HSPG) on the cell surface and in the extra cellular matrix (ECM) is considered to be of importance. Heparanase is believed to degrade heparan sulfate (HS). Our objectives were to determine if heparanase is responsible for endothelial injury and dysfunction in diabetes. <p>To determine if hyperglycemia and heparanase cause endothelial injury, high concentrations of glucose (30mM), mimicking hyperglycemia and optimal doses of heparinase I were used to treat cultured porcine aortic endothelial cells (PAECs). Cell injury was measured by determining live cell number and lactate dehydrogenase (LDH) release. To determine if heparanase is expressed in high glucose treated PAECs, reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify heparanase mRNA. In addition, heparanase activity was measured by incubating cell lysates with 35S-labelled ECM from cultured bovine corneal ECs, where released radioactive HS was analyzed by Sepharose gel filtration followed by â-scintillation counting. To help understand the mechanism of high glucose injury, heparanase mRNA and activity were also measured in PAECs treated with H2O2 or mannitol to determine if free radical injury or osmolarity caused effects similar to high glucose treatment. As well, high glucose or heparinase I treated PAECs were also treated with heparin (0.5 ìg/ml) and/or insulin (1 U/ml) and/or basic fibroblast growth factor (bFGF, 1 ng/ml) to determine if these compounds protected ECs from injury or inhibited heparanase expression induced by high glucose. p* PAECs injured by high glucose or heparinase I (0.3 U/ml in serum free medium) showed a significantly decreased live cell number and increased LDH release compared to control cells. High glucose or heparinase I treated ECs showed an increase in live cell number and decrease in LDH release when treated with heparin and/or insulin and bFGF. Heparanase mRNA and activity was expressed in PAECs treated with high glucose or H2O2. Heparin and/or insulin, but not bFGF prevented heparanase mRNA expression and activity in high glucose treated PAECs. Mannitol did not induce the upregulation of heparanase mRNA and activity. bFGF showed variable protection in cells treated with high glucose or heparinase I when combined with insulin or heparin. <p> From these results we conclude that hyperglycemia is a main cause of endothelial injury. Heparanase production induced by hyperglycemia is responsible for EC injury and vascular dysfunction likely through the degradation of HS, resulting in increased vascular permeability and detachment of cells from the basement membrane. The mechanism of heparanase upregulation may be related to the formation of reactive oxygen species, but not due to changes in osmolarity. Heparin and/or insulin and bFGF protect cells from injury caused by high glucose or heparinase I. Heparin and/or insulin but not bFGF inhibit heparanase mRNA upregulation induced by high glucose. This study provides new insight into the causes of vascular injury associated with diabetes and suggests possible treatments to reduce endothelial injury.

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.

No description available.

Diabetic cardiomyopathy is defined as changes in the structure and function of the myocardium that occur in diabetic patients in the absence of other cardiovascular risk factors. Our laboratory has shown that conjugated linoleic acid (CLA - a naturally-occurring polyunsaturated fatty acid with multiple health benefits) prevents endothelin-1-induced myocyte hypertrophy in vitro, as well as cardiac hypertrophy in vivo using a rodent model of spontaneously hypertensive heart failure. These cardioprotective effects of CLA were mediated through activation of peroxisome proliferator activated receptors (PPAR isomers α and γ) and stimulation of diacylglycerol kinase ζ (DGKζ). Thus, the aims of this study were to (i) determine the effect of CLA on hyperglycemia-induced structural and functional abnormalities of cardiomyocytes, and (ii) assess the role of PPAR-γ and DGK. High glucose treatment induced hypertrophy of primary adult cardiomyocytes, as indicated by augmented cell size and protein synthesis compared to untreated cardiomyocytes. The hyperglycemia-induced hypertrophy was attenuated by pretreatment with CLA (30 µM). The ability of CLA to prevent hyperglycemia-induced hypertrophy was suppressed by GW9662 (1 µM) and R59022 (10 μM), pharmacological inhibitors of PPAR-γ and DGK, respectively. In addition to structural abnormalities, high glucose impaired contractile function of adult cardiomyocytes as measured by maximal velocity of shortening, maximal velocity of relengthening, and peak shortening. Hyperglycemia-induced contractile dysfunction was likewise prevented by pretreatment with CLA (30 µM). Collectively, these findings support the idea that hyperglycemia is an independent risk factor for the development of diabetic cardiomyopathy. Hypertrophy and contractile dysfunction elicited by high glucose were prevented by CLA. The antihypertrophic actions of CLA are mediated, at least in part, by activation of PPAR-γ and DGK.

Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

Diabetic cardiomyopathy is defined as changes in the structure and function of the myocardium that occur in diabetic patients in the absence of other cardiovascular risk factors. Our laboratory has shown that conjugated linoleic acid (CLA - a naturally-occurring polyunsaturated fatty acid with multiple health benefits) prevents endothelin-1-induced myocyte hypertrophy in vitro, as well as cardiac hypertrophy in vivo using a rodent model of spontaneously hypertensive heart failure. These cardioprotective effects of CLA were mediated through activation of peroxisome proliferator activated receptors (PPAR isomers α and γ) and stimulation of diacylglycerol kinase ζ (DGKζ). Thus, the aims of this study were to (i) determine the effect of CLA on hyperglycemia-induced structural and functional abnormalities of cardiomyocytes, and (ii) assess the role of PPAR-γ and DGK. High glucose treatment induced hypertrophy of primary adult cardiomyocytes, as indicated by augmented cell size and protein synthesis compared to untreated cardiomyocytes. The hyperglycemia-induced hypertrophy was attenuated by pretreatment with CLA (30 µM). The ability of CLA to prevent hyperglycemia-induced hypertrophy was suppressed by GW9662 (1 µM) and R59022 (10 μM), pharmacological inhibitors of PPAR-γ and DGK, respectively. In addition to structural abnormalities, high glucose impaired contractile function of adult cardiomyocytes as measured by maximal velocity of shortening, maximal velocity of relengthening, and peak shortening. Hyperglycemia-induced contractile dysfunction was likewise prevented by pretreatment with CLA (30 µM). Collectively, these findings support the idea that hyperglycemia is an independent risk factor for the development of diabetic cardiomyopathy. Hypertrophy and contractile dysfunction elicited by high glucose were prevented by CLA. The antihypertrophic actions of CLA are mediated, at least in part, by activation of PPAR-γ and DGK.

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.