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In vivo phosphorylation of cardiac troponin I by PKCβ2 decreases cardiomyocyte calcium responsiveness and contractility in transgenic mouse hearts
The Second Annual Scientific Meeting
075 In g i v o Phosphorylation of Cardiac Troponin I by PKC~2 Decreases Cardiomyocyte Calcium R e s p o n s i v e n e s s and Contractility in Transgenic M o u s e Hearts
Yasuchika Takeishi, Guoxiang Chu, Darryl M. Kirkpatirck, Evangelia G. Kranias, George L. King, Richard A. Walsh, University of Cincinnati College of Medicine, Cincinnati, OH and Harvard Medical School, Boston, MA Recently, it has been reported that the protein kinase C (PKC) 13 isoforrn plays a critical role in the development of hypertrophy and heart failure. The purpose of the present study was to clarify the mechanism by which activation of PKCf~ led to depressed cardiac function. Thus, we used a PKCI32 overexpressing mouse, an animal model of heart failure, to examine mechanical properties and Ca2+ signals of isolated left ventricular cardiomyocytes. The percentage of shortening, rate of shortening, and rate of relengthening of cardiomyocytes were markedly reduced in PKC[~2 overexpression mice compared to wild type control mice, although the baseline level and amplitude of Caz+ signals were similar. These findings suggested a decreased myofilament responsiveness to Ca2+ in transgenic hearts. Therefore, the incorporation of [32p1 inorganic phosphate into cardiac myofibrillar proteins was studied in Langendorff-perfused hearts. There was a significant increase in the degree of phosphorylation of troponin I in PKCI32 overexpressing transgenic mice. The depressed cardiomyocyte function improved after the superfusion of a PKCf3 selective inhibitor. These findings indicate that in vivo PKC~2 mediated phosphorylation of troponin I decreases myofilament Caz+ responsiveness, and is responsible in part for cardiac chamber and cardiomyocyte dysfunction. Since chronic and excess activation of PKC~2 plays a direct and contributory role in the progression of cardiac dysfunction, the PKCf~ selective inhibitor may provide a new therapeutic modality in the setting of heart failure.
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HFSA
21
076 HLA Phenotype Antigens in Alcoholic Cardiomyopathy Elisabeth Coignard, Prrdrrique Jault, Benoit Blanchard, Richard Dorent*, Colette Raffoux**, Iradj Gandjbakhch*, Unit of Thoracic and Cardiovascular Surgery, Hospital La Piti6 Salprtri@re, Paris, France* Irmnunolgy and Histocompatibility Laboratory, Hospital Saint-Louis, Paris, France ** Background: HLA cell surfaceantigens, which are encoded by closely arranged genes on the short arm of the sixth chromosome,are one genetic factor which may affect an individual'spredispositionto diseases.
We studied the HLA types A, B and DR of 49 patients with alcoholic dilated cardiomyopathyreferredfor heart transplantationsince 1983. Methods: This groupconsistedof 48 men and one woman,all caucasien, a pair of aniovalar twins. Average age was 49, 14 +/- 8, 25 years. Chronical alcoholismwas defined as an alcohol intake greaterthan 80 graramesof ethanol a day for 10 years or more calculated after a cautiousand careful interrogation and an anonymous questionnatre. Patients with ischemic,valvular, hypertensive and congenital heart disease were excluded. None had viral myocarditis or systemic autoimmtme disease. The control group consisted of 181 normal adults. Patients and controls were typed for class I (HLA - A~ B) and class II (HLA - DR) antigens by routine
serological methods. Comparisonsof aUelic frequencies were performed by using chi-squaredanalysis,p values<0, 05 were consideredsignificant. Results: Our study shows that alcoholic cardiorayopathy is associated with a
statistically significant increased frequency of HLA-A32, HLA-B5, HLA-BI7 and HLA-B53 antigens. The percentagefrequenciesof antigens in patients with alcoholic cardiomyopathyare respectively7, 9% (13=0,04), 4, 3% (p=0, 02), 8, 5% (p=0, 00l) and2, 1% (p=0, 01). In conclusion these observations in alcoholic cardiorayopathysuggesta role for genetically determined immune-respansefactors in the susceptibilityto cardiac disease in alcoholics.
077
078
Endothelium Nitric Oxide Isoform Modulates Rat H i n d l i m b Vascular Resistance in Vivo
T h e Pattern of T i s s u e Cytokine Expression in Murine M o d e l s of Myocardial Infarction
Mohamed A. Gaballa, Steven Goldman. Tucson VA Medical Center and University of Arizona Heart Center, Tucson, AZ.
Karen Aitken, Fayez Dawood, Josef Penniger, Min Irwin, Wen-Hu Wen, Peter Liu. The Toronto Hospitals, University of Toronto, Toronto, Canada
To explore the in vivo biological role of nitric oxide synthase (eNOS) in resistance vessels, we transferred a recombinant eNOS transgene into rat hindlimb vasculature. In a constant flow perfused rat hindlimb, gene transfer to the vascular endothelium was accomplished by incubating a "firstgeneration" serotype 5, replication-deficient, adenoviral vector (1.2x109 plaque forming units/ml) containing either cDNA encoding the eNOS or the J3-galactosidase gene in the hindfimb vasculature for 30 minutes. Five days after infection, vascular resistance decreased (P
We have previously reported that tissue levels of the tumor necrosis factor-t~ (TNF-c0 is up regulated in the myocardium in models of rat myocardial infarction and canine pacing induced heart failure. To determine if this pattern is also seen in the novel murine model of myocardial infarction, and how it compares with other proinflammatory cytokines, we determine the tissue expression profile for 7 cytokines in this model. Murine infarction model was performed by LAD ligation under anesthesia. The hearts were removed at times of 0, 15, 30 minutes and 1,3 and 6 hours post infarction. Samples from the central zone of risk and remote normal zones were immediately frozen and extracted for RNA. RT-PCR was then performed for cytokine expression of lL-la, IL-2, IL-4, IL6, TNF-a, TNF-[3 (lymphotoxin) and interferon gamma (IFN- ¥). 13actin was amplified from all samples to act as controls. The results showed that the normal myocardial expression of lL-lcq IL-2, IL-4, TNF-13 and IFN- 7 are at very low levels, and does not change in response to myocardial infarction. In contrast, basal levels of TNF-c~ is detectable at low levels, however, after myocardial infarction, there was robust upregulation of TNF-c~ and IL-6 transcripts immediately following infarction, peaking at 1 hour, and beginning to diminish after 3 hours. We conclude that myocardial stress responsive cytokines TNF-c~ and IL-6 are uniquely up regulated following injury, and may regulate other downstream genes orchestrating the phenotypic change seen in infarction and failure.
075 In g i v o Phosphorylation of Cardiac Troponin I by PKC~2 Decreases Cardiomyocyte Calcium R e s p o n s i v e n e s s and Contractility in Transgenic M o u s e Hearts
Yasuchika Takeishi, Guoxiang Chu, Darryl M. Kirkpatirck, Evangelia G. Kranias, George L. King, Richard A. Walsh, University of Cincinnati College of Medicine, Cincinnati, OH and Harvard Medical School, Boston, MA Recently, it has been reported that the protein kinase C (PKC) 13 isoforrn plays a critical role in the development of hypertrophy and heart failure. The purpose of the present study was to clarify the mechanism by which activation of PKCf~ led to depressed cardiac function. Thus, we used a PKCI32 overexpressing mouse, an animal model of heart failure, to examine mechanical properties and Ca2+ signals of isolated left ventricular cardiomyocytes. The percentage of shortening, rate of shortening, and rate of relengthening of cardiomyocytes were markedly reduced in PKC[~2 overexpression mice compared to wild type control mice, although the baseline level and amplitude of Caz+ signals were similar. These findings suggested a decreased myofilament responsiveness to Ca2+ in transgenic hearts. Therefore, the incorporation of [32p1 inorganic phosphate into cardiac myofibrillar proteins was studied in Langendorff-perfused hearts. There was a significant increase in the degree of phosphorylation of troponin I in PKCI32 overexpressing transgenic mice. The depressed cardiomyocyte function improved after the superfusion of a PKCf3 selective inhibitor. These findings indicate that in vivo PKC~2 mediated phosphorylation of troponin I decreases myofilament Caz+ responsiveness, and is responsible in part for cardiac chamber and cardiomyocyte dysfunction. Since chronic and excess activation of PKC~2 plays a direct and contributory role in the progression of cardiac dysfunction, the PKCf~ selective inhibitor may provide a new therapeutic modality in the setting of heart failure.
•
HFSA
21
076 HLA Phenotype Antigens in Alcoholic Cardiomyopathy Elisabeth Coignard, Prrdrrique Jault, Benoit Blanchard, Richard Dorent*, Colette Raffoux**, Iradj Gandjbakhch*, Unit of Thoracic and Cardiovascular Surgery, Hospital La Piti6 Salprtri@re, Paris, France* Irmnunolgy and Histocompatibility Laboratory, Hospital Saint-Louis, Paris, France ** Background: HLA cell surfaceantigens, which are encoded by closely arranged genes on the short arm of the sixth chromosome,are one genetic factor which may affect an individual'spredispositionto diseases.
We studied the HLA types A, B and DR of 49 patients with alcoholic dilated cardiomyopathyreferredfor heart transplantationsince 1983. Methods: This groupconsistedof 48 men and one woman,all caucasien, a pair of aniovalar twins. Average age was 49, 14 +/- 8, 25 years. Chronical alcoholismwas defined as an alcohol intake greaterthan 80 graramesof ethanol a day for 10 years or more calculated after a cautiousand careful interrogation and an anonymous questionnatre. Patients with ischemic,valvular, hypertensive and congenital heart disease were excluded. None had viral myocarditis or systemic autoimmtme disease. The control group consisted of 181 normal adults. Patients and controls were typed for class I (HLA - A~ B) and class II (HLA - DR) antigens by routine
serological methods. Comparisonsof aUelic frequencies were performed by using chi-squaredanalysis,p values<0, 05 were consideredsignificant. Results: Our study shows that alcoholic cardiorayopathy is associated with a
statistically significant increased frequency of HLA-A32, HLA-B5, HLA-BI7 and HLA-B53 antigens. The percentagefrequenciesof antigens in patients with alcoholic cardiomyopathyare respectively7, 9% (13=0,04), 4, 3% (p=0, 02), 8, 5% (p=0, 00l) and2, 1% (p=0, 01). In conclusion these observations in alcoholic cardiorayopathysuggesta role for genetically determined immune-respansefactors in the susceptibilityto cardiac disease in alcoholics.
077
078
Endothelium Nitric Oxide Isoform Modulates Rat H i n d l i m b Vascular Resistance in Vivo
T h e Pattern of T i s s u e Cytokine Expression in Murine M o d e l s of Myocardial Infarction
Mohamed A. Gaballa, Steven Goldman. Tucson VA Medical Center and University of Arizona Heart Center, Tucson, AZ.
Karen Aitken, Fayez Dawood, Josef Penniger, Min Irwin, Wen-Hu Wen, Peter Liu. The Toronto Hospitals, University of Toronto, Toronto, Canada
To explore the in vivo biological role of nitric oxide synthase (eNOS) in resistance vessels, we transferred a recombinant eNOS transgene into rat hindlimb vasculature. In a constant flow perfused rat hindlimb, gene transfer to the vascular endothelium was accomplished by incubating a "firstgeneration" serotype 5, replication-deficient, adenoviral vector (1.2x109 plaque forming units/ml) containing either cDNA encoding the eNOS or the J3-galactosidase gene in the hindfimb vasculature for 30 minutes. Five days after infection, vascular resistance decreased (P
We have previously reported that tissue levels of the tumor necrosis factor-t~ (TNF-c0 is up regulated in the myocardium in models of rat myocardial infarction and canine pacing induced heart failure. To determine if this pattern is also seen in the novel murine model of myocardial infarction, and how it compares with other proinflammatory cytokines, we determine the tissue expression profile for 7 cytokines in this model. Murine infarction model was performed by LAD ligation under anesthesia. The hearts were removed at times of 0, 15, 30 minutes and 1,3 and 6 hours post infarction. Samples from the central zone of risk and remote normal zones were immediately frozen and extracted for RNA. RT-PCR was then performed for cytokine expression of lL-la, IL-2, IL-4, IL6, TNF-a, TNF-[3 (lymphotoxin) and interferon gamma (IFN- ¥). 13actin was amplified from all samples to act as controls. The results showed that the normal myocardial expression of lL-lcq IL-2, IL-4, TNF-13 and IFN- 7 are at very low levels, and does not change in response to myocardial infarction. In contrast, basal levels of TNF-c~ is detectable at low levels, however, after myocardial infarction, there was robust upregulation of TNF-c~ and IL-6 transcripts immediately following infarction, peaking at 1 hour, and beginning to diminish after 3 hours. We conclude that myocardial stress responsive cytokines TNF-c~ and IL-6 are uniquely up regulated following injury, and may regulate other downstream genes orchestrating the phenotypic change seen in infarction and failure.