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Novel Mechanistic Insights into the Role of Advanced Glycation End Products in the Development of Diabetic Cardiomyopathy

Hegab, Zeinab Sayed Mohammed el sayed

[Thesis]. Manchester, UK: The University of Manchester; 2012.

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Abstract

Advanced glycation end products (AGEs) are molecules formed through the nonenzymaticglycation of proteins and are central to the development of cardiovascularcomplications of diabetes including heart failure. AGEs influence cellular function throughthe cross-linking of cellular proteins as well as through actions on cell surface receptors,the most common of which is (RAGE). However, it is still unclear whether AGEs contributeto myocardial abnormalities observed in diabetes through direct myocardial actionsmediated through the RAGE receptor and if so, their underlying mechanisms of action. Wehave therefore investigated the effects of AGEs on calcium handling in isolated adultmouse cardiomyocytes and cultured neonatal rat cardiomyocytes (NRCM) andcharacterised their underlying mechanisms of action in NRCM.Standard molecular techniques were used. Western blot showed expression of RAGEreceptor in mouse whole heart tissue and in both NRCM and adult mouse cardiomyocytes.Incubation of NRCM for 24 hours with AGEs showed a dose dependant reduction ofcalcium transient amplitude with a maximum of 50% at 1 g/l (P<0.01) accompanied with32% reduction in SR calcium content with no detectable changes in calcium handlingproteins expression. We demonstrated a 24% increase (P<0.01) in the production ofreactive oxygen species (ROS) in AGE treated cardiomyocytes induced by enhancedNADPH oxidase activity (P<0.05) with subsequent activation and translocation of NF-kB, atranscriptional factor from the cytoplasm to the nucleus. Activation of NF-kB induced a56% increase in iNOS gene protein expression (P<0.01), a downstream target of NF-kBwhich was accompanied by a significant increase in NO production (P<0.05). Wedemonstrated nitrosylation of several key cellular proteins involved in excitationcontractioncoupling including the Ryanodine receptor and SERCA2a as detected byimmunofluorescence.In conclusion, our work provides insights into novel pathophysiological mechanisms thatunderlie the development of heart failure in diabetes. We demonstrate the presence andfunctionality of AGE receptors in myocardium and show that AGEs inhibit excitationcontractioncoupling through increased ROS production leading to activation andtranslocation of NF-kB from the cytoplasm to the nucleus resulting in increase in NOproduction. Concomitant increases in intracellular levels of ROS and NO favours theproduction of peroxynitrite with subsequent nitrosylation of key cellular proteins involved inthe process of excitation-contraction coupling such as the Ryanodine receptor andSERCA2a. This study provides novel insights into the role of AGEs in inducing myocardialdamage in diabetes mediated through RAGE receptor and independent from the vascular effects.

Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree type:
Doctor of Philosophy
Degree programme:
PhD Medicine (Biomedicine)
Publication date:
Location:
Manchester, UK
Total pages:
182
Abstract:
Advanced glycation end products (AGEs) are molecules formed through the nonenzymaticglycation of proteins and are central to the development of cardiovascularcomplications of diabetes including heart failure. AGEs influence cellular function throughthe cross-linking of cellular proteins as well as through actions on cell surface receptors,the most common of which is (RAGE). However, it is still unclear whether AGEs contributeto myocardial abnormalities observed in diabetes through direct myocardial actionsmediated through the RAGE receptor and if so, their underlying mechanisms of action. Wehave therefore investigated the effects of AGEs on calcium handling in isolated adultmouse cardiomyocytes and cultured neonatal rat cardiomyocytes (NRCM) andcharacterised their underlying mechanisms of action in NRCM.Standard molecular techniques were used. Western blot showed expression of RAGEreceptor in mouse whole heart tissue and in both NRCM and adult mouse cardiomyocytes.Incubation of NRCM for 24 hours with AGEs showed a dose dependant reduction ofcalcium transient amplitude with a maximum of 50% at 1 g/l (P<0.01) accompanied with32% reduction in SR calcium content with no detectable changes in calcium handlingproteins expression. We demonstrated a 24% increase (P<0.01) in the production ofreactive oxygen species (ROS) in AGE treated cardiomyocytes induced by enhancedNADPH oxidase activity (P<0.05) with subsequent activation and translocation of NF-kB, atranscriptional factor from the cytoplasm to the nucleus. Activation of NF-kB induced a56% increase in iNOS gene protein expression (P<0.01), a downstream target of NF-kBwhich was accompanied by a significant increase in NO production (P<0.05). Wedemonstrated nitrosylation of several key cellular proteins involved in excitationcontractioncoupling including the Ryanodine receptor and SERCA2a as detected byimmunofluorescence.In conclusion, our work provides insights into novel pathophysiological mechanisms thatunderlie the development of heart failure in diabetes. We demonstrate the presence andfunctionality of AGE receptors in myocardium and show that AGEs inhibit excitationcontractioncoupling through increased ROS production leading to activation andtranslocation of NF-kB from the cytoplasm to the nucleus resulting in increase in NOproduction. Concomitant increases in intracellular levels of ROS and NO favours theproduction of peroxynitrite with subsequent nitrosylation of key cellular proteins involved inthe process of excitation-contraction coupling such as the Ryanodine receptor andSERCA2a. This study provides novel insights into the role of AGEs in inducing myocardialdamage in diabetes mediated through RAGE receptor and independent from the vascular effects.
Thesis main supervisor(s):
Thesis advisor(s):
Funder(s):
Language:
en

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:153747
Created by:
Hegab, Zeinab
Created:
23rd January, 2012, 14:54:34
Last modified by:
Hegab, Zeinab
Last modified:
1st June, 2012, 12:53:12

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