Analysis Tools
cardiovascular system
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• by 10 months of age, the cross-sectional area of individual cardiomyocytes is significantly increased relative to wild-type controls
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• by 10 months of age, mutant hearts appear to be larger than wild-type
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• by 10 months of age, mutant hearts show increased interstitial cardiac fibrosis relative to wild-type controls
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• at 10 months of age, mutant hearts accumulate autophagosomes and autophagic flux, based on changes in LC3 II protein levels elicited by chloroquine treatment, is impaired
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• by 10 months of age, ejection fraction (EF) and percent fractional shortening (%FS) are reduced, suggesting impaired cardiac function
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• by 10 months of age, the end-diastolic left ventricular dimension (LVEDD) and the end-systolic left ventricular dimension (LVESD) are significantly increased relative to wild-type controls
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• by 10 months of age, mutant hearts show significantly increased apoptosis, as shown by TUNEL analysis
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• homozygotes are viable, fertile, and developmentally normal but develop age-related cardiomyopathy, evident by 10 months of age
• no alterations in cardiac structure are noted at 2 and 6 months of age
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• immunohistochemical staining of mutant hearts revealed that inflammatory cell markers CD45 (leucocytes) and CD45R (B cells) are significantly increased relative to wild-type hearts
• heart inflammation is already detectable at 6 months of age when cardiac structure and function are normal
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cellular
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• by 10 months of age, mutant hearts show increased interstitial cardiac fibrosis relative to wild-type controls
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• by 10 months of age, mutant hearts show significantly increased apoptosis, as shown by TUNEL analysis
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• at 10 months of age, mutant hearts display accumulation of lysosomal markers LAMP-1 and LAMP-2, suggesting that impaired autophagosome clearance is associated with increased lysosome numbers
• in contrast, cathepsin D, a lysosomal protease, is not significantly increased in mutant hearts
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• at 10 months of age, mutant hearts accumulate autophagosomes, as shown by enhanced protein abundance of LC3 II; LC3 immunofluorescence and ultrastructural analysis confirmed accumulation of autophagic vacuoles in mutant hearts
• p62 (a marker for autophagic flux), is significantly increased in mutant hearts, indicating impaired autophagic flux
• the observed increase of LC3 II protein levels in mutant hearts is not further enhanced by treatment with chloroquine (an inhibitor of autophagosome-lysosome fusion)
• mutant MEFs show significantly increased LC3 II protein levels; however, the increase in LC3 II protein levels is not further enhanced by bafilomycin A1 (an autophagosome-lysosome fusion inhibitor), suggesting that autophagic flux is impaired in mutant MEFs
• ultrastructural analysis of mutant MEFs revealed clustered double-membrane autophagosomes and complex vacuoles containing multiple layers of membrane
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immune system
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• immunohistochemical staining of mutant hearts revealed that inflammatory cell markers CD45 (leucocytes) and CD45R (B cells) are significantly increased relative to wild-type hearts
• heart inflammation is already detectable at 6 months of age when cardiac structure and function are normal
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• RT-PCR analysis showed significantly enhanced interleukin-6 (IL-6) mRNA expression in mutant hearts relative to wild-type controls
• however, no significant difference in interleukin-1beta (IL-1beta) mRNA expression is observed
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• RT-PCR analysis showed significantly enhanced TNF mRNA expression in mutant hearts relative to wild-type controls
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homeostasis/metabolism
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• at 10 months of age, mutant hearts accumulate autophagosomes, as shown by enhanced protein abundance of LC3 II; LC3 immunofluorescence and ultrastructural analysis confirmed accumulation of autophagic vacuoles in mutant hearts
• p62 (a marker for autophagic flux), is significantly increased in mutant hearts, indicating impaired autophagic flux
• the observed increase of LC3 II protein levels in mutant hearts is not further enhanced by treatment with chloroquine (an inhibitor of autophagosome-lysosome fusion)
• mutant MEFs show significantly increased LC3 II protein levels; however, the increase in LC3 II protein levels is not further enhanced by bafilomycin A1 (an autophagosome-lysosome fusion inhibitor), suggesting that autophagic flux is impaired in mutant MEFs
• ultrastructural analysis of mutant MEFs revealed clustered double-membrane autophagosomes and complex vacuoles containing multiple layers of membrane
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• RT-PCR analysis showed significantly enhanced interleukin-6 (IL-6) mRNA expression in mutant hearts relative to wild-type controls
• however, no significant difference in interleukin-1beta (IL-1beta) mRNA expression is observed
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• RT-PCR analysis showed significantly enhanced TNF mRNA expression in mutant hearts relative to wild-type controls
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muscle
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• by 10 months of age, the cross-sectional area of individual cardiomyocytes is significantly increased relative to wild-type controls
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• by 10 months of age, ejection fraction (EF) and percent fractional shortening (%FS) are reduced, suggesting impaired cardiac function
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• by 10 months of age, mutant hearts show significantly increased apoptosis, as shown by TUNEL analysis
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• homozygotes are viable, fertile, and developmentally normal but develop age-related cardiomyopathy, evident by 10 months of age
• no alterations in cardiac structure are noted at 2 and 6 months of age
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growth/size/body
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• by 10 months of age, mutant hearts appear to be larger than wild-type
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