Measuring Cardiac Autophagic Flux In Vitro and In Vivo

Gurney, Michael A.; Huang, Cai; Ramil, Jennifer; Ravindran, Nandini; Andres, Allen M.; Sin, Jon; Linton, Phyllis-Jean; Gottlieb, Roberta A.

doi:10.1007/978-1-4939-1661-0_14

Cited by 29 publications

(26 citation statements)

References 17 publications

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“…CQ inhibited autophagy by affecting lysosome acidification 21. CQ altered the lysosome pH, with the lysosomal neutralization inhibiting lysosome activities and can be used in assays of short-term autophagy flux 22. In addition, CQ decreased LC3-II/LC3-I protein ratio and undigested autophagosome observed by transmission electron microscopy in STZ-induced diabetic mice (Figure 5).…”

Section: Discussionmentioning

confidence: 99%

Chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice

Xiao¹,

Zhang²,

Hou³

et al. 2016

DDDT

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Diabetes is a potent risk factor for heart failure with preserved ejection fraction (HFpEF). Autophagy can be activated under pathological conditions, including diabetic cardiomyopathy. The therapeutic effects of chloroquine (CQ), an autophagy inhibitor, on left ventricle function in streptozotocin (STZ)-induced diabetic mice were investigated. The cardiac function, light chain 3 (LC3)-II/LC3-I ratio, p62, beclin 1, reactive oxygen species, apoptosis, and fibrosis were measured 14 days after CQ (ip 60 mg/kg/d) administration. In STZ-induced mice, cardiac diastolic function was decreased significantly with normal ejection fraction. CQ significantly ameliorated cardiac diastolic function in diabetic mice with HFpEF. In addition, CQ decreased the autophagolysosomes, cardiomyocyte apoptosis, and cardiac fibrosis but increased LC3-II and p62 expressions. These results suggested that CQ improved the cardiac diastolic function by inhibiting autophagy in STZ-induced HFpEF mice. Autophagic inhibitor CQ might be a potential therapeutic agent for HFpEF.

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Section: Discussionmentioning

confidence: 99%

Chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice

Xiao¹,

Zhang²,

Hou³

et al. 2016

DDDT

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“…The normal response to an autophagic stimulus involves an early increase in p62/SQSTM1 expression in the cytosol, followed by clearance of p62/SQSTM1 associated with aggregates or other cargo. While not indicated in every instance, assessment of soluble and detergent-insoluble p62/SQSTM1 pools can provide valuable information 48 . Like LC3, western blot analysis of p62/SQSTM1 is only a snapshot of a dynamic process.…”

Section: Methods To Measure Autophagy and Mitophagymentioning

confidence: 99%

“…For in vivo studies, chloroquine (10–50 mg/kg i.p.) 48, 69 , leupeptin (20–40 mg/kg i.p.) 67 , or bafilomycin A 1 (2.5 mg/kg i.p.…”

Section: Methods To Measure Autophagy and Mitophagymentioning

confidence: 99%

Untangling Autophagy Measurements

Gottlieb

Andres

Sin

et al. 2015

Circulation Research

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134

105

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Autophagy is an important physiological process in the heart, and alterations in autophagic activity can exacerbate or mitigate injury during various pathological processes. Methods to assess autophagy have changed rapidly as the field of research has expanded. As with any new field, methods and standards for data analysis and interpretation evolve as investigators acquire experience and insight. The purpose of this review is to summarize current methods to measure autophagy, selective mitochondrial autophagy (mitophagy), and autophagic flux. We will examine several published studies where confusion arose in in data interpretation, in order to illustrate the challenges. Finally we will discuss methods to assess autophagy in vivo and in patients.

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“…S4), suggesting that VEGFA could be directly degraded via autophagy. We pharmacologically blocked autophagic flux using Bafilomycin A1 (BafA1 hereafter), a universally established method to assay autophagic processes (75)(76)(77). Mechanistically, BafA1 inhibits the vacuolar H + ATPase to preclude mature autophagosomes from fusing with lysosomes, thereby giving a metric on the substrates undergoing autophagic degradation.…”

Section: Interfering With Basal Autophagic Flux Leads To An Accumulatmentioning

confidence: 99%

Catabolic degradation of endothelial VEGFA via autophagy

Neill

Chen

Buraschi

et al. 2020

Journal of Biological Chemistry

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Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

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Measuring Cardiac Autophagic Flux In Vitro and In Vivo

Cited by 29 publications

References 17 publications

Chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice

Chloroquine improves left ventricle diastolic function in streptozotocin-induced diabetic mice

Untangling Autophagy Measurements

Catabolic degradation of endothelial VEGFA via autophagy

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