Refined approach for quantification of in vivo ischemia-reperfusion injury in the mouse heart

Bohl, Steffen; Medway, Debra J.; Schulz‐Menger, Jeanette; Schneider, Jürgen; Neubauer, Stefan; Lygate, Craig A.

doi:10.1152/ajpheart.00836.2009

Cited by 80 publications

(70 citation statements)

References 12 publications

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“…Determination of Infarct Size-Myocardial infarct size was determined as described previously (43). Briefly, after reperfusion, animals were sacrificed, and the aorta was cannulated and perfused with saline to remove blood.…”

Section: Methodsmentioning

confidence: 99%

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Kundumani‐Sridharan

Hilgers

Owens

et al. 2016

Journal of Biological Chemistry

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Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.

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Section: Methodsmentioning

confidence: 99%

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Kundumani‐Sridharan

Hilgers

Owens

et al. 2016

Journal of Biological Chemistry

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“…Myocardial infarct size was determined by using Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) staining as described by Bohl et al (27). Briefly, the ascending aorta was cannulated with a 20-gauge tubing adapter, and 1% Evans blue was perfused into the aorta and coronary arteries to delineate the "area at risk" (AAR).…”

Section: Methodsmentioning

confidence: 99%

Cardiomyocyte-specific Prolyl-4-hydroxylase Domain 2 Knock Out Protects from Acute Myocardial Ischemic Injury

Hölscher

Silter

Krull

et al. 2011

Journal of Biological Chemistry

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Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the ␣-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd ؊/؊ ) When oxygen availability is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level (1). Hypoxia-inducible factors (HIFs) 2 are essential players in these responses because they regulate the transcription of a large number of genes that affect a myriad of cellular processes, including angiogenesis, metabolism, cell survival, and oxygen delivery (2). HIF is a heterodimeric protein comprising the oxygen-sensitive ␣-subunit HIF-1␣ or the more cell type-specifically expressed HIF-2␣ or HIF-3␣ and the oxygen-insensitive ␤-subunit (3). In the presence of oxygen, HIF␣ becomes hydroxylated at two critical proline residues by prolylhydroxylase domain (PHD) enzymes (4, 5). The PHD protein family responsible for HIF␣ regulation consists of three members called prolylhydroxylase domain (PHD)1, PHD2, and PHD3 (6, 7). Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein recognizes ⌯⌱F-1␣ subunits and targets them for rapid ubiquitination and proteasomal degradation (8 -10).Based on the ubiquitous expression pattern and its dominant effect in normoxia, it had to be assumed that PHD2 is the most critical HIF-1␣-regulating PHD isoform in most tissues (11)(12)(13). This notion, learned from in vitro studies, was confirmed by the up to now available genetically modified Phd2 mouse models (14). Phd2 knock-out embryos die between embryonic day (E) 12.5 and E14.5 (15). This time point coincides with the increased levels of PHD2 in wild-type (wt) mice starting from E9.0. A major role of PHD2 in regulating the HIF system is further underscored by mouse models with a somatic Phd2 Ϫ/Ϫ knock out, which enable to analyze the in vivo function of PHD2 in the adult mice. Two independent inducible Phd2 Ϫ/Ϫ mouse models were developed by Takeda et al. (16) and Minamishima et al. (17). The phenotype of these mice most obviously resembles the consequences of HIF␣ overexpression with increased angiogenesis, erythropoiesis, and extramedullar hematopoiesis (17,18). Most interestingly, these mice also develop a cardiac phenotype with symptoms of dilated cardiomyopathy. In the heart, HIF-1␣ and thereby also the PHDs are known to influence key components of heart development, morphogenesis, and function (19,20). Long term activation of HIF-1␣ in the heart seems to activate detrimental pathways resulting in the development of heart failure (21). Thus, it is tempting to speculate that loss of PHD2 in the heart is responsible for the dilated cardiomyopathy as observed in the inducible Phd2 Ϫ/Ϫ mice. However, because these mice also develop an inc...

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“…Myocardial I/R mouse model and infarct size determination were performed as previously reported (48). PA28α/tTA double-Tg mice and littermate tTA single-Tg mice at 8 weeks old were anesthetized with 1% isoflurane and ventilated through intubation via mouth.…”

Section: Sds-page and Western Blot Analysis Sds-page Andmentioning

confidence: 99%

Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice

et al. 2011

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The ubiquitin-proteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) has been observed in proteinopathies, such as desmin-related cardiomyopathy, and implicated in many common diseases, including dilated cardiomyopathy and ischemic heart disease. However, the pathogenic role of PFI has not been established. Here we created inducible Tg mice with cardiomyocyte-restricted overexpression of proteasome 28 subunit α (CR-PA28αOE) to investigate whether upregulation of the 11S proteasome enhances the proteolytic function of the proteasome in mice and, if so, whether the enhancement can rescue a bona fide proteinopathy and protect against ischemia/reperfusion (I/R) injury. We found that CR-PA28αOE did not alter the homeostasis of normal proteins and cardiac function, but did facilitate the degradation of a surrogate misfolded protein in the heart. By breeding mice with CR-PA28αOE with mice representing a well-established model of desmin-related cardiomyopathy, we demonstrated that CR-PA28αOE markedly reduced aberrant protein aggregation. Cardiac hypertrophy was decreased, and the lifespan of the animals was increased. Furthermore, PA28α knockdown promoted, whereas PA28α overexpression attenuated, accumulation of the mutant protein associated with desmin-related cardiomyopathy in cultured cardiomyocytes. Moreover, CR-PA28αOE limited infarct size and prevented postreperfusion cardiac dysfunction in mice with myocardial I/R injury. We therefore conclude that benign enhancement of cardiac proteasome proteolytic function can be achieved by CR-PA28αOE and that PFI plays a major pathogenic role in cardiac proteinopathy and myocardial I/R injury.

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Refined approach for quantification of in vivo ischemia-reperfusion injury in the mouse heart

Cited by 80 publications

References 12 publications

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Thioredoxin Uses a GSH-independent Route to Deglutathionylate Endothelial Nitric-oxide Synthase and Protect against Myocardial Infarction

Cardiomyocyte-specific Prolyl-4-hydroxylase Domain 2 Knock Out Protects from Acute Myocardial Ischemic Injury

Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice

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