The vestigial enzyme D-dopachrome tautomerase protects the heart against ischemic injury.
ABSTRACT: The cellular response to stress involves the recruitment and coordination of molecular signaling pathways that prevent cell death. D-dopachrome tautomerase (DDT) is an enzyme that lacks physiologic substrates in mammalian cells, but shares partial sequence and structural homology with macrophage migration inhibitory factor (MIF). Here, we observed that DDT is highly expressed in murine cardiomyocytes and secreted by the heart after ischemic stress. Antibody-dependent neutralization of secreted DDT exacerbated both ischemia-induced cardiac contractile dysfunction and necrosis. We generated cardiomyocyte-specific DDT knockout mice (Myh6-Cre Ddtfl/fl), which demonstrated normal baseline cardiac size and function, but had an impaired physiologic response to ischemia-reperfusion. Hearts from Myh6-Cre Ddtfl/fl mice exhibited more necrosis and LV contractile dysfunction than control hearts after coronary artery ligation and reperfusion. Furthermore, treatment with DDT protected isolated hearts against injury and contractile dysfunction after ischemia-reperfusion. The protective effect of DDT required activation of the metabolic stress enzyme AMP-activated protein kinase (AMPK), which was mediated by a CD74/CaMKK2-dependent mechanism. Together, our data indicate that cardiomyocyte secretion of DDT has important autocrine/paracrine effects during ischemia-reperfusion that protect the heart against injury.
Project description:Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown.To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice.Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Following 1 day of reperfusion, hearts subjected to ischemia at the sleep-to-wake transition (zeitgeber time [ZT]12) resulted in 3.5-fold increases in infarct size compared to hearts subjected to ischemia at the wake-to-sleep transition (ZT0). Following 1 month of reperfusion, prior ischemic event at ZT12 versus ZT0 resulted in significantly greater infarct volume, fibrosis, and adverse remodeling, as well as greater depression of contractile function. Genetic ablation of the cardiomyocyte circadian clock (termed cardiomyocyte-specific circadian clock mutant [CCM] mice) attenuated/abolished time-of-day variations in I/R outcomes observed in wild-type hearts. Investigation of Akt and glycogen synthase kinase-3beta in wild-type and CCM hearts identified these kinases as potential mechanistic ties between the cardiomyocyte circadian clock and I/R tolerance.We expose a profound time-of-day dependence for I/R tolerance, which is mediated by the cardiomyocyte circadian clock. Further understanding of I/R tolerance rhythms will potentially provide novel insight regarding the etiology and treatment of ischemia-induced cardiac dysfunction.
Project description:Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that also modulates physiologic cell signaling pathways. MIF is expressed in cardiomyocytes and limits cardiac injury by enhancing AMPK activity during ischemia. Reperfusion injury is mediated in part by activation of the stress kinase JNK, but whether MIF modulates JNK in this setting is unknown. We examined the role of MIF in regulating JNK activation and cardiac injury during experimental ischemia/reperfusion in mouse hearts. Isolated perfused Mif-/- hearts had greater contractile dysfunction, necrosis, and JNK activation than WT hearts, with increased upstream MAPK kinase 4 phosphorylation, following ischemia/reperfusion. These effects were reversed if recombinant MIF was present during reperfusion, indicating that MIF deficiency during reperfusion exacerbated injury. Activated JNK acts in a proapoptotic manner by regulating BCL2-associated agonist of cell death (BAD) phosphorylation, and this effect was accentuated in Mif-/- hearts after ischemia/reperfusion. Similar detrimental effects of MIF deficiency were observed in vivo following coronary occlusion and reperfusion in Mif-/- mice. Importantly, excess JNK activation also was observed after hypoxia-reoxygenation in human fibroblasts homozygous for the MIF allele with the lowest level of promoter activity. These data indicate that endogenous MIF inhibits JNK pathway activation during reperfusion and protects the heart from injury. These findings have clinical implications for patients with the low-expression MIF allele.
Project description:Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.
Project description:The mechanisms contributing to heart failure remain incompletely understood. d-dopachrome tautomerase (DDT) is a member of the macrophage migration inhibitory factor family of cytokines and is highly expressed in cardiomyocytes. This study examined the role of cardiomyocyte DDT in the setting of heart failure. Patients with advanced heart failure undergoing transplantation demonstrated decreased cardiac DDT expression. To understand the effect of loss of cardiac DDT in experimental heart failure, cardiomyocyte-specific DDT-KO (DDT-cKO) and littermate control mice underwent surgical transverse aortic constriction (TAC) to induce cardiac pressure overload. DDT-cKO mice developed more rapid cardiac contractile dysfunction, greater cardiac dilatation, and pulmonary edema after TAC. Cardiomyocytes from DDT-cKO mice after TAC had impaired contractility, calcium transients, and reduced expression of the sarcoplasmic reticulum calcium ATPase. The DDT-cKO hearts also exhibited diminished angiogenesis with reduced capillary density and lower VEGF-A expression after TAC. In pharmacological studies, recombinant DDT (rDDT) activated endothelial cell ERK1/2 and Akt signaling and had proangiogenic effects in vitro. The DDT-cKO hearts also demonstrated more interstitial fibrosis with enhanced collagen and connective tissue growth factor expression after TAC. In cardiac fibroblasts, rDDT had an antifibrotic action by inhibiting TGF-β-induced Smad-2 activation. Thus, endogenous cardiomyocyte DDT has pleiotropic actions that are protective against heart failure.
Project description:AMP-activated kinase (AMPK) is a stress responsive kinase that regulates cellular metabolism and protects against cardiomyocyte injury during ischemia-reperfusion (IR). Mitochondria play an important role in cell survival, but the specific actions of activated AMPK in maintaining mitochondrial integrity and function during reperfusion are unknown. Thus, we assessed the consequences of AMPK inactivation on heart mitochondrial function during reperfusion. Mouse hearts expressing wild type (WT) or kinase-dead (KD) AMPK were studied. Mitochondria isolated from KD hearts during reperfusion had intact membrane integrity, but demonstrated reduced oxidative capacity, increased hydrogen peroxide production and decreased resistance to mitochondrial permeability transition pore opening compared to WT. KD hearts showed increased activation of the mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) and greater necrosis during reperfusion after coronary occlusion. Transgenic expression of mitochondrial catalase (MCAT) prevented the excessive cardiac JNK activation and attenuated the increased myocardial necrosis observed during reperfusion in KD mice. Inhibition of JNK increased the resistance of KD hearts to mPTP opening, contractile dysfunction and necrosis during IR. Thus, intrinsic activation of AMPK is critical to prevent excess mitochondrial reactive oxygen production and consequent JNK signaling during reperfusion, thereby protecting against mPTP opening, irreversible mitochondrial damage and myocardial injury.
Project description:Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury.Constitutive overexpression of iNOS in transgenic mice (alpha-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[(3)H]-glucose. Reperfusion-induced loss of NAD(+) and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition-induced NAD(+) loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A.Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.
Project description:Mitochondria constitute 30% of myocardial mass. Mitochondrial fusion and fission appear essential for health of most tissues. Mitochondrial fission occurs in neonatal cardiomycyte and is implicated in cardiomyocyte death. Mitochondrial fusion has not been observed in postmitotic myocytes of adult hearts, and its occurrence and function in this context are controversial.Determine the consequences on organelle and organ function of disrupting cardiomyocyte mitochondrial fusion in vivo.The murine mfn1 and mfn2 genes, encoding mitofusins (Mfn) 1 and 2 that mediate mitochondrial tethering and outer mitochondrial membrane fusion, were interrupted by Cre-mediated excision of essential exons in neonatal (Nkx2.5-Cre) and adult (MYH6 modified estrogen receptor-Cre-modified estrogen receptor plus tamoxifen or Raloxifene) hearts. Embryonic combined Mfn1/Mfn2 ablation was lethal after e9.5. Conditional combined Mfn1/Mfn2 ablation in adult hearts induced mitochondrial fragmentation, cardiomyocyte and mitochondrial respiratory dysfunction, and rapidly progressive and lethal dilated cardiomyopathy. Before heart failure developed, cardiomyocyte shortening and calcium cycling were unaffected by absence of Mfn1 and Mfn2. Based on the time course over which fusion-defective mitochondrial size decreases, a mitochondrial fusion/fission cycle in adult mouse hearts occurs approximately every 16 days.Mitochondrial fusion in adult cardiac myocytes is necessary to maintain normal mitochondrial morphology and is essential for normal cardiac respiratory and contractile function. Interruption of mitochondrial fusion causes lethal cardiac failure at a time corresponding to 3 or 4 cycles of unopposed mitochondrial fission.
Project description:Ischemic heart disease is characterized by contractile dysfunction and increased cardiomyocyte death, induced by necrosis and apoptosis. Increased cell survival after an ischemic insult is critical and depends on several cellular pathways, which have not been fully elucidated.To test the hypothesis that the anti-apoptotic hematopoietic lineage substrate-1-associated protein X-1 (HAX-1), recently identified as regulator of cardiac Ca cycling, also may ameliorate cellular injury with an ischemic insult.We report that cardiac ischemia/reperfusion injury is associated with significant decreases in HAX-1 levels ex vivo and in vivo. Accordingly, overexpression of HAX-1 improved contractile recovery, coupled with reduced infarct size, plasma troponin I level, and apoptosis. The beneficial effects were associated with decreased endoplasmic reticulum (ER) stress response through specific inhibition of the inositol-requiring enzyme (IRE-1) signaling pathway, including its downstream effectors caspase-12 and the transcription factor C/EBP homologous protein. Conversely, HAX-1 heterozygous-deficient hearts exhibited increases in infarct size and IRE-1 activity. The inhibitory effects of HAX-1 were mediated by its binding to the N-terminal fragment of the heat shock protein 90 (Hsp90). Moreover, HAX-1 sequestered Hsp90 from IRE-1 to the phospholamban-sarcoplasmic/endoplasmic reticulum calcium ATPase complex. The HAX-1 regulation was further supported by loss of IRE-1 inhibition in presence of the Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin.Cardiac ischemia-reperfusion injury is associated with decreases in HAX-1 levels. Consequently, overexpression of HAX-1 promotes cardiomyocyte survival, mediated by its interaction with Hsp90 and specific inhibition of IRE-1 signaling at the ER/sarcoplasmic reticulum.
Project description:Urocortin 2 (Ucn2), a peptide of the corticotropin-releasing factor (CRF) family, binds with high affinity to type 2 CRF receptors (CRFR2) on cardiomyocytes and confers protection against ischemia/reperfusion. The mechanisms by which the Ucn2-CRFR2 axis mitigates against ischemia/reperfusion injury remain incompletely delineated. Activation of AMP-activated protein kinase (AMPK) also limits cardiac damage during ischemia/reperfusion. AMPK is classically activated by alterations in cellular energetics; however, hormones, cytokines, and additional autocrine/paracrine factors also modulate its activity. We examined the effects of both the endogenous cardiac Ucn2 autocrine/paracrine pathway and Ucn2 treatment on AMPK regulation. Ucn2 treatment increased AMPK activation and downstream acetyl-CoA carboxylase phosphorylation and glucose uptake in isolated heart muscles. These actions were blocked by the CRFR2 antagonist anti-sauvagine-30 and by a PKC? translocation-inhibitor peptide (?V1-2). Hypoxia-induced AMPK activation was also blunted in heart muscles by preincubation with either anti-sauvagine-30, a neutralizing anti-Ucn2 antibody, or ?V1-2. Treatment with Ucn2 in vivo augmented ischemic AMPK activation and reduced myocardial injury and cardiac contractile dysfunction after regional ischemia/reperfusion in mice. Ucn2 also directly activated AMPK in ex vivo-perfused mouse hearts and diminished injury and contractile dysfunction during ischemia/reperfusion. Thus, both Ucn2 treatment and the endogenous cardiac Ucn2 autocrine/paracrine pathway activate AMPK signaling pathway, via a PKC?-dependent mechanism, defining a Ucn2-CRFR2-PKC?-AMPK pathway that mitigates against ischemia/reperfusion injury.
Project description:Ischemia and reperfusion (I/R) of the heart is associated with biochemical and ionic changes that result in cardiac contractile and electrical dysfunction. In rabbits, platelet-rich plasma activated using nanosecond pulsed electric fields (nsPRP) has been shown to improve left ventricular pumping. Here, we demonstrate that nsPRP causes a similar improvement in mouse left ventricular function. We also show that nsPRP injection recovers electrical activity even before reperfusion begins. To uncover the mechanism of nsPRP action, we studied whether the enhanced left ventricular function in nsPRP rabbit and mouse hearts was associated with increased expression of heat-shock proteins and altered mitochondrial function under conditions of oxidative stress. Mouse hearts underwent 30 min of global ischemia and 1 h of reperfusion in situ. Rabbit hearts underwent 30 min of ischemia in vivo and were reperfused for 14 days. Hearts treated with nsPRP expressed significantly higher levels of Hsp27 and Hsp70 compared to hearts treated with vehicle. Also, pretreatment of cultured H9c2 cells with nsPRP significantly enhanced the "spare respiratory capacity (SRC)" also referred to as "respiratory reserve capacity" and ATP production in response to the uncoupler FCCP. These results suggest a cardioprotective effect of nsPRP on the ischemic heart during reperfusion.