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The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis. Cancer Res; 71(21); 6708–17. ©2011 AACR.

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Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of heart failure (HF) and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion ofSirt2(Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specificSirt2deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion ofNrf2in the hearts ofSirt2-/-mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitors reduces cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in the progression of HF and cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of this disorder.

All living cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here, we report a universal eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of genes encoding high-affinity leucine transporters and RAPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency (ID) supersedes other nutrient inputs into mTORC1. This process occurs in vivo, and is not an indirect effect by canonical iron-utilizing pathways. Elevated expression of KDM3B targets is associated with reduced survival in a subset of human cancers and ID represses mTORC1 in patient-derived tumor cells and sensitizes cancer cells to chemotherapy. These data demonstrate a novel mechanism of eukaryotic iron sensing through dynamic chromatin remodeling and repression of mTORC1 mediated anabolism. Due to ancestral eukaryotes sharing homologues of KDMs and mTORC1 core components, this pathway likely predated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrate that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, feroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.

Chronic loss of Augmenter of Liver Regeneration (ALR) results in mitochondrial myopathy with cataracts; however, the mechanism for this disorder remains unclear. Here, we demonstrate that loss of ALR, a principal component of the MIA40/ALR protein import pathway, results in impaired cytosolic Fe/S cluster biogenesis in mammalian cells. Mechanistically, MIA40/ALR facilitates the mitochondrial import of ATP-binding cassette (ABC)-B8, an inner mitochondrial membrane protein required for cytoplasmic Fe/S cluster maturation, through physical interaction with ABCB8. Downregulation of ALR impairs mitochondrial ABCB8 import, reduces cytoplasmic Fe/S cluster maturation, and increases cellular iron through the iron regulatory protein-iron response element system. Our finding thus provides a mechanistic link between MIA40/ALR import machinery and cytosolic Fe/S cluster maturation through the mitochondrial import of ABCB8, and offers a potential explanation for the pathology seen in patients with ALR mutations.

Chronic loss of Augmenter of Liver Regeneration (ALR) results in mitochondrial myopathy with cataracts, however, the mechanism for this disorder remains unclear. Here, we demonstrate that loss of ALR, a principal component of the MIA40/ALR protein import pathway, results in impaired cytosolic Fe/S cluster biogenesis in mammalian cells. Mechanistically, MIA40/ALR facilitates the mitochondrial import of ATP binding cassette (ABC)-B8, an inner mitochondrial membrane protein required for cytoplasmic Fe/S cluster maturation, through physical interaction with ABCB8. Downregulation of ALR impairs mitochondrial ABCB8 import, reduces cytoplasmic Fe/S cluster maturation, and increases cellular iron through the iron regulatory protein-iron response element system. Our finding provides a mechanistic link between MIA40/ALR import machinery and cytosolic Fe/S cluster maturation through the mitochondrial import of ABCB8, and offers a potential explanation for the pathology seen in patients with ALR mutations.

Introduction: A loss of function mutation in the mRNA binding protein ZFP36L2 has been associated with congenital heart diseases in humans, although the mechanism remains unclear. Objectives: We sought to elucidate the role of ZFP36L2 in the heart using mice with cardiac-specific ZFP36L2 deletion. Results: Cardiac-specific Zfp36l2 knockout (cs-L2KO) female mice exhibit a dramatic phenotype in which the majority of mice die after repeated pregnancies due to heart failure. We performed RNA-seq in H9c2 cells with ZFP36L2 KD, and found the expression of multiple mTOR pathway genes were altered, including marked repression of Redd1 and Sesn2 . Consistent with the RNA-seq data, the activity of mTORC1 was increased in cs-L2KO mouse hearts and in ZFP36L2 KD H9c2 cells. Loss of TSC2 mitigated mTORC1 hyperactivity in ZFP36L2 KD cells, indicating that ZFP36L2 regulates mTORC1 activity through the TSC2 complex. Overexpression of REDD1 rescued increased mTORC1 activity with ZFP36L2 KD, suggesting that ZFP36L2 regulates mTORC1, in-part through REDD1. Under total amino acid starvation, we observed higher p-S6K T389 levels in ZFP36L2 KD cells, and SESN2 overexpression mitigated p-S6K T389 , indicating loss of ZFP36L2 also regulates mTORC1 through amino acid sensing. p53 is a transcriptional activator of both REDD1 and SESN2, and we found that ZFP36L2 was required to maintain p53 levels by directly destabilizing MDM2 mRNA. Accordingly, administration of Nutlin3, an MDM2 inhibitor, reversed the reduction in p53, REDD1 and SESN2 protein levels, and prevented the increase in S6K phosphorylation in response to ZFP36L2 KD. Next, we treated pregnant mice with rapamycin, and cs-L2KO mice treated with rapamycin displayed significantly improved cardiac function after delivery. Finally, we analyzed human heart samples from patients with peri-partum cardiomyopathy and found ZFP36L2 protein to be significantly decreased and p-S6K T389 levels increased, compared to healthy controls. Conclusions: Our studies demonstrate that ZFP36L2 regulates mTORC1 activity through modifying p53 stability, and that the disruption of this pathway induces peri-partum cardiomyopathy. Inhibition of mTORC1 hyperactivity with rapamycin can be a potential therapy for this disease.

The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative pathways, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. However, it is not known what determines the fate of G6P. HK1 contains an N-terminal mitochondrial–binding domain, but its physiologic significance remains unclear. We overexpressed full-length and truncated HK1 in tissue culture and generated mice lacking the HK1 mitochondrial-binding domain (ΔE1HK1). Although ΔE1HK1 mice displayed no overt phenotype, HK1 dislocation from the mitochondria increased glucose flux through the PPP, decreased flux below the level of GAPDH, and induced a hyper-inflammatory response to lipopolysaccharide. The mechanism for the increased PPP flux is through glycolytic block at GAPDH, which is mediated by binding of cytosolic HK1 with S100A8/A9 and increased GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed an increase in cytosolic HK1 and cytokine production, along with reduced GAPDH activity. Our data indicate that HK1 mitochondrial-binding alters glucose metabolism through regulation of GAPDH.

Introduction: Sirtuins are NAD+ dependent deacetylases and critical regulators of energy metabolism and response to oxidative stress. Sirtuin2 (SIRT2) is a cytoplasmic member of the sirtuin family, and has been shown to regulate cellular iron homeostasis through deacetylation of nuclear factor erythroid-derived 2-related factor 2 (NRF2). However, whether SIRT2-NRF2 pathway is involved in the development of heart failure remains unknown. Methods and results: To investigate the functional role of SIRT2 in the response to cardiac stress, SIRT2 knockout (KO) mice and their littermate controls were subjected to pressure overload by transverse aortic constriction (TAC). SIRT2 KO had normal appearance and cardiovascular parameters at baseline. However, in response to TAC, Sirt2 -/- mice displayed resistance to the pathological hypertrophic response, whereas wild type (WT) mice developed cardiac hypertrophy and heart failure. In addition, SIRT2 KO mice displayed less cardiac damage after /reperfusion injury. SIRT2 knockdown in neonatal rat cardiomyocytes (NRCM) reduced reactive oxygen species (ROS) production and cell death after H2O2 treatment. Since cellular oxidative stress is one of major contributor of cardiac dysfunction caused by both I/R injury and pressure overload, we examined whether NRF2 is associated with SIRT2-mediated cardiac response to oxidative stress. Levels of NRF2 was upregulated in NRCM with SIRT2 knockdown and treated with H2O2 compared to wild type (WT) cells. Moreover, NRF2 is translocated into the nucleus and its anti-oxidant target proteins are upregulated in NRCM with SIRT2 knockdown. SIRT2 was also found to bind and deacetylate NRF2 directly as determined by co-immunoprecipitation studies. This led to a reduction of its nuclear translocation and transcriptional activity. Finally, knockdown of both SIRT2 and NRF2 diminished the effects of SIRT2 knockdown on ROS production and cellular damage. Conclusion: These results indicate that SIRT2 contributes to pressure overload and I/R injury induced heart impairment in mice, and promotes oxidative stress injury in cardiomyocytes via deacetylating NRF2 and altering its activity.

Introduction: A loss of function mutation in the mRNA binding protein ZFP36L2 has been associated with congenital heart diseases in humans, although the mechanism remains unclear. Objectives: We sought to elucidate the role of ZFP36L2 in the heart using mice with cardiac-specific ZFP36L2 deletion. Results: Cardiac-specific Zfp36l2 knockout (cs-L2KO) female mice exhibit a dramatic phenotype in which the majority of mice die after repeated pregnancies due to heart failure. We performed RNA-seq in H9c2 cells with ZFP36L2 KD, and found the expression of multiple mTOR pathway genes were altered, including marked repression of Redd1 and Sesn2 . Consistent with the RNA-seq data, the activity of mTORC1 was increased in cs-L2KO mouse hearts and in ZFP36L2 KD H9c2 cells. Loss of TSC2 mitigated mTORC1 hyperactivity in ZFP36L2 KD cells, indicating that ZFP36L2 regulates mTORC1 activity through the TSC2 complex. Overexpression of REDD1 rescued increased mTORC1 activity with ZFP36L2 KD, suggesting that ZFP36L2 regulates mTORC1, in-part through REDD1. Under total amino acid starvation, we observed higher p-S6K T389 levels in ZFP36L2 KD cells, and SESN2 overexpression mitigated p-S6K T389 , indicating loss of ZFP36L2 also regulates mTORC1 through amino acid sensing. p53 is a transcriptional activator of both REDD1 and SESN2, and we found that ZFP36L2 was required to maintain p53 levels by directly destabilizing MDM2 mRNA. Accordingly, administration of Nutlin3, an MDM2 inhibitor, reversed the reduction in p53, REDD1 and SESN2 protein levels, and prevented the increase in S6K phosphorylation in response to ZFP36L2 KD. Next, we treated pregnant mice with rapamycin, and cs-L2KO mice treated with rapamycin displayed significantly improved cardiac function after delivery. Finally, we analyzed human heart samples from patients with peri-partum cardiomyopathy and found ZFP36L2 protein to be significantly decreased and p-S6K T389 levels increased, compared to healthy controls. Conclusions: Our studies demonstrate that ZFP36L2 regulates mTORC1 activity through modifying p53 stability, and that the disruption of this pathway induces peri-partum cardiomyopathy. Inhibition of mTORC1 hyperactivity with rapamycin can be a potential therapy for this disease.

Introduction: Altered DNA damage response (DDR) can result in phenotypic changes that share features with age-associated pathological conditions, including metabolic and cardiovascular abnormalities. Snf1-related kinase (SNRK) is a serine/threonine kinase that belongs to the AMPK family. Our recent data demonstrates that SNRK expression is upregulated in heart failure (HF) and low energy states and that SNRK improves cardiac mitochondrial efficiency and cell survival. Here, we assessed the hypothesis that in response to stress, SNRK translocates to the nucleus where it modulates nuclear processes to enhance DNA repair and ultimately reduces cardiomyocyte death. Results: We first showed that the nuclear distribution of endogenous SNRK in cardiomyoblast is enhanced under low nutrient and oxidative stress. Next, we performed global phosphoproteomics analysis on cells with overexpression of wild type SNRK and mutant kinase-dead SNRK (D158A). We found significant increase of several phosphopeptides involved in nuclear processes, in particular in the regulation of chromatin remodeling and DDR. SNRK downregulation resulted in an increase in nucleus volume and heterochromatin content, with increased nuclear dysmorphia when subjected to oxidative stress. Despite having lower expression of DDR signal effectors p53 and Mdm2, these cells accumulated higher levels of pH2AX (DDR marker) and displayed higher cellular death after oxidative stress, consistent with increased DNA damage. Etoposide treatment in SNRK knockdown cells failed to induce p53 levels despite pH2AX accumulation. Additionally, pharmacological stabilization of p53 protein through nutlin-3A treatment did not increase p53 in SNRK knockdown cells. These findings suggest that SNRK regulates p53 expression, thereby acting as a new modulator of DDR. Conclusions: Our results demonstrate increased nuclear localization of SNRK under stress conditions, and that SNRK regulates phosphorylation of nuclear proteins involved in chromatin remodelling and DDR. Thus, SNRK may induce its pro-survival cardiac functions through regulation of chromatin architecture and enhancing DDR. Those novel findings can contribute to development of new therapies against HF.

SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2-related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2-/- mice had decreased iron levels, while this effect was reversed in Sirt2-/- Nrf2-/- double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.

Iron is an essential nutrient and is critical for cellular growth and metabolism. Here, we delineate a novel mechanism by which iron alters amino acid homeostasis and mTOR activity by remodeling the cellular epigenetic landscape. We find that iron deficiency inactivates Jumonji-C domain containing histone-demethylases, resulting in histone hyper-methylation and silencing of the leucine transporter LAT3 and obligatory mTORC1 cofactor RAPTOR. Additionally, we identify that mTOR-mediated regulation of RNA stability through tristetraprolin (TTP) is a novel and requisite step in selective-autophagy. In the absence of TTP, mitochondria damaged by the loss of iron cannot undergo fission, rendering the mitochondria too large for engulfment and subsequent recycling. Accumulation of damaged mitochondria leads to defective oxidative metabolism and impairs hepatic gluconeogenesis in response to fasting. These studies uncover a novel pathway that integrates iron sensing with cellular metabolism, mitochondrial dynamics and autophagy.

Excess cellular iron increases reactive oxygen species (ROS) production and causes cellular damage. Mitochondria are the major site of iron metabolism and ROS production; however, few studies have investigated the role of mitochondrial iron in the development of cardiac disorders, such as ischemic heart disease or cardiomyopathy (CM). We observe increased mitochondrial iron in mice after ischemia/reperfusion (I/R) and in human hearts with ischemic CM, and hypothesize that decreasing mitochondrial iron protects against I/R damage and the development of CM. Reducing mitochondrial iron genetically through cardiac‐specific overexpression of a mitochondrial iron export protein or pharmacologically using a mitochondria‐permeable iron chelator protects mice against I/R injury. Furthermore, decreasing mitochondrial iron protects the murine hearts in a model of spontaneous CM with mitochondrial iron accumulation. Reduced mitochondrial ROS that is independent of alterations in the electron transport chain's ROS producing capacity contributes to the protective effects. Overall, our findings suggest that mitochondrial iron contributes to cardiac ischemic damage, and may be a novel therapeutic target against ischemic heart disease.

The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.

Cells respond to iron deficiency by activating iron-regulatory proteins to increase cellular iron uptake and availability. However, it is not clear how cells adapt to conditions when cellular iron uptake does not fully match iron demand. Here, we show that the mRNA-binding protein tristetraprolin (TTP) is induced by iron deficiency and degrades mRNAs of mitochondrial Fe/S-cluster-containing proteins, specifically Ndufs1 in complex I and Uqcrfs1 in complex III, to match the decrease in Fe/S-cluster availability. In the absence of TTP, Uqcrfs1 levels are not decreased in iron deficiency, resulting in nonfunctional complex III, electron leakage, and oxidative damage. Mice with deletion of Ttp display cardiac dysfunction with iron deficiency, demonstrating that TTP is necessary for maintaining cardiac function in the setting of low cellular iron. Altogether, our results describe a pathway that is activated in iron deficiency to regulate mitochondrial function to match the availability of Fe/S clusters.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

The prevalence of obesity is rising globally, and the United States has one of the highest obesity rates in the world: ≈17% of the young and >33% of adults are obese.1 Obesity is associated with chronic low-grade systemic inflammation, which is considered a critical underlying factor in the development of insulin resistance (IR).2 IR is a major risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease.3 In the development of obesity, white adipose tissue, particularly the abdominal adipose tissue, is the key site that mediates systemic inflammation and IR, though other organs, such as skeletal muscle and liver, have also been implicated.4 Adipose tissue is a highly vascularized organ where every adipocyte is connected to at least one capillary.5 To maintain normal adipose tissue function, the proper signaling between adipocytes and endothelial cells (ECs) from the surrounding vasculature is important.6 There is a growing body of evidence suggesting that EC dysfunction contributes to the pathogenesis of atherosclerosis, obesity, and T2DM.7,8 Therefore, it is of key interest to further study the role of the crosstalk between adipose tissue ECs and adipocytes in obesity-associated IR and to identify potential therapeutic targets for novel interventions. Recently, several reports suggested that microRNAs (miRs) are important mediators of the development of inflammation and IR in obese adipose tissue.9 Subsequently, numerous studies explored targeting specific miRs in diabetic complications to mitigate the pathological sequela of T2DM.9 Given these points, using miRs to modulate adipocyte–EC axis in adipose tissue may offer new tools to combat the growing epidemic of obesity and its associated comorbidities. Article, see p 810 Over the past 2 decades, several studies elucidated the underlying molecular mechanisms linking inflammation to obesity-associated IR. Hotamisligil et al10 was the first to demonstrate …

Background Sex‐related differences in cardiac function and iron metabolism exist in humans and experimental animals. Male patients and preclinical animal models are more susceptible to cardiomyopathies and heart failure. However, whether similar differences are seen in iron‐overload cardiomyopathy is poorly understood. Methods and Results Male and female wild‐type and hemojuvelin‐null mice were injected and fed with a high‐iron diet, respectively, to develop secondary iron overload and genetic hemochromatosis. Female mice were completely protected from iron‐overload cardiomyopathy, whereas iron overload resulted in marked diastolic dysfunction in male iron‐overloaded mice based on echocardiographic and invasive pressure‐volume analyses. Female mice demonstrated a marked suppression of iron‐mediated oxidative stress and a lack of myocardial fibrosis despite an equivalent degree of myocardial iron deposition. Ovariectomized female mice with iron overload exhibited essential pathophysiological features of iron‐overload cardiomyopathy showing distinct diastolic and systolic dysfunction, severe myocardial fibrosis, increased myocardial oxidative stress, and increased expression of cardiac disease markers. Ovariectomy prevented iron‐induced upregulation of ferritin, decreased myocardial SERCA2a levels, and increased NCX1 levels. 17β‐Estradiol therapy rescued the iron‐overload cardiomyopathy in male wild‐type mice. The responses in wild‐type and hemojuvelin‐null female mice were remarkably similar, highlighting a conserved mechanism of sex‐dependent protection from iron‐overload‐mediated cardiac injury. Conclusions Male and female mice respond differently to iron‐overload‐mediated effects on heart structure and function, and females are markedly protected from iron‐overload cardiomyopathy. Ovariectomy in female mice exacerbated iron‐induced myocardial injury and precipitated severe cardiac dysfunction during iron‐overload conditions, whereas 17β‐estradiol therapy was protective in male iron‐overloaded mice.

Iron chelation rather than supplementation would be beneficial to patients with heart diseases. More effort should be devoted to the development of iron chelators that could target mitochondrial iron. Iron is an essential trace mineral for normal mammalian physiology. It is incorporated into iron/sulfur (Fe/S) clusters that serve as important cofactors for many enzymes, including the mitochondrial respiratory chain, and it gives blood and muscle their signature red color through its presence in heme.1 Because of its involvement in many important cellular processes, dysregulation of iron often causes disease. There are 2 broad categories of diseases associated with iron dysregulation—iron overload and iron deficiency. Each category can be further subdivided based on whether the changes in iron occur at the cellular or at the systemic level. Iron is critical for developmental processes, and studies have linked severe systemic iron deficiency to impaired brain development in children.2 However, most of these studies were conducted in developing countries where iron deficiency tends to be more severe. In developed countries, the major disorder associated with iron deficiency is anemia. Although systemic iron deficiency and anemia may have been used interchangeably, these 2 terms refer to different conditions. Systemic iron deficiency refers to low amounts of iron in circulation because of limited iron absorption or excess iron loss. Anemia is defined as low hemoglobin content within red blood cells and can be caused by low systemic iron, mutations in hemoglobin genes, vitamin B12 deficiency, or underlying chronic diseases. Limited oxygen-carrying capacity secondary to anemia can cause fatigue; however, it generally does not cause mortality or major damage to vital organs. Except for high-output heart failure (HF) associated with severe anemia, iron deficiency and anemia are not associated with any other major cardiac disorders. Cellular iron deficiency is a rare phenomenon, and except …

Objectives The goal of this study was to characterize the regulation of heme and non-heme iron in human failing hearts. Background Iron is an essential molecule for cellular physiology, but in excess it facilitates oxidative stress. Mitochondria are the key regulators of iron homeostasis through heme and iron-sulfur cluster synthesis. Because mitochondrial function is depressed in failing hearts and iron accumulation can lead to oxidative stress, we hypothesized that iron regulation may also be impaired in heart failure (HF). Methods We measured mitochondrial and cytosolic heme and non-heme iron levels in failing human hearts retrieved during cardiac transplantation surgery. In addition, we examined the expression of genes regulating cellular iron homeostasis, the heme biosynthetic pathway, and micro-RNAs that may potentially target iron regulatory networks. Results Although cytosolic non-heme iron levels were reduced in HF, mitochondrial iron content was maintained. Moreover, we observed a significant increase in heme levels in failing hearts, with corresponding feedback inhibition of the heme synthetic enzymes and no change in heme degradation. The rate-limiting enzyme in heme synthesis, delta-aminolevulinic acid synthase 2 (ALAS2), was significantly upregulated in HF. Overexpression of ALAS2 in H9c2 cardiac myoblasts resulted in increased heme levels, and hypoxia and erythropoietin treatment increased heme production through upregulation of ALAS2. Finally, increased heme levels in cardiac myoblasts were associated with excess production of reactive oxygen species and cell death, suggesting a maladaptive role for increased heme in HF. Conclusions Despite global mitochondrial dysfunction, heme levels are maintained above baseline in human failing hearts.

SummaryIron is an essential cofactor with unique redox properties. Iron-regulatory proteins 1 and 2 (IRP1/2) have been established as important regulators of cellular iron homeostasis, but little is known about the role of other pathways in this process. Here we report that the mammalian target of rapamycin (mTOR) regulates iron homeostasis by modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. Mechanistic studies identify tristetraprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA. We also show that TTP is strongly induced by iron chelation, promotes downregulation of iron-requiring genes in both mammalian and yeast cells, and modulates survival in low-iron states. Taken together, our data uncover a link between metabolic, inflammatory, and iron-regulatory pathways, and point toward the existence of a yeast-like TTP-mediated iron conservation program in mammals.

Ischaemic heart disease limits oxygen and metabolic substrate availability to the heart, resulting in tissue death. Here, we demonstrate that the AMP-activated protein kinase (AMPK)-related protein Snf1-related kinase (SNRK) decreases cardiac metabolic substrate usage and mitochondrial uncoupling, and protects against ischaemia/reperfusion. Hearts from transgenic mice overexpressing SNRK have decreased glucose and palmitate metabolism and oxygen consumption, but maintained power and function. They also exhibit decreased uncoupling protein 3 (UCP3) and mitochondrial uncoupling. Conversely, Snrk knockout mouse hearts have increased glucose and palmitate oxidation and UCP3. SNRK knockdown in cardiac cells decreases mitochondrial efficiency, which is abolished with UCP3 knockdown. We show that Tribbles homologue 3 (Trib3) binds to SNRK, and downregulates UCP3 through PPARα. Finally, SNRK is increased in cardiomyopathy patients, and SNRK reduces infarct size after ischaemia/reperfusion. SNRK also decreases cardiac cell death in a UCP3-dependent manner. Our results suggest that SNRK improves cardiac mitochondrial efficiency and ischaemic protection., The Snf1-related kinase (SNRK) is widely expressed and yet its function is poorly understood. Here the authors show that SNRK regulates mitochondrial coupling via the Trib3-PPARα-UCP3 pathway and that cardiac overexpression of SNRK decreases metabolic substrate usage and oxygen consumption but maintains cardiac function and energy in mice.

Introduction: Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. Hypothesis: We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. Results: We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to non-transgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death by ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to non-ischemic cardiomyopathy. Conclusions: Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the development of heart failure.

Introduction: Iron can catalyze the formation of reactive oxygen species (ROS) and promote tissue damage. While some studies suggested benefits with iron chelation therapy in ischemic heart disease (IHD), several others failed to show any benefits. Mitochondria are a major site of iron utilization and ROS production, and mitochondrial iron accumulation has been associated with increased oxidative stress. We therefore hypothesized that mitochondrial iron plays a causative role in ischemia/reperfusion (I/R) damage, and a decrease in mitochondrial iron (as opposed to cytoplasmic iron) would be sufficient to protect against I/R injury. Results: We observed an increase in cardiac mitochondrial iron in mice after I/R injury. Using two iron chelators with distinct mitochondrial permeability, i.e., 2,2’-bipyridyl (BPD, a mitochondria-accessible iron chelator) and deferoxamine (DFO, an iron chelator that does not modulate mitochondrial iron), we demonstrated that mice pretreated with BPD but not DFO were protected against I/R injury. Similar results were obtained in vitro . Since these two iron chelators also modulate iron in other subcellular compartments, we used transgenic (TG) mice with cardiomyocyte-specific overexpression of the mitochondrial iron export protein ATP-binding cassette (ABC)-B8 to confirm that modulation of mitochondrial iron alone is sufficient to confer protection. ABCB8 TG mice had significantly lower mitochondrial iron (but normal cytosolic iron) in the heart compared to nontransgenic (NTG) littermates at baseline, but exhibited normal cardiac function. After I/R, ABCB8 TG mice displayed significantly less apoptosis and lower levels of markers of ROS and better preserved cardiac function than NTG littermates, suggesting that a reduction in mitochondrial iron protects against I/R injury, most likely through a reduction in ROS. Conclusions: Our findings demonstrate that selective reduction in mitochondrial iron is sufficient to protect against I/R injury. Thus, targeting mitochondrial iron with selective iron chelators may provide a novel approach for the treatment of IHD.

Introduction: Heart failure (HF) is associated with the accumulation of branched-chain amino acids (BCAAs) in heart and plasma. Besides, BCAAs are essential for normal cell growth and function. Therefore, tight regulation of BCAA metabolism is crucial to maintain normal cellular homeostasis. Tristetraprolin (TTP), an mRNA binding protein that promotes the degradation of its targets and regulates a number of biological processes including cellular metabolism. In in silico analyses, we identified the E2 subunit of branched-chain α-ketoacid dehydrogenase complex (BCKDC), the rate-limiting enzyme complex in BCAA catabolism, as a potential target of TTP. Here we assess the hypothesis that TTP plays a regulatory role in BCAA catabolism through regulating BCKDC-E2 in the heart. We also investigate TTP’s contribution in the pathogenesis of HF by modulating BCAA catabolism. Results: We observed a significant increase in TTP levels in cardiac tissue of human HF and mouse myocardial infarct induced HF samples. We also showed that deletion of TTP significantly decreases BCAA levels in vitro. As BCAA accumulation in the heart is associated with the pathogenesis of HF, we investigated whether TTP can regulate cardiac BCAA catabolism. Computational analysis of mRNAs of enzymes, which are involved in BCAA catabolism, revealed highly conserved TTP binding sites in BCKDC-E2 mRNA, suggesting that it is a potential target of TTP. BCKDC-E2 mRNA levels were significantly higher in the heart of TTP knock-out mice and TTP siRNA treated cardiomyocytes. Additionally, the mRNA stability of BCKDC-E2 was higher in cardiomyocytes with TTP downregulation, further confirming that TTP regulates the stability of this mRNA. Moreover, RNA co-immunoprecipitation studies demonstrated that TTP physically interacts with the BCKDC-E2 mRNA. Finally, we showed that BCKDC-E2 mRNA levels were significantly decreased in human HF samples. Conclusion: Our data indicate that TTP levels are increased in HF samples and TTP regulates BCAA catabolism in the heart by binding to and degrading BCKDC-E2 mRNA. These findings suggest that alterations in TTP expression may play a role in the development of HF based on its effect upon BCKDC-E2 and BCAA catabolism.

The erbB family of receptor tyrosine kinases are known to play important roles in normal epithelial development and epithelial neoplasia. Considerable evidence also suggests that signaling through the epidermal growth factor receptor (EGFR) plays an important role in multistage skin carcinogenesis in mice; however, less is known about the role of erbB2. In this study, to further examine the role of both erbB2 and EGFR in epithelial carcinogenesis, we examined the effect of a dual erbB2/EGFR tyrosine kinase inhibitor, GW2974, given in the diet on skin tumor promotion during two-stage carcinogenesis in wild-type and BK5.erbB2 mice. In BK5.erbB2 mice, erbB2 is overexpressed in the basal layer of epidermis and leads to heightened sensitivity to skin tumor development. GW2974 effectively inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in wild-type and BK5.erbB2 mice, although a more marked effect was seen in BK5.erbB2 mice. In addition, this inhibitory effect was reversible when GW2974 treatment was withdrawn. GW2974 inhibited 12-O-tetradecanoylphorbol-13-acetate–induced epidermal hyperproliferation, which correlated with reduced activation of both the EGFR and erbB2. These results support the hypothesis that both the EGFR and erbB2 play an important role in the development of skin tumors during two-stage skin carcinogenesis, especially during the tumor promotion stage. Furthermore, the marked sensitivity of BK5.erbB2 mice to the inhibitory effects of GW2974 during tumor promotion suggest greater efficacy for this compound when erbB2 is overexpressed or amplified as an early event in the carcinogenic process. Cancer Prev Res; 3(8); 940–52. ©2010 AACR.

Background Heme is an essential iron‐containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate‐limiting enzyme in heme synthesis, δ‐aminolevulinic acid synthase 2 ( ALAS 2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS 2) leads to increased oxidative stress and cell death in the heart. Methods and Results We first showed that ALAS 2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac‐specific overexpression of ALAS 2. While ALAS 2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS 2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS 2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS 2‐overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS 2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS 2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. Conclusions Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the development of heart failure.