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The cardiovascular system requires iron to maintain its high energy demands and metabolic activity. Iron plays a critical role in oxygen transport and storage, mitochondrial function, and enzyme activity. However, excess iron is also cardiotoxic due to its ability to catalyze the formation of reactive oxygen species and promote oxidative damage. While mammalian cells have several redundant iron import mechanisms, they are equipped with a single iron-exporting protein, which makes the cardiovascular system particularly sensitive to iron overload. As a result, iron levels are tightly regulated at many levels to maintain homeostasis. Iron dysregulation ranges from iron deficiency to iron overload and is seen in many types of cardiovascular disease, including heart failure, myocardial infarction, anthracycline-induced cardiotoxicity, and Friedreich’s ataxia. Recently, the use of intravenous iron therapy has been advocated in patients with heart failure and certain criteria for iron deficiency. Here, we provide an overview of systemic and cellular iron homeostasis in the context of cardiovascular physiology, iron deficiency, and iron overload in cardiovascular disease, current therapeutic strategies, and future perspectives.

Heart failure with preserved ejection fraction (HFpEF) is a common cause of morbidity and mortality worldwide, but the underlying pathophysiology is not well-understood and treatment options are limited. Hexokinase-1 (HK1) mitochondrial-binding and protein O-GlcNAcylation are both altered in conditions with risk factors for HFpEF. Here we report a novel mouse model of HFpEF and show that HK1 mitochondrial-binding in endothelial cells (EC) is critical for the development of HFpEF. We demonstrate increased mitochondrial dislocation of HK1 in ECs from HFpEF mice. Mice with deletion of the mitochondrial-binding-domain of HK1 spontaneously develop HFpEF, and their ECs display impaired angiogenic potential. Mitochondrial-bound HK1 associates with dolichyl-diphosphooligosaccharide-protein-glycosyltransferase (DDOST) and its mitochondrial dislocation decreases protein N-glycosylation. We also show that the spatial proximity of dislocated HK1 and O-linked N-acetylglucosamine-transferase (OGT) increases protein O-GlcNAcylation by shifting the balance of the hexosamine-biosynthetic-pathway intermediate supply into the O-GlcNAcylation machinery. Pharmacological inhibition of OGT or EC-specific overexpression of O-GlcNAcase reverses angiogenic defects in ECs and the HFpEF phenotype, indicating that increased protein O-GlcNAcylation is responsible for the development of HFpEF. Our study demonstrates a new mechanism for HFpEF through HK1 cellular localization and resultant protein O-GlcNAcylation in ECs, and provides a potential new therapy for 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.

Background & Aims Nonalcoholic steatohepatitis (NASH) is commonly observed in patients with type 2 diabetes, and thiazolidinediones (TZD) are considered a potential therapy for NASH. Although TZD increase insulin sensitivity and partially reduce steatosis and alanine aminotransferase, the efficacy of TZD on resolving liver pathology is limited. In fact, TZD may activate peroxisome proliferator-activated receptor gamma (PPARγ) in hepatocytes and promote steatosis. Therefore, we assessed the role that hepatocyte-specific PPARγ plays in the development of NASH, and how it alters the therapeutic effects of TZD on the liver of mice with diet-induced NASH. Methods Hepatocyte-specific PPARγ expression was knocked out in adult mice before and after the development of NASH induced with a high fat, cholesterol, and fructose (HFCF) diet. Results HFCF diet increased PPARγ expression in hepatocytes, and rosiglitazone further activated PPARγ in hepatocytes of HFCF-fed mice in vivo and in vitro. Hepatocyte-specific loss of PPARγ reduced the progression of HFCF-induced NASH in male mice and increased the benefits derived from the effects of TZD on extrahepatic tissues and non-parenchymal cells. RNAseq and metabolomics indicated that HFCF diet promoted inflammation and fibrogenesis in a hepatocyte PPARγ–dependent manner and was associated with dysregulation of hepatic metabolism. Specifically, hepatocyte-specific loss of PPARγ plays a positive role in the regulation of methionine metabolism, and that could reduce the progression of NASH. Conclusions Because of the negative effect of hepatocyte PPARγ in NASH, inhibition of mechanisms promoted by endogenous PPARγ in hepatocytes may represent a novel strategy that increases the efficiency of therapies for NAFLD., Graphical abstract

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: 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.

Glucokinase (GCK) is the principal hexokinase (HK) in the liver, operating as a glucose sensor to regulate glucose metabolism and lipid homeostasis. Recently, we proposed HK domain-containing 1 (HKDC1) to be a fifth HK with expression in the liver. Here, we reveal HKDC1 to have low glucose-phosphorylating ability and demonstrate its association with the mitochondria in hepatocytes. As we have shown previously that genetic deletion of HKDC1 leads to altered hepatic triglyceride levels, we also explored the influence of overexpression of HKDC1 in hepatocytes on cellular metabolism, observing reduced glycolytic capacity and maximal mitochondrial respiration with concurrent reductions in glucose oxidation and mitochondrial membrane potential. Furthermore, we found that acute in vivo overexpression of HKDC1 in the liver induced substantial changes in mitochondrial dynamics. Altogether, these findings suggest that overexpression of HKDC1 causes mitochondrial dysfunction in hepatocytes. However, its overexpression was not enough to alter energy storage in the liver but led to mild improvement in glucose tolerance. We next investigated the conditions necessary to induce HKDC1 expression, observing HKDC1 expression to be elevated in human patients whose livers were at more advanced stages of nonalcoholic fatty liver disease (NAFLD) and similarly, found high liver expression in mice on diets causing high levels of liver inflammation and fibrosis. Overall, our data suggest that HKDC1 expression in hepatocytes results in defective mitochondrial function and altered hepatocellular metabolism and speculate that its expression in the liver may play a role in the development of NAFLD.

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.