Konrad T. Sawicki

Taking Diabetes to Heart—Deregulation of Myocardial Lipid Metabolism in Diabetic Cardiomyopathy

Heart disease is the leading cause of death in patients with diabetes.[1][1] Although advances in medical management and lifestyle interventions have reduced cardiovascular mortality in diabetic patients by as much as 40% over the last decade, the actual number of deaths is predicted to rise as a

Role of Interferon α (IFNα)-inducible Schlafen-5 in Regulation of Anchorage-independent Growth and Invasion of Malignant Melanoma Cells

IFNα exerts potent inhibitory activities against malignant melanoma cells in vitro and in vivo, but the mechanisms by which it generates its antitumor effects remain unknown. We examined the effects of interferon α (IFNα) on the expression of human members of the Schlafen (SLFN) family of genes, a group of cell cycle regulators that mediate growth-inhibitory responses. Using quantitative RT-real time PCR, we found detectable basal expression of all the different human SLFN genes examined (SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14), in malignant melanoma cells and primary normal human melanocytes, but SLFN5 basal expression was suppressed in all analyzed melanoma cell lines. Treatment of melanoma cells with IFNα resulted in induction of expression of SLFN5 in malignant cells, suggesting a potential involvement of this gene in the antitumor effects of IFNα. Importantly, stable knockdown of SLFN5 in malignant melanoma cells resulted in increased anchorage-independent growth, as evidenced by enhanced colony formation in soft agar assays. Moreover, SLFN5 knockdown also resulted in increased invasion in three-dimensional collagen, suggesting a dual role for SLFN5 in the regulation of invasion and anchorage-independent growth of melanoma cells. Altogether, our findings suggest an important role for the SLFN family of proteins in the generation of the anti-melanoma effects of IFNα and for the first time directly implicate a member of the human SLFN family in the regulation of cell invasion.

Molecular and cellular basis of viable dysfunctional myocardium

Heart failure is a leading cause of healthcare expenditures, hospitalization, and mortality in developed countries, and its burden is growing globally.1 With the aging of the population and increasing prevalence of chronic diseases, including hypertension, diabetes mellitus, and obesity, the current heart failure epidemic is guaranteed to significantly worsen in the near future. Thus, new disease-modifying treatments for heart failure are needed urgently and represent an area of intense investigation.2 Multiple studies in humans and animals have shown that the functionality of myocardial tissue of a failing heart can be restored. First, in ischemic heart failure because of severe coronary artery stenosis, revascularization therapy is known to improve heart function in a proportion of patients.3,4 Second, the mechanical unloading of the heart by left ventricular assist device (LVAD) is associated with improvements in cardiac function. In a recent report of 80 patients with heart failure who underwent implantation of a continuous-flow LVAD, the ejection fraction increased by >50% in about one third of the patients, with corresponding improvements in LV end-systolic and end-diastolic volumes and decreases in LV mass at 6 months after LVAD unloading.5 In addition, normalization of echocardiographic parameters has obviated completely the need for continuing LVAD support or cardiac transplantation in several patients.6,7 Importantly, the positive effects of mechanical unloading were noted in patients with both ischemic and nonischemic heart failure,5 suggesting that dysfunctional but potentially salvageable segments of myocardium exist in the failing heart regardless of pathogenesis. Third, in patients with broken heart syndrome (also known as Takotsubo cardiomyopathy), characterized by a rapid and severe loss of cardiac contractility secondary to emotional stress, myocardial function normalized spontaneously, again arguing for the reversibility of heart failure.8 In summary, although maladaptive changes observed in failing hearts were …

Sirtuin 2 regulates cellular iron homeostasis via deacetylation of transcription factor NRF2

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.

Role of Heme in Cardiovascular Physiology and Disease

Heme is an essential molecule for living aerobic organisms and is involved in a remarkable array of diverse biological processes. In the cardiovascular system, heme plays a major role in gas exchange, mitochondrial energy production, antioxidant defense, and signal transduction. Although heme, as

Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury

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 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. Methods and 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 nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with 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 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.

Reduction in mitochondrial iron alleviates cardiac damage during injury.

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 chains 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.

mRNA-binding protein tristetraprolin is essential for cardiac response to iron deficiency by regulating mitochondrial function

Significance Iron deficiency is the most common nutrient deficiency, yet cardiomyopathy rarely develops in these patients. We solved this paradox and identified a protective mechanism involving the mRNA-binding protein tristetraprolin (TTP), which adjusts mitochondrial function in response to iron deficiency. Mice lacking TTP in their hearts were phenotypically normal at baseline but developed spontaneous cardiomyopathy under iron deficiency and exhibited increased reactive oxygen species (ROS)-mediated damage in their hearts. We further demonstrate that down-regulation of specific iron-containing mitochondrial complexes by TTP is the mechanism preventing the formation of dysfunctional mitochondria, subsequent ROS production, and cellular damage. In summary, we show that activation of TTP is part of a required pathway protecting critical organ function when iron becomes scarce. 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.

Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21

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.