Rongxue Wu

Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation

Doxorubicin is an effective anticancer drug with known cardiotoxic side effects. It has been hypothesized that doxorubicin-dependent cardiotoxicity occurs through ROS production and possibly cellular iron accumulation. Here, we found that cardiotoxicity develops through the preferential accumulation of iron inside the mitochondria following doxorubicin treatment. In isolated cardiomyocytes, doxorubicin became concentrated in the mitochondria and increased both mitochondrial iron and cellular ROS levels. Overexpression of ABCB8, a mitochondrial protein that facilitates iron export, in vitro and in the hearts of transgenic mice decreased mitochondrial iron and cellular ROS and protected against doxorubicin-induced cardiomyopathy. Dexrazoxane, a drug that attenuates doxorubicin-induced cardiotoxicity, decreased mitochondrial iron levels and reversed doxorubicin-induced cardiac damage. Finally, hearts from patients with doxorubicin-induced cardiomyopathy had markedly higher mitochondrial iron levels than hearts from patients with other types of cardiomyopathies or normal cardiac function. These results suggest that the cardiotoxic effects of doxorubicin develop from mitochondrial iron accumulation and that reducing mitochondrial iron levels protects against doxorubicin-induced cardiomyopathy.

Reduction in Hexokinase II Levels Results in Decreased Cardiac Function and Altered Remodeling After Ischemia/Reperfusion Injury

Rationale: Cardiomyocytes switch substrate utilization from fatty acid to glucose under ischemic conditions; however, it is unknown how perturbations in glycolytic enzymes affect cardiac response to ischemia/reperfusion (I/R). Hexokinase (HK)II is a HK isoform that is expressed in the heart and can bind to the mitochondrial outer membrane. Objective: We sought to define how HKII and its binding to mitochondria play a role in cardiac response and remodeling after I/R. Methods and Results: We first showed that HKII levels and its binding to mitochondria are reduced 2 days after I/R. We then subjected the hearts of wild-type and heterozygote HKII knockout (HKII+/−) mice to I/R by coronary ligation. At baseline, HKII+/− mice have normal cardiac function; however, they display lower systolic function after I/R compared to wild-type animals. The mechanism appears to be through an increase in cardiomyocyte death and fibrosis and a reduction in angiogenesis; the latter is through a decrease in hypoxia-inducible factor–dependent pathway signaling in cardiomyocytes. HKII mitochondrial binding is also critical for cardiomyocyte survival, because its displacement in tissue culture with a synthetic peptide increases cell death. Our results also suggest that HKII may be important for the remodeling of the viable cardiac tissue because its modulation in vitro alters cellular energy levels, O2 consumption, and contractility. Conclusions: These results suggest that reduction in HKII levels causes altered remodeling of the heart in I/R by increasing cell death and fibrosis and reducing angiogenesis and that mitochondrial binding is needed for protection of cardiomyocytes.

Disruption of Hexokinase II–Mitochondrial Binding Blocks Ischemic Preconditioning and Causes Rapid Cardiac Necrosis

Rationale: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. Objective: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. Methods and Results: Ex vivo perfused HKII+/− hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 &mgr;mol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. Conclusions: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

Disruption of ATP-binding cassette B8 in mice leads to cardiomyopathy through a decrease in mitochondrial iron export

Mitochondrial iron levels are tightly regulated, as iron is essential for the synthesis of Fe/S clusters and heme in the mitochondria, but high levels can cause oxidative stress. The ATP-binding cassette (ABC) transporter ABCB8 is a mitochondrial inner membrane protein with an unknown function. Here, we show that ABCB8 is involved in mitochondrial iron export and is essential for baseline cardiac function. Induced genetic deletion of ABCB8 in mouse hearts resulted in mitochondrial iron accumulation and cardiomyopathy, as assessed by echocardiography and invasive hemodynamics. Mice with ABCB8 deletion in the heart also displayed mitochondrial damage, and higher levels of reactive oxygen species and cell death. Down-regulation of ABCB8 in vitro resulted in decreased iron export from isolated mitochondria, whereas its overexpression had the opposite effect. Furthermore, ABCB8 is needed for the maturation of the cytosolic Fe/S proteins, as its deletion in vitro and in vivo led to decreased activity of cytosolic, but not mitochondrial, iron–sulfur-containing enzymes. These results indicate that ABCB8 is essential for normal cardiac function, maintenance of mitochondrial iron homeostasis and maturation of cytosolic Fe/S proteins. In summary, this report provides characterization of a protein involved in mitochondrial iron export.

Hexokinase II knockdown results in exaggerated cardiac hypertrophy via increased ROS production

Hexokinase‐II (HKII) is highly expressed in the heart and can bind to the mitochondrial outer membrane. Since cardiac hypertrophy is associated with a substrate switch from fatty acid to glucose, we hypothesized that a reduction in HKII would decrease cardiac hypertrophy after pressure overload. Contrary to our hypothesis, heterozygous HKII‐deficient (HKII+/−) mice displayed increased hypertrophy and fibrosis in response to pressure overload. The mechanism behind this phenomenon involves increased levels of reactive oxygen species (ROS), as HKII knockdown increased ROS accumulation, and treatment with the antioxidant N‐acetylcysteine (NAC) abrogated the exaggerated response. HKII mitochondrial binding is also important for the hypertrophic effects, as HKII dissociation from the mitochondria resulted in de novo hypertrophy, which was also attenuated by NAC. Further studies showed that the increase in ROS levels in response to HKII knockdown or mitochondrial dissociation is mediated through increased mitochondrial permeability and not by a significant change in antioxidant defenses. Overall, these data suggest that HKII and its mitochondrial binding negatively regulate cardiac hypertrophy by decreasing ROS production via mitochondrial permeability.

Regulation and cytoprotective role of hexokinase III.

Background Hexokinases (HKs) catalyze the first step in glucose metabolism. Of the three mammalian 100-kDa HK isoforms, HKI and II can bind to mitochondria and protect against cell death. HKIII does not bind mitochondria, and little is known about its regulation or cytoprotective effects. We studied the regulation of HKIII at the transcriptional and protein levels and investigated its role in cellular protection. Methodology/Principal Findings We show that like HKII, HKIII expression is regulated by hypoxia, but other factors that regulate HKII expression have no effect on HKIII levels. This transcriptional regulation is partially dependent on hypoxia-inducible factor (HIF) signaling. We also demonstrate regulation at the protein level, as mutations in putative N-terminal substrate binding residues altered C-terminal catalytic activity, suggesting that HKIII activity is governed, in part, by interactions between these two domains. Overexpression of HKIII reduced oxidant-induced cell death, increased ATP levels, decreased the production of reactive oxygen species (ROS), and preserved mitochondrial membrane potential. HKIII overexpression was also associated with higher levels of transcription factors that regulate mitochondrial biogenesis, and greater total mitochondrial DNA content. Attempts to target HKIII to the mitochondria by replacing its N-terminal 32-amino-acid sequence with the mitochondrial-targeting sequence of HKII led to protein aggregation, suggesting that this region is necessary to maintain proper protein folding and solubility. Conclusions/Significance These results suggest that HKIII is regulated by hypoxia and there are functional interactions between its two halves. Furthermore, HKIII exerts protective effects against oxidative stress, perhaps by increasing ATP levels, reducing oxidant-induced ROS production, preserving mitochondrial membrane potential, and increasing mitochondrial biogenesis.

ATP-binding cassette B10 regulates early steps of heme synthesis

Rationale: Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia–reperfusion injury in the heart; however, its primary function has not been established. Objective: The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. Methods and Results: Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with &dgr;-aminolevulinic acid reversed these defects. Overexpression of mitochondrial &dgr;-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of &dgr;-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. Conclusions: ABCB10 plays a critical role in heme synthesis pathway by facilitating &dgr;-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

ABCB10 Regulates Early Steps of Heme Synthesis

Rationale: Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia–reperfusion injury in the heart; however, its primary function has not been established. Objective: The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells. Methods and Results: Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with &dgr;-aminolevulinic acid reversed these defects. Overexpression of mitochondrial &dgr;-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of &dgr;-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy. Conclusions: ABCB10 plays a critical role in heme synthesis pathway by facilitating &dgr;-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy.

Patients with type 2 diabetes often present with cardiovascular complications; however, it is not clear how diabetes promotes cardiac dysfunction. In murine models, deletion of the gene encoding aryl hydrocarbon nuclear translocator (ARNT, also known as HIF1β) in the liver or pancreas leads to a diabetic phenotype; however, the role of ARNT in cardiac metabolism is unknown. Here, we determined that cardiac-specific deletion of Arnt in adult mice results in rapid development of cardiomyopathy (CM) that is characterized by accumulation of lipid droplets. Compared with hearts from ARNT-expressing mice, ex vivo analysis of ARNT-deficient hearts revealed a 2-fold increase in fatty acid (FA) oxidation as well as a substantial increase in the expression of PPARα and its target genes. Furthermore, deletion of both Arnt and Ppara preserved cardiac function, improved survival, and completely reversed the FA accumulation phenotype, indicating that PPARα mediates the detrimental effects of Arnt deletion in the heart. Finally, we determined that ARNT directly regulates Ppara expression by binding to its promoter and forming a complex with HIF2α. Together, these findings suggest that ARNT is a critical regulator of myocardial FA metabolism and that its deletion leads to CM and an increase in triglyceride accumulation through PPARα.

MicroRNA‐210 Decreases heme Levels by Targeting Ferrochelatase in Cardiomyocytes

Background MicroRNA‐210 (miR‐210) increases in hypoxia and regulates mitochondrial respiration through modulation of iron‐sulfur cluster assembly proteins (ISCU1/2), a protein that is involved in Fe/S cluster synthesis. However, it is not known how miR‐210 affects cellular iron levels or production of heme, another iron containing molecule that is also needed for cellular and mitochondrial function. Methods and Results To screen for micro‐ribonucleic acids (miRNAs) regulated by iron, we performed a miRNA gene array in neonatal rat cardiomyocytes treated with iron chelators. Levels of miR‐210 are significantly increased with iron chelation, however, this response was mediated entirely through the hypoxia‐inducible factor (HIF) pathway. Furthermore, miR‐210 reduced cellular heme levels and the activity of mitochondrial and cytosolic heme‐containing proteins by modulating ferrochelatase (FECH), the last enzyme in heme biosynthesis. Mutation of the 2 miR‐210 binding sites in the 3′ untranslated region (UTR) of FECH reversed the miR‐210 response, while mutation of either binding site in isolation did not exert any effects. Changes mediated by miR‐210 in heme and FECH were independent of ISCU, as overexpression of an ISCU construct lacking the 3′ UTR does not alter miR‐210 regulation of heme and FECH. Finally, FECH levels increased in hypoxia, and this effect was not reversed by miR‐210 knockdown, suggesting that the effects of miR‐210 on heme are restricted to normoxic conditions, and that the pathway is overriden in hypoxia. Conclusions Our results identify a role for miR‐210 in the regulation of heme production by targeting and inhibiting FECH under normoxic conditions.