Tejaswitha J Naik

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.

Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II

ABSTRACT Alterations in glucose metabolism have been demonstrated for diverse disorders ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. HKI and II can bind to mitochondria through their N-terminal hydrophobic regions, and their overexpression in tissue culture protects against cell death. In order to determine the relative contributions of mitochondrial binding and glucose-phosphorylating activities of HKs to their overall protective effects, we expressed full-length HKI and HKII, their truncated proteins lacking the mitochondrial binding domains, and catalytically inactive proteins in tissue culture. The overexpression of full-length proteins resulted in protection against cell death, decreased levels of reactive oxygen species, and possibly inhibited mitochondrial permeability transition in response to H2O2. However, the truncated and mutant proteins exerted only partial effects. Similar results were obtained with primary neonatal rat cardiomyocytes. The HK proteins also resulted in an increase in the phosphorylation of voltage-dependent anion channel (VDAC) through a protein kinase Cε (PKCε)-dependent pathway. These results suggest that both glucose phosphorylation and mitochondrial binding contribute to the protective effects of HKI and HKII, possibly through VDAC phosphorylation by PKCε.

Blockade of the erbB2 receptor induces cardiomyocyte death through mitochondrial and reactive oxygen species-dependent pathways

Overexpression of the receptor tyrosine kinase erbB2 (Her2 in humans) is correlated with a poor prognosis in breast and ovarian cancers. Treatment with trastuzumab (a monoclonal antibody against erbB2) improves survival; however, it also causes cardiomyopathy. We hypothesized that blockade of the erbB2 receptor induces cardiomyocyte death through a mitochondrial pathway that is dependent on the production of reactive oxygen species (ROS). We first showed that levels of erbB2 receptor are significantly decreased in an animal model of ischemic heart disease and in human ischemic cardiomyopathy. We treated neonatal rat cardiomyocytes with an inhibitory erbB2 antibody to study the mechanism behind the deleterious effects of erbB2 blockade. These cells displayed a dose-dependent increase in ROS production and cell death compared with control IgG-treated cells; these processes were reversed by the antioxidant, N-acetylcysteine. The effects of erbB2 antibody on both cell death and ROS production were also reversed by cyclosporine A and diazoxide, chemicals that regulate the pro- and anti-apoptotic channels in the mitochondria, respectively. Furthermore, mouse embryonic fibroblasts lacking Bax and Bak (proteins that mediate cell death through a mitochondrial pathway) were resistant to the deleterious effects of erbB2 antibody. These effects of erbB2 blockade appear to occur through a pathway involving AKT and PKC-α. Our results suggest that erbB2 plays a role in cardiomyocyte survival, and that the deleterious effects of trastuzumab on the heart occur through a mitochondrial pathway and is mediated by ROS production. Manipulation of redox signaling may be beneficial in cancer patients receiving trastuzumab.

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.

Phosphorylation of contractile proteins in response to α- and β-adrenergic stimulation in neonatal cardiomyocytes

alpha- and beta-Adrenergic receptor agonists induce an inotropic response in the adult heart by promoting the phosphorylation of several regulatory proteins, including myosin-binding protein C (MyBP-C), cardiac troponin I (cTnI), and phospholamban (PLB). However, the adrenergic-induced phosphorylation of these proteins has not been characterized in the developing heart. Accordingly, we evaluated MyBP-C, cTnI, and PLB phosphorylation in cultured neonatal rat cardiomyocytes (NRCMs) after alpha- and beta-receptor activation with phenylephrine and isoproterenol. alpha-Receptor stimulation increased, whereas beta-receptor activation reduced MyBP-C phosphorylation. Isoelectric-focusing experiments indicated that the amount of monophosphorylated MyBP-C was sensitive to alpha-adrenergic activation, but diphosphorylated and triphosphorylated MyBP-C levels were largely unaffected. The phosphorylation of cTnI and PLB was consistent with the mechanism observed in adult hearts: alpha- and beta-Receptor stimulation phosphorylated both proteins. For cTnI, the greatest difference associated with beta-receptor activation was observed in the diphosphorylated state, whereas alpha-receptor activation was associated with a marked increase in the tetraphosphorylated protein and absence of the unphosphorylated state. Despite these apparent changes in cTnI and PLB phosphorylation, beta-receptor activation failed to alter calcium transients in NRCMs. Collectively, these findings suggest that, unlike cTnI and PLB, MyBP-C and inotropy are not coupled to beta-adrenergic stimulation in NRCMs. Therefore, cTnI and PLB probably play a more central role in modulating contractile function in NRCMs in response to catecholamines than does MyBP-C, and MyBP-C may have a structural role in stabilizing thick filament assembly rather than influencing cross-bridge formation in developing hearts.

63 MITOCHONDRIAL BINDING AND GLUCOSE PHOSPHORYLATION ARE BOTH NEEDED FOR THE PROTECTIVE EFFECTS OF HEXOKINASE I AND II.

Alterations in glucose metabolism have been demonstrated in diverse disorders, ranging from heart disease to cancer. The first step in glucose metabolism is carried out by the hexokinase (HK) family of enzymes. Overexpression of HKI and HKII in tissue culture protects against oxidant-induced cell death. The protective effects of these enzymes are thought to be due to either an increase in glucose phosphorylation or closure of the mitochondrial permeability transition pore (mPTP) as a result of HK binding to the voltage-dependent anion channel (VDAC) on the mitochondria. VDAC is believed to form part of mPTP, the opening of which causes cellular injury. The relative contribution of HK binding to the mitochondria and the increase in glucose phosphorylation to the overall protective effects of HKs are not clear. Furthermore, there is no clear evidence supporting the hypothesis that HK binding to mitochondria inhibits mPTP. To better understand the mechanism(s) behind the protective effects of HKs, we overexpressed full-length HKI and HKII (FL-HKI and FL-HKII, respectively), their truncated proteins lacking the N-terminal hydrophobic domains (Tr-HKI and Tr-HKII, respectively), and catalytically inactive proteins (Mut-HKI and mut-HKII, respectively) in human embryonic kidney (HEK293) cells. The truncated enzymes cannot bind to mitochondria but can phosphorylate glucose, whereas the catalytically inactive enzymes can bind to the mitochondria but do not phosphorylate glucose. The cells overexpressing these constructs were subjected to oxidant stress followed by measurement of mitochondrial membrane potential and cell death. Overexpression of FL-HKI and FL-HKII resulted in complete protection against oxidant-induced loss of mitochondrial membrane potential and cell death (survival percentage of 96 ± 9 and 95 ± 5 for FL-HKI and FL-HKII, respectively). Although overexpression of the truncated and mutant proteins reduced cell death, the degree of protection was about 40 to 50% less than that of the full-length proteins. Furthermore, FL-HKI and FL-HKII inhibited mitochondrial permeability transition (MPT) in the presence of H2O2, whereas the truncated and mutant forms only caused partial inhibition. Similar results were obtained when these proteins were expressed in primary neonatal rat cardiomyocytes using an adenoviral technique. To understand the mechanism for the protective effects of HKs, we measured VDAC phosphorylation in cells overexpressing these proteins. Overexpression of FL-HKI and FL-HKII resulted in a 5- to 10-fold increase in VDAC phosphorylation. The mechanism for VDAC phosphorylation appears to be through PKC-e as inhibitors of this enzyme led to a reversal of this process. These results suggest that both glucose phosphorylation and inhibition of mPTP contribute to the protective effects of HKI and HKII. Furthermore, overexpression of HKI and HKII leads to VDAC phosphorylation in a PKC-e-dependent pathway. These findings bear implications of HK overexpression and binding to the mitochondria as a potential clinical treatment strategy for various forms of human disease.