R. Mark Payne

Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix.

Background: The mechanisms initiating protein acylation in mitochondria are unknown. Results: The pH and acyl-CoA concentrations of the mitochondrial matrix are sufficient to cause protein lysine acetylation and succinylation. Conclusion: Protein acylation in mitochondria may be a nonenzymatic event facilitated by the alkaline pH and high acyl-CoA concentrations. Significance: The mitochondrial deacylases SIRT3 and SIRT5 may have evolved to regulate nonenzymatic protein acylation. Alterations in mitochondrial protein acetylation are implicated in the pathophysiology of diabetes, the metabolic syndrome, mitochondrial disorders, and cancer. However, a viable mechanism responsible for the widespread acetylation in mitochondria remains unknown. Here, we demonstrate that the physiologic pH and acyl-CoA concentrations of the mitochondrial matrix are sufficient to cause dose- and time-dependent, but enzyme-independent acetylation and succinylation of mitochondrial and nonmitochondrial proteins in vitro. These data suggest that protein acylation in mitochondria may be a chemical event facilitated by the alkaline pH and high concentrations of reactive acyl-CoAs present in the mitochondrial matrix. Although these results do not exclude the possibility of enzyme-mediated protein acylation in mitochondria, they demonstrate that such a mechanism may not be required in its unique chemical environment. These findings may have implications for the evolutionary roles that the mitochondria-localized SIRT3 deacetylase and SIRT5 desuccinylase have in the maintenance of metabolic health.

Acute Doxorubicin Cardiotoxicity Is Associated With p53-Induced Inhibition of the Mammalian Target of Rapamycin Pathway

Background— Doxorubicin is used to treat childhood and adult cancer. Doxorubicin treatment is associated with both acute and chronic cardiotoxicity. The cardiotoxic effects of doxorubicin are cumulative, which limits its chemotherapeutic dose. Free radical generation and p53-dependent apoptosis are thought to contribute to doxorubicin-induced cardiotoxicity. Methods and Results— Adult transgenic (MHC-CB7) mice expressing cardiomyocyte-restricted dominant-interfering p53 and their nontransgenic littermates were treated with doxorubicin (20 mg/kg cumulative dose). Nontransgenic mice exhibited reduced left ventricular systolic function (predoxorubicin fractional shortening [FS] 61±2%, postdoxorubicin FS 45±2%, mean±SEM, P<0.008), reduced cardiac mass, and high levels of cardiomyocyte apoptosis 7 days after the initiation of doxorubicin treatment. In contrast, doxorubicin-treated MHC-CB7 mice exhibited normal left ventricular systolic function (predoxorubicin FS 63±2%, postdoxorubicin FS 60±2%, P>0.008), normal cardiac mass, and low levels of cardiomyocyte apoptosis. Western blot analyses indicated that mTOR (mammalian target of rapamycin) signaling was inhibited in doxorubicin-treated nontransgenic mice but not in doxorubicin-treated MHC-CB7 mice. Accordingly, transgenic mice with cardiomyocyte-restricted, constitutively active mTOR expression (MHC-mTORca) were studied. Left ventricular systolic function (predoxorubicin FS 64±2%, postdoxorubicin FS 60±3%, P>0.008) and cardiac mass were normal in doxorubicin-treated MHC-mTORca mice, despite levels of cardiomyocyte apoptosis similar to those seen in doxorubicin-treated nontransgenic mice. Conclusions— These data suggest that doxorubicin treatment induces acute cardiac dysfunction and reduces cardiac mass via p53-dependent inhibition of mTOR signaling and that loss of myocardial mass, and not cardiomyocyte apoptosis, is the major contributor to acute doxorubicin cardiotoxicity.

A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta

Mutations in nuclear and mitochondrial genomes can lead to defects in mitochondrial function. To date, repair of these defects with exogenous proteins or gene transfer has been difficult with either viral or nonviral vectors. We hypothesized that TAT fusion proteins would cross both mitochondrial membranes and that incorporation of a mitochondrial signal sequence into a TAT fusion protein would allow processing and localization of exogenous proteins in mitochondria. A TAT-mitochondrial malate dehydrogenase signal sequence (mMDH)-enhanced green fluorescent protein (eGFP) fusion protein was constructed. TAT-mMDH-eGFP allowed rapid transduction and localization of fusion protein into mitochondria of multiple cell types. In contrast, TAT-GFP, without a mitochondrial signal sequence, rapidly transduced into cells and mitochondria, displayed pseudo-first-order kinetics, but did not remain there. Mice injected 5 days prior with TAT-mMDH-eGFP had detectable eGFP activity in multiple tissue types. Western blotting of cytosolic and mitochondrial fractions isolated from their livers confirmed eGFP localization to mitochondria and that the mMDH transit peptide was recognized and processed. Furthermore, TAT-mMDH-eGFP fusion protein injected into pregnant mice crossed the placenta and was detectable in both the fetus and the newborn pups. TAT fusion proteins containing a mitochondrial signal sequence are a viable method to localize proteins to mitochondria.

Octamer Formation and Coupling of Cardiac Sarcomeric Mitochondrial Creatine Kinase Are Mediated by Charged N-terminal Residues

Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.

A TAT–Frataxin fusion protein increases lifespan and cardiac function in a conditional Friedreich's ataxia mouse model

Friedreichs ataxia (FRDA) is the most common inherited human ataxia and results from a deficiency of the mitochondrial protein, frataxin (FXN), which is encoded in the nucleus. This deficiency is associated with an iron-sulfur (Fe-S) cluster enzyme deficit leading to progressive ataxia and a frequently fatal cardiomyopathy. There is no cure. To determine whether exogenous replacement of the missing FXN protein in mitochondria would repair the defect, we used the transactivator of transcription (TAT) protein transduction domain to deliver human FXN protein to mitochondria in both cultured patient cells and a severe mouse model of FRDA. A TAT-FXN fusion protein bound iron in vitro, transduced into mitochondria of FRDA deficient fibroblasts and reduced caspase-3 activation in response to an exogenous iron-oxidant stress. Injection of TAT-FXN protein into mice with a conditional loss of FXN increased their growth velocity and mean lifespan by 53% increased their mean heart rate and cardiac output, increased activity of aconitase and reversed abnormal mitochondrial proliferation and ultrastructure in heart. These results show that a cell-penetrant peptide is capable of delivering a functional mitochondrial protein in vivo to rescue a very severe disease phenotype, and present the possibility of TAT-FXN as a protein replacement therapy.

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth.

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (αMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These αMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. αMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in αMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the αMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.

Analysis of Ventricular Hypertrabeculation and Noncompaction Using Genetically Engineered Mouse Models

Ventricular trabeculation and compaction are two of the many essential steps for generating a functionally competent ventricular wall. A significant reduction in trabeculation is usually associated with ventricular compact zone deficiencies (hypoplastic wall), which commonly lead to embryonic heart failure and early embryonic lethality. In contrast, hypertrabeculation and lack of ventricular wall compaction (noncompaction) are closely related defects in cardiac embryogenesis associated with left ventricular noncompaction, a genetically heterogeneous disorder. Here we summarize our recent findings through the analyses of several genetically engineered mouse models that have defects in cardiac trabeculation and compaction. Our data indicate that cellular growth and differentiation signaling pathways are keys in these ventricular morphogenetic events.

Friedreich’s Ataxia reveals a mechanism for coordinate regulation of oxidative metabolism via feedback inhibition of the SIRT3 deacetylase

Friedreichs ataxia (FRDA) is the most common inherited human ataxia and is caused by a deficiency in the mitochondrial protein frataxin. Clinically, patients suffer from progressive spinocerebellar degeneration, diabetes and a fatal cardiomyopathy, associated with mitochondrial respiratory chain defects. Recent findings have shown that lysine acetylation regulates mitochondrial function and intermediary metabolism. However, little is known about lysine acetylation in the setting of pathologic energy stress and mitochondrial dysfunction. We tested the hypothesis that the respiratory chain defects in frataxin deficiency alter mitochondrial protein acetylation. Using two conditional mouse models of FRDA, we demonstrate marked hyperacetylation of numerous cardiac mitochondrial proteins. Importantly, this biochemical phenotype develops concurrently with cardiac hypertrophy and is caused by inhibition of the NAD(+)-dependent SIRT3 deacetylase. This inhibition is caused by an 85-fold decrease in mitochondrial NAD(+)/NADH and direct carbonyl group modification of SIRT3, and is reversed with excess SIRT3 and NAD(+) in vitro. We further demonstrate that protein hyperacetylation may be a common feature of mitochondrial disorders caused by respiratory chain defects, notably, cytochrome oxidase I (COI) deficiency. These findings suggest that SIRT3 inhibition and consequent protein hyperacetylation represents a negative feedback mechanism limiting mitochondrial oxidative pathways when respiratory metabolism is compromised, and thus, may contribute to the lethal cardiomyopathy in FRDA.

Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

Noonan syndrome (NS) is an autosomal dominant congenital disorder characterized by multiple birth defects including heart defects and myeloproliferative disease (MPD). Approximately 50% of NS patients have germline gain-of-function mutations in PTPN11, which encodes the protein-tyrosine phosphatase, Shp2. We provide evidence that conditional ablation of Stat3 in hematopoietic cells and cardiac valvular tissues leads to myeloid progenitor hyperplasia and pulmonary stenosis due to the leaflet thickening, respectively. Consistently, STAT3 activation is significantly compromised in peripheral blood cells from NS patients bearing Shp2-activating mutations. Biochemical and functional analyses demonstrate that activated Shp2 is able to down-regulate Tyr(P)-Stat3 and that constitutively active Stat3 rescues activating mutant Shp2-induced granulocyte-macrophage colony-stimulating factor hypersensitivity in bone marrow cells. Collectively, our work demonstrates that Stat3 is an essential signaling component potentially contributing to the pathogenesis of NS and juvenile myelomonocytic leukemia caused by PTPN11 gain-of-function mutations.

Mitochondrial trifunctional protein defects: Clinical implications and therapeutic approaches

The mitochondrial trifunctional protein (MTP) is a heterotrimeric protein that consists of four alpha-subunits and four beta-subunits and catalyzes three of the four chain-shortening reactions in the mitochondrial beta-oxidation of long-chain fatty acids. Families with recessively inherited MTP defects display a spectrum of maternal and fetal phenotypes. Current management of patients with MTP defects include long-term dietary therapy of fasting avoidance, low-fat/high-carbohydrate diet with restriction of long-chain fatty acid intake and substitution with medium-chain fatty acids. These dietary approaches appear promising in the short-term, but the long-term outcome of patients treated with dietary intervention is largely unknown. Potential therapeutic approaches targeted at correcting the metabolic defect will be discussed. We will discuss the potential use of protein transduction domains that cross the mitochondrial membranes for the treatment of mitochondrial disorders. In addition, we discuss the phenotypes of MTP in a heterozygous state and potential ways to intervene to increase hepatic fatty acid oxidative capacity.