A Barth Syndrome Patient-Derived D75H Point Mutation in TAFAZZIN Drives Progressive Cardiomyopathy in Mice.

Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (TazPM) that phenocopies BTHS clinical traits. As TazPM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile TazPM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile TazPM hearts. However, adult TazPM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult TazPM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.

Biomarkers of fibrosis and portal hypertension in Fontan-associated liver disease in children and adults.

BACKGROUND: Fontan-associated liver disease (FALD) refers to structural and functional changes of the liver caused by the physiology of the Fontan palliation. Currently, liver biopsy is the gold standard to assess liver fibrosis of FALD. AIM: Investigate biomarkers correlating with severity of liver biopsy fibrosis in FALD. METHODS: A retrospective study of post-Fontan patients ≥ 10 years of age who underwent liver biopsy was conducted. Advanced liver disease (ALD) was defined as bridging fibrosis and/or cirrhosis on liver biopsy. AST-to-platelet ratio index (APRI), Fibrosis-4 (FIB-4) and Liver Stiffness Measurement (LSM) from FibroScan were used as non-invasive fibrosis scores. RESULTS: Sixty-six patients (26/47; 55.3% adults and 13/19 children; 68.4%) had ALD on biopsy. ALD was associated with lower platelet count (151 vs. 198 K/uL, p = 0.003), higher APRI (0.64 vs. 0.32, p = 0.01), higher FIB-4 (0.64 vs. 0.32, p = 0.02). Liver fibrosis score correlated with APRI (0.34, p = 0.02) and FIB-4 (0.47, p = 0.001) in adults. LSM had a high sensitivity at 81.3% with 45.5% specificity at a cut-off 18.5 kPa. CONCLUSIONS: APRI and FIB-4 had modest discrimination to identify adults with advanced liver disease, but not children, indicating that these values may be followed as a marker of FALD progression in older patients.

Biomarker sST2 in Adults with Transposition of the Great Arteries Palliated by Mustard Procedure: A Five-Year Follow-up.

The Mustard procedure was an early cardiac surgery for transposition of the great arteries (TGA). Despite being successful, it has been associated with long-term arrhythmias and heart failure. A key factor complicating management in adults with congenital heart disease (CHD) is the deficiency of biomarkers predicting outcome. Soluble suppression of tumorogenicity-2 (sST2) is secreted by cardiomyocytes in response to mechanical strain and fibrosis. We hypothesized that adults with a Mustard procedure would have higher levels of sST2 than healthy individuals, and this would correlate with clinical outcome. We performed a single-center study in patients managed during childhood with a Mustard procedure versus age-matched controls. Clinical and demographic data were collected and biomarkers (sST2, cTnI, BNP, lipid panel, insulin, and glucose) were obtained. There were 18 patients (12 male) in the Mustard cohort and 18 patients (6 male) in the control group (22-49 years, mean of 35.8 vs. mean 32.6 years, respectively, p = ns). Nine Mustard subjects were NYHA class II, and 9 subjects were class III. The control group was asymptomatic. sST2 in the Mustard group was elevated in 56% vs. 17% in controls (p = 0.035). Of the Mustard subjects with elevated sST2, 60% had elevated cTnI and BNP, and 90% had low HDL. Over five years, the Mustard patients with elevated sST2 values had greater medication use, arrhythmias, hospitalizations, and ablation/pacer implantations than Mustard subjects with normal sST2. Mustard subjects with elevated sST2 had other biomarker abnormalities and clinically worse outcomes. Thus, sST2 may add a predictive value to cardiac-related morbidity and mortality.

Post-translational modifications in mitochondria: protein signaling in the powerhouse.

There is an intimate interplay between cellular metabolism and the pathophysiology of disease. Mitochondria are essential to maintaining and regulating metabolic function of cells and organs. Mitochondrial dysfunction is implicated in diverse diseases, such as cardiovascular disease, diabetes and metabolic syndrome, neurodegeneration, cancer, and aging. Multiple reversible post-translational protein modifications are located in the mitochondria that are responsive to nutrient availability and redox conditions, and which can act in protein-protein interactions to modify diverse mitochondrial functions. Included in this are physiologic redox signaling via reactive oxygen and nitrogen species, phosphorylation, O-GlcNAcylation, acetylation, and succinylation, among others. With the advent of mass proteomic screening techniques, there has been a vast increase in the array of known mitochondrial post-translational modifications and their protein targets. The functional significance of these processes in disease etiology, and the pathologic response to their disruption, are still under investigation. However, many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondrial-targeted therapeutic strategies. This review addresses the current knowledge of post-translational processing and signaling mechanisms in mitochondria, and their implications in health and disease.

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

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.

Mitochondrial acetylation and diseases of aging.

In recent years, protein lysine acetylation has emerged as a prominent and conserved regulatory posttranslational modification that is abundant on numerous enzymes involved in the processes of intermediary metabolism. Well-characterized mitochondrial processes of carbon utilization are enriched in acetyl-lysine modifications. Although seminal discoveries have been made in the basic biology of mitochondrial acetylation, an understanding of how acetylation states influence enzyme function and metabolic reprogramming during pathological states remains largely unknown. This paper will examine our current understanding of eukaryotic acetate metabolism and present recent findings in the field of mitochondrial acetylation biology. The implications of mitochondrial acetylation for the aging process will be discussed, as well as its potential implications for the unique and localized metabolic states that occur during the aging-associated conditions of heart failure and cancer growth.

Calcification after myocardial infarction is independent of amniotic fluid stem cell injection.

Ischemic heart disease remains one of the most common causes of mortality in developed countries. Recently, stem cell therapy is being considered for treating ischemic heart diseases. On the other hand, there has been evidence of chondro-osteogenic mass formation after stem cell injection in the heart. In a recent publication, Chiavegato et al. (J Mol Cell Cardiol. 42 (2007) 746-759) has suggested that amniotic fluid-derived stem (AFS) cells cause chondro-osteogenic masses in the infarcted heart. The goal of the current study was to further examine the formation of such masses, specifically, the role of AFS cells in this process. Our results confirm the presence of similar bone-like masses in the left ventricular wall of infarcted rats; however, this phenomenon occurred independent of AFS cell injection into the myocardium. Moreover, AFS cell injection did not increase the presence of chondro-osteogenic masses. Echocardiographic analysis of large infarctions in rats frequently revealed the presence of echogenic structures in the left ventricular wall. We further demonstrated a significant relationship between the infarction size and chondro-osteogenic formation and subsequent decrease in cardiac function. Collectively, our study indicates that chondro-osteogenic differentiation can take place in infarcted rat heart independent of cell injection. These results have significant implications for future design and testing of stem cell therapy for treatment of cardiac muscle diseases.

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.

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 mouse model for juvenile doxorubicin-induced cardiac dysfunction.

Doxorubicin (DOX) is a potent antitumor agent. DOX can also induce cardiotoxicity, and high cumulative doses are associated with recalcitrant heart failure. Children are particularly sensitive to DOX-induced heart failure. The ability to genetically modify mice makes them an ideal experimental system to study the molecular basis of DOX-induced cardiotoxicity. However, most mouse DOX studies rely on acute drug administration in adult animals, which typically are analyzed within 1 wk. Here, we describe a juvenile mouse model of chronic DOX-induced cardiac dysfunction. DOX treatment was initiated at 2 wk of age and continued for a period of 5 wk (25 mg/kg cumulative dose). This resulted in a decline in cardiac systolic function, which was accompanied by marked atrophy of the heart, low levels of cardiomyocyte apoptosis, and decreased growth velocity. Other animals were allowed to recover for 13 wk after the final DOX injection. Cardiac systolic function improved during this recovery period but remained depressed compared with the saline injected controls, despite the reversal of cardiac atrophy. Interestingly, increased levels of cardiomyocyte apoptosis and concomitant myocardial fibrosis were observed after DOX withdrawal. These data suggest that different mechanisms contribute to cardiac dysfunction during the treatment and recovery phases.

The pathogenesis of pulmonary contusion: an open chest model in the rat.

BACKGROUND: Chemokines direct leukocytes to areas of inflammation or injury. In general, CC chemokines (MCP-1, MIP-1alpha, RANTES) are chemoattractants for mononuclear cells and CXC (CINC-1, MIP-2alpha) for polymorphonuclear cells (PMNs). Herein we describe an open chest model of pulmonary contusion (PC) in a rodent (rat) and have identified a possible role for CC and CXC chemokines in the pathogenesis of PC. METHODS: Sprague-Dawley rats (350 g) underwent thoracotomy. The exposed lung was struck with a piston at 5.2 m/s (150 J/M2). Blood, bronchoalveolar lavage (BAL), and lung tissue were collected at 3 hours and 24 hours after injury. PaO2/FiO2 (P/F) ratio was calculated at 15-minute intervals for 3 hours after contusion. Serum was evaluated for cytokine and chemokine expression using ELISA. Cell count/differential was performed on BAL, and lung tissue was obtained for histologic analysis, protein expression and wet to dry weights. Data are reported as pg/mL +/- SE. Data were analyzed using Student's t test to identify significant differences (p < or = 0.05 significant) between sham and injured animals. RESULTS: Piston impact caused PC based upon morphologic and histologic criteria. BAL cell count and lung wet to dry weights were increased and P/F ratio was decreased after PC. Systemic levels of IL-ra, MCP-1, and the CXC chemokines MIP-2alpha and CINC-1 were significantly elevated at 3 hours when sham and injured animals were compared. All chemokines were found to be significantly elevated at 24 hours, consistent with the early PMN and subsequent mononuclear infiltration observed in the contused lung. Pulmonary expression of TNF-alpha, IL-1beta, CINC-1, MIP-2alpha, ICAM-1, and elastase were increased and activated systemic neutrophils showed increased CD-11b. CONCLUSION: A model of PC is described in which innate inflammation is activated locally and systemically. Systemic levels of CC and CXC chemokines are increased after PC. This correlates with elevated PMN CD-11b expression, enhanced pulmonary ICAM-1 expression, and mononuclear and PMN infiltration into the lung and alveolar space. Elevated levels of CC and CXC chemokines are seen after PC and may be involved in the lung's inflammatory response to injury.

Transactivator of transcription fusion protein transduction causes membrane inversion.

The transactivator of transcription (TAT) protein transduction domain is an 11-amino acid positively charged peptide that has been shown to pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and have been shown to cross intracellular membranes as well. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Here, we report that TAT transduction causes membrane phospholipid rearrangement as evidenced by detection of phosphatidylserine on the outer surface of the cell membrane. Furthermore, these rearrangements can be blocked by positively charged polylysine, further implicating electrostatic interactions as a part of the mechanism. Neither apoptosis nor necrosis is induced in these cells after exposure to TAT. We conclude that the process of TAT.GFP transduction causes phosphatidylserine to translocate from the inner to the outer leaflet of the plasma membrane. These results provide insight into the mechanism of TAT protein transduction domain transduction.

Video-assisted ductal ligation in premature infants.

BACKGROUND: Video-assisted thoracic surgery has been shown to be a safe and effective method of closing the patent ductus arteriosus in infants and children. We have applied this technique in low birth weight premature infants and now report our experience. METHODS: Since 1996, we have used video-assisted thoracic surgery ligation as the treatment of choice for all patent ductus arteriosus, including 100 performed on premature infants (23 to 31 weeks' gestation, mean 25.6 weeks; 0.420 to 1.5 kg, mean 0.859 kg). A modification of our previously described technique was used with a three-port approach. All patients had some degree of symptoms of congestive failure with failure to wean from ventilatory support or oxygen dependency. Five infants had associated patent foramen, and 1 had a small ventricular septal defect. RESULTS: All 100 procedures were performed in the operating room. One infant was found to have a coarctation, and the procedure was aborted. The remaining 99 were successfully ligated, although three were converted to an open procedure (3%) because of coagulopathy, poor pulmonary compliance, or hemodynamic instability. There were no procedure-related deaths; however, 15 infants subsequently died of complications of prematurity, including enterocolitis, sepsis, and late respiratory failure. Six infants had chest tubes left in place for coagulopathy, effusions, suspected air leak, and existing empyema. There were six residual pneumothoraces, four requiring treatment. CONCLUSIONS: Video-assisted thoracic surgery is a safe and effective technique for patent ductus arteriosus ligation in premature infants, including those with very low and extremely low birth weight.

Targeting proteins to mitochondria using TAT.

Disorders of mitochondrial function cause significant human disease and suffering. To date, correction of these mitochondria defects has depended on biochemical approaches and has not been achieved via gene therapies. Using previously described fusion proteins containing the transactivator of transcription (TAT) region from the HIV virus and green fluorescent protein (GFP), with and without a mitochondrial targeting sequence (MTS) from mitochondrial malate dehydrogenase (mMDH), we have investigated transduction across mitochondrial membranes. Both TAT-GFP and TAT-mMDH-GFP fusion proteins are protected from externally added protease when incubated with isolated mitochondria. Furthermore, both TAT fusion proteins rapidly enter cultured cells and transduce into mitochondria. However, the MTS allows processing of the fusion protein and is necessary for persistence in mitochondria over time. Neither degradation of import receptors nor disruption of the mitochondrial membrane potential or pH gradient inhibits protein transduction of either fusion protein. Furthermore, when injected into pregnant mice, TAT-mMDH-GFP is detectable throughout fetal and neonatal pups. These results indicate that TAT fusion proteins are able to traverse mitochondrial membranes through mechanisms that do not involve the regular import pathway, and that the addition of a MTS allows persistence of the fusion protein within mitochondria. TAT-MTS fusion proteins may represent a viable option as potential mitochondrial protein therapies.

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.