Human dilated cardiomyopathy (DCM) is characterized by congestive heart failure and altered myocardial gene expression. Epigenetic changes, including DNA methylation, are implicated in the development of DCM but have not been studied extensively. Clinical human DCM and nonfailing control left ventricle samples were individually analyzed for DNA methylation and expressional changes. Expression microarrays were used to identify 393 overexpressed and 349 underexpressed genes in DCM (GEO accession number: GSE43435). Gene promoter microarrays were utilized for DNA methylation analysis, and the resulting data were analyzed by two different computational methods. In the first method, we utilized subtractive analysis of DNA methylation peak data to identify 158 gene promoters exhibiting DNA methylation changes that correlated with expression changes. In the second method, a two-stage approach combined a particle swarm optimization feature selection algorithm and a discriminant analysis via mixed integer programming classifier to identify differentially methylated gene promoters. This analysis identified 51 hypermethylated promoters and six hypomethylated promoters in DCM with 100% cross-validation accuracy in the group assignment. Generation of a composite list of genes identified by subtractive analysis and two-stage computation analysis revealed four genes that exhibited differential DNA methylation by both methods in addition to altered gene expression. Computationally identified genes (AURKB, BTNL9, CLDN5, and TK1) define a central set of differentially methylated gene promoters that are important in classifying DCM. These genes have no previously reported role in DCM. This study documents that rigorous computational analysis applied to microarray analysis of healthy and diseased human heart samples helps to define clinically relevant DNA methylation and expressional changes in DCM.

Transgenic mice (TG) were used to define mitochondrial oxidative stress and cardiomyopathy (CM) induced by zidovudine (AZT), an antiretroviral used to treat HIV/AIDS. Genetically engineered mice either depleted or overexpressed mitochondrial superoxide dismutase (SOD2 þ /À KOs and SOD2-OX, respectively) or expressed mitochondrially targeted catalase (mCAT). TGs and wild-type (WT) littermates were treated (oral AZT, 35 days). Cardiac mitochondrial H 2 O 2 , aconitase activity, histology and ultrastructure were analyzed. Left ventricle (LV) mass and LV end-diastolic dimension were determined echocardiographically. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Cardiac mitochondrial H 2 O 2 increased and aconitase was inactivated in SOD2 þ /À KOs, and cardiac dysfunction was worsened by AZT. Conversely, the cardiac function in SOD2-OX and mCAT hearts was protected. In SOD2-OX and mCAT TG hearts, mitochondrial H 2 O 2 , LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H 2 O 2 in SOD2 þ /À KO. Conversely, both SOD2-OX and mCAT TGs prevent or attenuate AZT-induced cardiac oxidative stress and LV dysfunction. As dysfunctional changes are ameliorated by decreasing and worsened by increasing H 2 O 2 abundance, oxidative stress from H 2 O 2 is crucial pathogenetically in AZT-induced mitochondrial CM.

Purpose To establish the phenotype of reproductive aging in our mouse model. To test the hypotheses that reproductive aging is associated with a decrease in mitochondrial abundance that could ultimately reflect dysfunction in oocytes. Methods Breeding studies were performed in young and aged female virgin wild type C57BL6J mice to establish their reproductive phenotype by measuring time to conception, litter size, and live birth per dam. Individual oocytes were analyzed for mtDNA content. Transmission electron microscopy was used to study ultrastructure of mitochondria in oocytes. Results Old females were found to have significantly prolonged time to conception and fewer surviving pups in their litters. Oocytes from old mice had 2.7-fold less mtDNA compared to younger controls (p<0.001; 95 % CI 2.1-3.5). Decrease in mitochondrial organelle abundance in old animal's oocytes was confirmed with transmission electron microscopy. Distinct morphological changes were noted in mitochondria, suggesting altered mitochondrial biogenesis in the old animals' oocytes. Conclusions Reproductive aging in mice is associated with reduced reproductive competence. Aging is associated with a significant decrease in number of mitochondria in oocytes. Our data support mitochondrial organelle loss and dysfunction in oocytes as a potential etiology for reproductive senescence.

Background: Sickle hemoglobin (HbS) under conditions of deoxygenation polymerizes to cause sickling of red blood cells (RBCs) and other rheological abnormalities. Voxelotor has been previously shown in a preclinical model of sickle cell disease (SCD) to increase HbS affinity to oxygen, thus reducing its polymerization and sickling with subsequent increase in the half-life of RBCs. We hypothesized that given this mechanism of action, we would observe improvements in RBC physiology in patients receiving voxelotor. In the Phase 3 GBT HOPE trial, the use of voxelotor in patients with SCD caused a significant reduction in markers of hemolysis and anemia. Ektacytometry is considered the gold standard to study deformability of RBCs with membrane protein disorders. The deformability of RBCs can be assessed using a defined value of shear stress with an increasing osmotic gradient (osmoscan) as well as with a newer technology to subject these cells to gradual deoxygenation (oxygenscan). Both assays can be measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA, RR Mechatronics, NL). In this pilot study, we analyzed samples from patients with SCD receiving voxelotor, before and 12 weeks after starting therapy to assess the benefits of voxelotor on RBC physiology. Methods: Our pilot study obtained whole blood from children ages 4-11 years with SCD, who were enrolled in the IRB approved GBT 440-007 clinical trial (NCT02850406; a study evaluating multiple doses of voxelotor at 1500 mg/day equivalent exposure to adults based on body weight) at Emory University/Children's Healthcare of Atlanta. All participants in this cohort continued their stable, optimal hydroxyurea dose during treatment with voxelotor. The below measurements were performed on the pre-dose and Week 12 visit samples. Deformability of RBCs was performed at a shear stress of 30 Pa and varying osmolality gradients (0-600 mOsm/Kg) for Osmoscan. Omin corresponds to the value of the hypotonic osmolality, where 50% of the cells hemolyze in an osmotic fragility assay and provides information on the initial surface area:volume ratio. Maximal deformability or Elongation Index (EImax) near isotonic osmolality informs us of the RBC cytoskeleton mechanics and Ohyper, the osmolality corresponding to 50% of the Elmax, provides information regarding the cytoplasmic viscosity. Oxygenscan was performed but under controlled deoxygenation using nitrogen. Point of Sickling (POS) is a point on the curve during deoxygenation when sickling begins, and EImin corresponds when sickle RBCs can least elongate. Oxygen dissociation curves were obtained using a HemOx Analyzer (TCS Scientific). Complete blood count parameters were determined on a clinical laboratory hematology analyzer (ADVIA, Siemens). Data was analyzed with Prism using a paired T-test. Results: Both pre-dose and Week 12 visit samples were available for 10 participants. Mean hemoglobin at baseline was 9.0 g/dL (7.6-10.0) and at 12 weeks, 10.3 g/dL (8.2-12.3). Six out of 10 participants had a hemoglobin response at Week 12 (defined as an increase in Hb from baseline by >1 g/dL), of which 5 had hemoglobin over 10 g/dL. Mean % change in percentage of reticulocytes was -17.0%. Significant improvement in EImax on osmoscan was noted at Week 12 (p=0.0147), suggesting RBCs were more deformable with improved cytoskeleton mechanics. In addition, oxygenscan curves shifted upwards towards normal with a significant increase in EImax (p=0.0347) and EImin (p=0.0079). These findings combined with a decrease in POS (p=0.0001) during deoxygenation suggests that at low oxygen tension, voxelotor treated RBCs were more deformable possibly from reduced HbS polymer inside these cells. Significant reductions in P50 (p=0.0011) and P20 (p=0.0001) with a left shift of the oxygen dissociation curve further demonstrates the effect of voxelotor on RBCs. Discussion: Voxelotor therapy in children with HbSS is associated with reductions in anemia and reticulocyte response, and recovery in RBC health as early as 12 weeks of treatment. Voxelotor's ability to inhibit HbS polymerization and RBC sickling is associated with specific modulation in red cell rheology at normoxic and deoxygenating conditions. Left shifted oxygen dissociation curves confirm voxelotor's ability to increase oxygen affinity. These findings suggest that voxelotor improves RBC deformability and anemia and delays the initiation of RBC sickling. Figure Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Baratz:Prolong Pharmacuticals: Honoraria; Global Blood Therapeutics: Research Funding. Pochron:Global Blood Therapeutics: Employment, Equity Ownership. Dixon:Global Blood Therapeutics: Employment, Equity Ownership. Tonda:Global Blood Therapeutics: Employment, Equity Ownership. Lehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Brown:Pfizer: Research Funding; Novartis, Inc: Research Funding; Imara, Inc: Consultancy, Research Funding; Global Blood Therapeutics, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Archer:AstraZeneca: Research Funding; Prolong Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding.