Although increased risk for adverse pregnancy outcomes has been well characterized in women with sickle cell disease (SCD), there remains unexplored value in the characterization of a preclinical model which could minimize human risk. This study aimed to characterize pregnancy outcomes in the SCD mouse model with emphasis on analogous clinical correlates and biological contributors. As such, we identified worsened outcomes including reduced litter sizes (haemoglobin HbSS (SS) 5.18 ± 1.25 embryos vs. haemoglobin HbAA (AA) 6.86 ± 1.51**), fetal weight (SS 0.38 ± 0.16 g vs. AA 0.49 ± 0.14 g**), viability of embryos (SS 20.00% interquartile range (IQR) 33.33 vs. AA 100.00% IQR 0.0***) and maternal mortality (SS 7.14% (2/28) vs. AA 0.00% (0/27) odds ratio (OR) = 4.82 ns). We further noted a significant reduction in vascular density and impaired uterine and umbilical artery blood flow within SCD placentae. Assessments of soluble growth factors revealed evidence of angiogenic dysregulation but maintained limited translational utility due to the multiparous nature of mouse pregnancy. These results serve as a cornerstone characterization of pregnancy outcomes in the SCD mouse model while highlighting the implications of placental vascular insufficiency. They further demonstrate both the utility and limitations of the model, emphasizing the need for continued clinical assessment.

Despite being the first genetic disease described, sickle cell disease (SCD) continues to result in severe complications. Of these complications, acute chest syndrome (ACS), a form of acute lung injury, leads all-cause mortality. However, the pathophysiology of ACS remains incompletely understood, resulting in patients with ACS receiving only supportive measures. Here, we found that ACS is accompanied by activation of the complement pathway, an evolutionarily ancient innate immune system responsible for eliminating microbes. Using a well-defined preclinical model of SCD, hemolysis, a precursor of ACS, not only induced ACS but also drove robust complement activation. Artificial activation of complement alone similarly induced ACS, whereas genetic removal or pharmacological inhibition of complement rendered SCD mice resistant to ACS even after induction of hemolysis. These results demonstrate that complement drives ACS, establishing a link between SCD and this ancient form of immunity that provides an opportunity for targeted treatment of this complication.

Background: Despite being the first genetic disease described, sickle cell disease (SCD) continues to afflict millions of individuals worldwide. Patients with SCD can experience severe complications, including vasoocclusive crisis (VOC), which can progress to acute chest syndrome (ACS), a form of acute lung injury. While VOC can cause significant morbidity, ACS is the leading cause of mortality in patients with SCD. Despite the devastating consequences of ACS, no interventions exist that directly treat this complication. As a result, when patients present with ACS, options often remain limited to supportive care. The lack of optimal treatment options for patients with SCD experiencing ACS directly contributes to their morbidity and mortality. Using a combined approach of prospective observational studies in patients with SCD that experienced ACS and a novel preclinical model specifically designed to define the role of complement in SCD pathophysiology, we tested the hypothesis that complement plays a central role in the pathophysiology of ACS. Methods: Samples from patients with SCD experiencing ACS were compared to baseline measurements for the same patient obtained at outpatient follow up 4-6 weeks later. ELISA based assays were used to measure Ba, Bb, C3a, C5a, C5b-9 (membrane attack complex (MAC)) in each sample. SCD mice (HbSS) and control mice (HbAA) were injected with 7.5 units of cobra venom factor (CVF), a potent complement activator, followed by evaluation of hematocrit (Hct) measured by a Sysmex veterinary hematology analyzer, free heme by calorimetric assay, and complement deposition on red blood cells (RBCs) by flow cytometry or pulmonary endothelial cells by confocal analysis. Respiratory rate and O2 saturation were measured using a MouseOx pulse oximeter. HbSS or HbAA mice were crossed with complement component 3 (C3) knock out (KO) mice to generate HbSS x C3 KO and HbAA x C3 KO mice. Hemin (70 to 210 mmol/kg concentration) or CVF (7.5 units/mouse) were injected into HbSS, HbAA, HbSS x C3 KO or HbAA x C3 KO followed by evaluation of hemolysis, C3 deposition and pulmonary function. A one-way ANOVA with a Tukey's post hoc with a p value <0.05 was considered significant. Results: During ACS in patients, significant elevations in Ba and Bb, complement activation products unique to the alternative pathway, and downstream activation of complement, including C3a, C5a and MAC, were observed (p<0.0001). Changes in complement activation were accompanied by a drop in hemoglobin during ACS when compared to baseline (p<0.001), suggesting that complement activation results in hemolysis. To formally test this, the impact of complement activation in a preclinical model of SCD was defined. Injection of CVF or hemin resulted in rapid C3 deposition on RBCs and pulmonary endothelium in HbSS, but not HbAA, recipients. In addition, CVF or hemin injection was accompanied by increased hemolysis and ACS manifested by increased respiratory rate, drop in oxygen saturation and death within 2 hours in the majority of recipients (p<0.001). In contrast, control Hb AA mice were unaffected by CVF or hemin injection. Given the role of free heme in the development of ACS, we next defined the role of complement in hemin-induced lung injury. To accomplish this, SCD mice were crossed with C3 KO mice to generate HbSS x C3 KO. HbSS x C3 KO mice displayed a mild increase in Hb values at baseline (p<0.01), suggesting a role of C3 even in steady-state SCD-related hemolysis. Equally important, injection of HbSS x C3 KO mice with CVF or hemin failed to result in the same levels of C3 deposition, hemolysis, compromised pulmonary function, or mortality (p<0.001) as HbSS recipients. Discussion: These results demonstrate a critical role for C3 in the development of acute lung injury in SCD. The sensitivity of HbSS RBCs to complement-induced hemolysis and C3 deposition observed following hemin injection in SCD recipients suggests that C3 plays a key role in decreased hematocrits and pulmonary injury that accompanies ACS. As treatment of ACS largely relies on supportive care, these results suggest that approaches aimed at targeting complement activation may represent a useful prophylactic or treatment strategy for ACS. In doing so, these results hold promise in providing a more direct approach at reducing one of the most severe complications of patients with SCD.

Abstract Introduction: Sickle haemoglobin (HbS) polymerisation perturbs red blood cell (RBC) rheology and drives sickle cell disease (SCD) pathophysiology. Voxelotor is an HbS polymerisation inhibitor that increases haemoglobin (Hb)–oxygen affinity. Methods/Results: In this 48‐week, prospective, single‐centre translational study, 10 children aged 4–11 years with SCD were treated with voxelotor. Improvements in RBC deformability were observed using osmotic/oxygen gradient ektacytometry, with increases in minimal and maximal elongation index and reductions in point of sickling. Increased Hb and reduced markers of haemolysis were also observed. Conclusion: These findings suggest that voxelotor treatment is associated with reduced RBC sickling and haemolysis in children with SCD.

Introduction: Sickle Cell Disease (SCD) is a group of inherited red blood cell disorders commonly characterized by abnormal hemoglobin production resulting in the deformation of red blood cells into a rigid, sickle-shape under conditions of hypoxia, acidosis, and increased physiological stress such as pregnancy. Pregnancy in SCD severely increases the likelihood of obstetric complications including veno-thromboembolism, preterm labor, intrauterine growth restriction, and maternal and fetal mortality. During placental development, the impact of SCD becomes evident as vaso-occlusion and vasculopathy resulting from sickling of red blood cells contribute to hemolysis, endothelial dysfunction, pro-inflammatory responses, and increased cellular adhesion. These mechanisms lead to diminished placental health and impaired nutrient and oxygen exchange to the fetus. Through our previous studies, we have identified worsened pregnancy outcomes in the Townes humanized mouse model of SCD. This study now aims to characterize the ramifications of SCD on the placenta using the same translational model. Methods: Townes SCD (SS) females underwent timed breeding with healthy (AA) males along with AA male and female breeding pairs as controls. At the end of gestation (18.5 days post conception (dpc)) dams were sacrificed and placentas collected and processed for histology. Frozen sections underwent appropriate staining (H&E, PAS, Masson's trichrome) and were digitized and coded for blind assessment by a pathologist. Identified pathologies were scored for severity using the scale 0.0 - Absent, 1.0 - Mild, 2.0 - Moderate, and 4.0 - Severe. For all studies we analyzed one (1) placenta per litter for seven (7) SS and seven (7) AA dams. Data is reported as mean ± SD and p-values were calculated with α = 0.05 and significant threshold set at p < 0.05 denoted by (*). Highly significant p-values were further denoted as: p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****); ns = not significant. Results: When compared to healthy controls, placentas from mice with SCD display significant evidence of vascular trauma including maternal and fetal vessel ectasia (AA 1.3 ± 1.1 vs SS 2.6 ± 0.8**), fibrosis (AA 0.4 ± 1.1 vs SS 1.9 ± 1.2**), thrombosis (AA 0.4 ± 0.8 vs SS 1.6 ± 0.9*), hemorrhage (AA 1.1 ± 1.1 vs SS 2.9 ± 1.4*), acute necrosis (AA 0.3 ± 1.4 vs SS 1.7 ± 1.5*), and calcification (AA 0.6 ± 0.6 vs SS 1.6 ± 0.6**). Furthermore, SCD placentas displayed irregularities in overall structure such as decreased labyrinth area (AA 55.6 ± 8.6% vs SS 44.7 ± 6.0%*), placental architectural loss (AA 0.4 ± 1.4 vs SS 2.9 ± 1.3****), and invasion of glycogenated cells (AA 0.4 ± 1.2 vs SS 1.7 ± 0.9*) and spongiotrophoblasts (AA 1.3 ± 0.9 vs SS 2.1 ± 0.0*) into the labyrinth acting to damage the placenta's ability to effectively participate in maternal-fetal nutrient, gas, and waste exchange. Additionally, placentas from mice with SCD displayed an increased number of decidual natural killer cells which are cytotoxic to the maternal-fetal interface and implicated in preterm delivery (Peak Section Count AA 5.6 ± 2.8 vs SS 9.1 ± 3.3**) Conclusion: With the growing adoption of improved care plans along with increased availability of interventions such as hydroxyurea, blood transfusion therapy, and recent anti-sickling agents the overall health and survival of individuals with SCD has improved significantly, allowing more women to reach sexual maturity and consider reproduction. It is the objective of our investigations to address the severe obstetric disparities faced by these women to ensure a safer childbearing experience. This study helps to further characterize pregnancy in the humanized mouse model of SCD by identifying placental pathologies which likely contribute to worsened maternal and fetal outcomes. These abnormalities are highly reflective of the systemic inflammatory and ischemic environment of SCD and highlight the unique milieu's ability to compromise the integrity and functionality of the placenta. We believe these studies establish the importance of the maternal-fetal interface in overall pregnancy outcomes in SCD and support continued investigation of placental insufficiency in both clinical and translational applications. We further believe these results support the utility of the Townes mouse model in preclinical investigations of pregnancy in SCD, a condition where clinical vulnerabilities remain high.

Sickle cell disease (SCD) is an inherited hemolytic anemia due to a single point mutation in the gene encoding for the beta-globin subunit of hemoglobin (Hgb) and resulting in the production of hemoglobin S (HbS). The clinical manifestations of SCD are due to HbS polymerization under low oxygen conditions, which causes RBC sickling and intravascular hemolysis, leading to anemia, expansion of erythropoiesis, inflammation, vaso-occlusion, and eventually, organ failure. Polymerization of HbS is highly dependent on HbS concentration within the RBC. Case reports and observational studies showed that iron deficiency in patients with SCD is associated with a reduction of HbS concentration, intravascular hemolysis, and vaso-occlusive crises (Haddy, 1982; Lionnet, 2012; Padaro, 2019). Iron restriction causing a reduction of HbS concentration in RBCs may therefore be a viable therapeutic strategy for patients with SCD. Hepcidin is a master regulator of iron homeostasis. The expression of hepcidin is downregulated by the transmembrane serine protease 6 (TMPRSS6) through the HJV/BMP/SMAD signaling pathway. DISC-3405 is a humanized monoclonal antibody blocking the biological action of TMPRSS6 with subsequent upregulation of hepcidin, leading to decreased iron absorption by enterocytes and decreased iron release from stores. In preclinical studies, the murine analog of DISC-3405, r4K12B, significantly increased hepcidin production, suppressed serum iron levels, and demonstrated efficacy in mouse models of beta-thalassemia and polycythemia vera (Chen, 2023; Chen, 2021). DISC-3405 is currently being evaluated in a Phase 1 study of healthy volunteers (NCT06050915). In this study, we tested the efficacy of r4K12B in the Townes mouse model of SCD. r4K12B was intraperitoneally administered to homozygous SS mice at 2 dose levels (3 and 10 mg/kg) weekly for 8 weeks. A vehicle treatment group was included as a control. At the end of the study, complete blood count, bilirubin, lactate dehydrogenase (LDH), and spleen index were measured. While the dose of 3 mg/kg of r4K12B did not have a significant effect in the Townes mice, the 10 mg/kg dose showed greater evidence of iron restriction and decreased hemolysis. Treatment with 10 mg/kg of r4K12B showed a significant reduction in the cellular hemoglobin concentration mean (CHCM) from 25.1±0.7 g/dL in vehicle-treated mice to 24.2±0.9 g/dL (p=0.029) in 10 mg/kg rK12B-treated mice. r4K12B treatment at 10 mg/kg resulted in a significant decrease in hemolysis markers. LDH levels decreased from 2054±525 U/L in vehicle-treated mice to 1076±910 U/L in 10 mg/kg r4K12B-treated mice, and total bilirubin decreased from 2.6 ± 0.2 mg/dL in vehicle-treated mice to 1.9±0.3 mg/dL in 10 mg/kg r4K12B-treated mice. A significant decrease was also observed for Hgb (6.8±1.3 g/dL vs 5.4±0.8 g/dL), hematocrit (HCT) (32.4±5.7% vs 26.8±3.6%), mean corpuscular volume (MCV) (47.0±3.9 fL vs 39.6±5.9 fL), and mean corpuscular hemoglobin (MCH) (10.1±0.5 pg vs 8.0±1.0 pg) in vehicle-treated mice compared to r4K12B-treated mice, respectively. Importantly, no changes in RBC numbers were observed in mice treated with vehicle compared to the r4K12B-treated group, suggesting that the observed iron-restricted erythropoiesis was balanced by decreased RBC hemolysis. Importantly, r4K12B treatment at 10 mg/kg significantly decreased leukocytosis, with white blood cell numbers decreasing from 20.8±7.8 x 109/L in the vehicle group to 10.7±6.8 x 109/L in the treatment group, suggesting decreased inflammation following treatment. r4K12B treatment at 10 mg/kg significantly reduced the spleen index compared to the vehicle group, indicating decreased extramedullary erythropoiesis. Taken together, these results suggest that iron restriction, through inhibition of TMPRSS6, may provide therapeutic benefits to SCD patients by reducing HbS concentration within the RBCs. References: Chen B, Wang J, Zheng B, et al. Blood. 2021;138(Suppl 1):941. Chen B, Wang J, Huang L, et al. Blood. 2023;142(Suppl 1):3837. Haddy TB, Castro O. Arch Intern Med. 1982;142(9):1621-1624. Lionnet F, Hammoudi N, Stojanovic KS, et al. Haematologica. 2012;97(8):1136-1141. Padaro E, Kueviakoe IMD, Agbétiafa K, et al. Med Sante Trop. 2019;29(1):106-107.

Background: Sickle cell disease (SCD) is a group of hereditary disorders commonly associated with the distortion of red blood cells (RBC) at low oxygen levels. It is characterized by complications such as vaso-occlusion, anemia, and hemolysis. These complications are lifelong and become extremely relevant during periods of increased physiological stress such as pregnancy. Pregnancy in sickle cell disease (SCD) is associated with an increased risk of complications including, pre-term delivery, deep vein thrombosis, intra-uterine growth restriction, pre-eclampsia, and intra-uterine death. Recent meta-analyses have shown that women with SCD are at 26x greater risk for maternal mortality than their healthy counterparts; a figure which, despite the adoption of improved care plans, has shown little to no significant improvement over the last two decades. While impaired vascular maintenance underlies multiple complications of pregnancy outside of sickle cell disease, it is especially relevant to, and characteristic of sickle cell-related pathologies at baseline and is highly likely to be exacerbated as pregnancy proceeds. Through this study we aimed to characterize the effects of SCD-related vasculopathy on pregnancy outcomes and overall placental health using the Townes humanized mouse model of SCD. Methods: SS (sickle hemoglobin) and AA (normal hemoglobin) mice underwent timed breeding to produce pregnant dams. These dams were housed under normal conditions until 17.5 days post conception (dpc), at which point they began undergoing procedures for analysis including 24-hour urine collection, high-resolution ultrasound analysis (18.5dpc), and post-mortem analysis of pregnancy outcomes and tissue collection (18.5dpc). Included in this report are gross pregnancy outcomes, histological assessments of placental tissue, and reports from ultrasound analysis including 3-D volume measures, placental vascularization renders, and laser-Doppler assessment of uterine and umbilical arteries. Continuing research efforts will further utilize tissues from this study to evaluate the biochemical interactions influencing poor pregnancy outcomes related to SCD. Results: These studies show that the SCD mice recapitulate many similarly poor outcomes as women. We observed: reduced litter sizes (AA 6.9 ± 1.5 embryos vs SS 5.2 ± 1.2 embryos**), fetal weight (AA 0.49 ± 0.14g vs SS 0.38 ± 0.16g*), viability of embryos (AA 100.0% IQR 0.0 vs SS 20.0% IQR 33.3****), and maternal mortality (AA 0.0% vs SS 8.7%, OR = 4.3 ns). In assessing placental histology, we identified evidence of vasculopathy common to end organ damage in SCD such as increased presence of avascular areas, reduced relative labyrinth area (AA 55.6 ± 8.6% vs SS 44.7 ± 6.0%*) and reduced vascular density (AA 38.9 ± 6.2% vs SS 34.4 ± 7.0%*) which are likely responsible for worsening outcomes. Additional pathologies include increased occurrence of placental ectasia, calcification of the labyrinth, vascular thrombi, and perivascular fibrosis when compared to controls. Further, ultrasound analysis revealed several measures of placental insufficiency amongst SS mice that were significantly impaired including uterine blood flow and an overall reduction in placental vascularization and oxygenation. Conclusions: SCD mice demonstrate many similar pregnancy outcomes as humans, allowing for increased translational application. We've shown these outcomes to be severe and largely driven by vascular impairment specifically within the placenta, the main maternal-fetal interface. This study serves as the groundwork for future studies aimed at investigating the underlying mechanisms of these outcomes while also establishing baseline measures and novel methods of approach towards the broader goal of improving obstetric disparities faced by women with SCD.

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.

Cardiomyopathy (CM) is an intrinsic weakening of myocardium with contractile dysfunction and congestive heart failure (CHF). CHF has been postulated to result from decreased mitochondrial energy production and oxidative stress. Effects of decreased mitochondrial oxygen consumption also can accelerate with aging. We previously showed DNA methylation changes in human hearts with CM. This was associated with mitochondrial DNA depletion, being another molecular marker of CM. We examined the relationship between mitochondrial dysfunction and cardiac epigenetic DNA methylation changes in both young and old mice. We used genetically engineered C57Bl/6 mice transgenic for a cardiac-specific mutant of the mitochondrial polymerase-γ (termed Y955C). Y955C mice undergo left ventricular hypertrophy (LVH) at a young age (∼ 94 days old), and LVH decompensated to CHF at old age (∼ 255 days old). Results found 95 genes differentially expressed as a result of Y955C expression, while 4,452 genes were differentially expressed as a result of aging hearts. Moreover, cardiac DNA methylation patterns differed between Y955C (4,506 peaks with 68.5% hypomethylation) and aged hearts (73,286 peaks with 80.2% hypomethylated). Correlatively, of the 95 Y955C-dependent differentially expressed genes, 30 genes (31.6%) also displayed differential DNA methylation; in the 4,452 age-dependent differentially expressed genes, 342 genes (7.7%) displayed associated DNA methylation changes. Both Y955C and aging demonstrated significant enrichment of CACGTG-associated E-box motifs in differentially methylated regions. Cardiac mitochondrial polymerase dysfunction alters nuclear DNA methylation. Furthermore, aging causes a robust change in cardiac DNA methylation that is partially associated with mitochondrial polymerase dysfunction.

Methamphetamine (METH) is a widely abused illicit drug that is highly addictive and affects cardiac metabolism and contraction. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 mice. Parallel DNA methylation changes were identified using tiled DNA microarrays to implicate epigenetic reprogramming and highlight long‐term cardiac impact of METH. Gene expression arrays identified 485 genes that were differentially expressed following METH (expression fold change>1.5, p<0.05). Using pathway enrichment analysis, families relating to calcium signaling and contractility were altered in the LV by METH. With DNA methylation analysis, 10.6% of probed regions of LV DNA exhibited significant changes following METH exposure; nearly 75% of those changes were hypomethylation. By comparing gene expression and DNA methylation changes, 27 genes revealed differences by both methods of analysis. Among these, only the promoter for CACNA1C, the gene encoding L‐type calcium channel Cav1.2, was hypomethylated by METH, and CACNA1C gene expression was increased by METH on microarray. For confirmation, quantitative PCR verified Cav1.2 LV mRNA increased due to METH. Correlative immunoblots revealed a 3.5‐fold increase in Cav1.2 protein abundance in METH LVs compared to LVs of vehicle controls. Histopathologically, contraction band necrosis was abundant in METH LVs. These results implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH and suggest possible mechanisms for METH cardiac toxicity in humans.

MDMA (ecstasy) is an illicit drug that stimulates monoamine neurotransmitter release and inhibits reuptake. MDMA's acute cardiotoxicity includes tachycardia and arrhythmia which are associated with cardiomyopathy. MDMA acute cardiotoxicity has been explored, but neither long-term MDMA cardiac pathological changes nor epigenetic changes have been evaluated. Microarray analyses were employed to identify cardiac gene expression changes and epigenetic DNA methylation changes. To identify permanent MDMA-induced pathogenetic changes, mice received daily 10- or 35-day MDMA, or daily 10-day MDMA followed by 25-day saline washout (10 + 25 days). MDMA treatment caused differential gene expression (p < .05, fold change >1.5) in 752 genes following 10 days, 558 genes following 35 days, and 113 genes following 10-day MDMA + 25-day saline washout. Changes in MAPK and circadian rhythm gene expression were identified as early as 10 days. After 35 days, circadian rhythm genes (Per3, CLOCK, ARNTL, and NPAS2) persisted to be differentially expressed. MDMA caused DNA hypermethylation and hypomethylation that was independent of gene expression; hypermethylation of genes was found to be 71% at 10 days, 68% at 35 days, and 91% at 10 + 25 days washout. Differential gene expression paralleled DNA methylation in 22% of genes at 10-day treatment, 17% at 35 days, and 48% at 10 + 25 days washout. We show here that MDMA induced cardiac epigenetic changes in DNA methylation where hypermethylation predominated. Moreover, MDMA induced gene expression of key elements of circadian rhythm regulatory genes. This suggests a fundamental organism-level event to explain some of the etiologies of MDMA dysfunction in the heart.

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change>1.5, p<0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects.

Mitochondrial dysfunction causes oxidative stress and cardiomyopathy. Oxidative stress also is a side effect of dideoxynucleoside antiretrovirals (NRTI) and is observed in NRTI-induced cardiomyopathy. We show here that treatment with the NRTI AZT {1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione} modulates cardiac gene expression epigenetically through production of mitochondrially derived reactive oxygen species. Transgenic mice with ubiquitous expression of mitochondrially targeted catalase (MCAT) and C57Bl/6 wild-type mice littermates (WT) were administered AZT (0.22 mg/day po, 35 days), and cardiac DNA and mRNA were isolated. In AZT-treated WT, 95 cardiac genes were differentially expressed compared with vehicle-treated WTs. When MCAT mice were treated with AZT, each of those 95 genes reverted toward the expression of vehicle-treated WTs. In AZT-treated WT hearts, Mthfr [5,10-methylenetetrahydrofolate reductase; a critical enzyme in synthesis of methionine cycle intermediates including S-adenosylmethionine (SAM)], was overexpressed. Steady-state abundance of SAM in cardiac extracts from AZT-treated MCAT mice increased 60% above that of vehicle-treated MCAT. No such change occurred in WT. AZT caused hypermethylation (47%) and hypomethylation (53%) of differentially methylated DNA regions in WT cardiac DNA. AZT-treated MCAT heart DNA exhibited greater hypermethylation (91%) and less hypomethylation (9%) compared with vehicle-treated MCAT controls. The gene encoding protein kinase C-α displayed multifocal epigenetic regulation caused by oxidative stress. Results show that mitochondrially derived oxidative stress in the heart hinders cardiac DNA methylation, alters steady-state abundance of SAM, alters cardiac gene expression, and promotes characteristic pathophysiological changes of cardiomyopathy. This mechanism for NRTI toxicity offers insight into long-term side effects from these commonly used antiviral agents.

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.

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle ( n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls ( n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435 ). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.

A mitochondrial matrix-specific p53 construct (termed p53-290) in HepG2 cells was utilized to determine the impact of p53 in the mitochondrial matrix following oxidative stress. H2O2 exposure reduced cellular proliferation similarly in both p53-290 and vector cells, and p53-290 cells demonstrating decreased cell viability at 1 mM H2O2 (~ 85% viable). Mitochondrial DNA (mtDNA) abundance was decreased in a dose-dependent manner in p53-290 cells while no change was observed in vector cells. Oximetric analysis revealed reduced maximal respiration and reserve capacity in p53-290 cells. Our results demonstrate that mitochondrial matrix p53 sensitizes cells to oxidative stress by reducing mtDNA abundance and mitochondrial function.