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INTRODUCTION The contribution of coagulation activation to the pathogenesis of sickle cell disease (SCD) remains incompletely defined. We evaluated the efficacy and safety of rivaroxaban, an oral direct factor Xa inhibitor, in subjects with sickle cell anemia. MATERIALS AND METHODS In this pilot, single-center, randomized, double-blind, placebo-controlled, crossover study, eligible subjects with sickle cell anemia received rivaroxaban or placebo. The effect of rivaroxaban on coagulation activation, endothelial activation, inflammation, and microvascular blood flow was evaluated. RESULTS Fourteen patients (HbSS - 14; females - 9) with mean age of 38 ± 10.6 years were randomized to receive rivaroxaban 20 mg daily or placebo for 4 weeks and, following a 2-week washout phase, were "crossed-over" to the treatment arm opposite to which they were initially assigned. Mean adherence to treatment with rivaroxaban, assessed by pill counts, was 85.6% in the first treatment period and 93.6% in the second period. Treatment with rivaroxaban resulted in a decrease from baseline of thrombin-antithrombin complex versus placebo (-34.4 ug/L [95% CI: -69.4, 0.53] vs. 0.35 ug/L [95% CI: -3.8, 4.5], p = .08), but the difference was not statistically significant. No significant differences were observed in changes from baseline of D-dimer, inflammatory, and endothelial activation markers or measures of microvascular blood flow. Rivaroxaban was well tolerated. CONCLUSIONS Rivaroxaban was safe but did not significantly decrease coagulation activation, endothelial activation, or inflammation. Rivaroxaban did not improve microvascular blood flow. Adequately powered studies are required to further evaluate the efficacy of rivaroxaban in SCD. Clinicaltrials.gov Identifier: NCT02072668.

Introduction : Chronic kidney disease is common in patients with sickle cell disease (SCD) and is associated with increased mortality. As albuminuria and changes in estimated glomerular filtration rate (eGFR) occur well after substantial structural and functional tissue damage have ensued, biomarkers that predict the development of CKD are needed. Metabolite profiling and the bioinformatics tools to study them may aid in the discovery of novel associations and uncover pathophysiological pathways in SCD-related glomerulopathy. In this study, we have performed an exploratory study to identify urinary biomarkers, which may be predictive of kidney damage in children with SCD. Methods: Consecutive patients with SCD seen at a pediatric chronic care clinic at Kamuzu Central Hospital in Lilongwe, Malawi, between January and May 2015 were evaluated. Patient urine samples were analyzed for metabolites using NMR-based methodologies. Spot urine measurements for albumin/creatinine ratio (UACR) were required to ascertain the degree of albuminuria (normal albuminuria [UACR < 30 mg/ g creatinine] or albuminuria [UACR ≥ 30 mg/ g creatinine]). Descriptive statistics (mean and standard deviation for continuous variables, and count and percentage for discrete variables) were calculated for each of the baseline variables. T-test and Fisher's exact test were performed to compare the baseline measurements between those with and without albuminuria for continuous and discrete variables, respectively. Metabolite measurements were Pareto scaled and quantile normalized. To detect differential metabolites between the normal albuminuria and albuminuria groups, Wilcoxon rank sum test was performed. Results: Spot urine samples were obtained in 92 children, 32 (46.4%) of whom were female, average age of 40.6 (±0.98) months, and 23 (25%) patients with normal albuminuria. The patients in both categories were generally similar (age, sex, WBC count, hemoglobin, bilirubin and serum creatinine), except for higher baseline levels of lactate dehydrogenase (p < 0.05) in patients in the albuminuria category. From 1078 bins, we deleted those with low measurements across the majority (>95%) of samples and applied the differential metabolite analysis to 723 bins. Among these bins, 43 metabolites were different in both albuminuria categories (p ≤ 0.05). Although none of the metabolites were significantly different after adjustment for multiple comparisons, the top list is preliminarily identified as ethylmalonic acid, an acidogenic and a metabotoxic branched fatty acid, as an important urinary metabolite differentiating the two groups. High levels of ethylmalonic acid are also found in the urine of patients with short-chain acyl-coenzyme A dehydrogenase deficiency, a fatty acid metabolism disorder. The results remain largely unchanged after important covariates, age, sex, WBC count, hemoglobin, lactate dehydrogenase, bilirubin and creatinine were adjusted. In addition, principal component analysis (PCA) and partial least square discrimination analysis (PLS-DA) were performed (Figures 1A and 1B). Despite the lack of distinct clusters on the score plots, 15 metabolites were identified as "important features" in PLS-DA, based on their variance of importance projection (VIP) score (Figure 2). Conclusion: Our exploratory study shows that urinary ethylmalonic acid is a potentially important biomarker for the early detection of kidney damage in children with SCD. More studies of larger patient populations with SCD are required to identify urine metabolites which may predict the development of albuminuria. Disclosures Key: UniQure BV: Research Funding. Ataga:Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Novartis: Consultancy, Honoraria; Modus Therapeutics: Honoraria; Bioverativ: Honoraria, Membership on an entity's Board of Directors or advisory committees.

BACKGROUND The pathogenesis of albuminuria in SCD remains incompletely understood. We evaluated the association of albuminuria with measures of endothelial function, and explored associations of both albuminuria and measures of endothelial function with selected biological variables (vascular endothelial growth factor [VEGF], endothelin-1 [ET-1], soluble fms-like tyrosine kinase-1 [sFLT-1], soluble vascular cell adhesion molecule-1 [soluble VCAM-1] and plasma hemoglobin). METHODS Spot urine measurements for albumin-creatinine ratio (UACR) and 24-hour urine protein were obtained. Endothelial function was assessed using brachial artery ultrasound with measurements of flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NTMD) and hyperemic velocity. RESULTS Twenty three subjects with varying degrees of albuminuria were evaluated. UACR was significantly correlated with FMD (ρ = -0.45, p = 0.031). In univariate analysis, UACR was correlated with VEGF (ρ = -0.49; 95% CI: -0.75 --0.1, p = 0.015), plasma hemoglobin (ρ = 0.50; 95% CI: 0.11-0.75, p = 0.013) and ET-1 (ρ = 0.40; 95% CI: -0.03-0.69, p = 0.06). Multivariable analysis showed significant associations of ET-1 (estimate: 455.1 [SE: 198.3], p = 0.02), VEGF (estimate: -1.1 [SE: 0.53], p = 0.04) and sFLT-1 (estimate: -1.14 [SE: 0.49], p = 0.02) with UACR. Only ET-1 (estimate: -8.03 [SE: 3.87], p = 0.04) was significantly associated with FMD in multivariable analyses. Finally, UACR was correlated with both 24-hour urine protein (ρ = 0.90, p < 0.0001) and urine aliquots for albumin-creatinine ratio obtained from the 24-hour urine collection (ρ = 0.97, p < 0.0001). CONCLUSION This study provides more definitive evidence for the association of albuminuria with endothelial dysfunction in SCD. Elevated circulating levels of ET-1 may contribute to SCD-related glomerulopathy by mediating endothelial dysfunction.

Background Glomerulopathy is an increasingly identified complication in young patients with sickle cell disease (SCD). Hyperfiltration and albuminuria followed by declining glomerular filtration rates and eventual end-stage renal disease (ESRD) is assumed to be the typical progression of glomerular disease. There are only a few reported biomarkers to identify early-stage renal disease in SCD. Procedures We detail the renal profile of 101 children with SCD in Malawi and propose a novel urinary biomarker for the identification of early renal disease. Results Among children with sickle cell anemia, 24.8% had a urine albumin-creatinine ratio of 30 mg/g or above. In univariate analysis, only patients with higher urinary nephrin, a urinary marker of glomerular injury, had significantly greater odds of having albuminuria. In multivariable analysis, nephrin remained significantly associated with albuminuria. A nephrin-creatinine ratio (NCR) cut-point of 622 ng/mg, the 50th percentile, was associated with a 45.8 times greater odds of having albuminuria in children with nephrinuria above this value. Further analysis revealed this urinary NCR cut-point to have 96% sensitivity, 64% specificity, 47% positive predictive value, and 98% negative predictive value for the presence of albuminuria. Conclusions These data suggest that a substantial number of children with SCD in Malawi have renal disease and could be at risk for worsening nephropathy and ESRD as they age. Our data suggest that urinary nephrin could be utilized as an early marker of glomerular disease in SCD.

Vinculin is a ubiquitously expressed multiliganded protein that links the actin cytoskeleton to the cell membrane. In myocytes, it is localized in protein complexes which anchor the contractile apparatus to the sarcolemma. Its function in the myocardium remains poorly understood. Therefore, we developed a mouse model with cardiac-myocyte-specific inactivation of the vinculin (Vcl) gene by using Cre-loxP technology. Sudden death was found in 49% of the knockout (cVclKO) mice younger than 3 months of age despite preservation of contractile function. Conscious telemetry documented ventricular tachycardia as the cause of sudden death, while defective myocardial conduction was detected by optical mapping. cVclKO mice that survived through the vulnerable period of sudden death developed dilated cardiomyopathy and died before 6 months of age. Prior to the onset of cardiac dysfunction, ultrastructural analysis of cVclKO heart tissue showed abnormal adherens junctions with dissolution of the intercalated disc structure, expression of the junctional proteins cadherin and beta1D integrin were reduced, and the gap junction protein connexin 43 was mislocalized to the lateral myocyte border. This is the first report of tissue-specific inactivation of the Vcl gene and shows that it is required for preservation of normal cell-cell and cell-matrix adhesive structures.

The concept of myocardial remodeling links an initial pathological insult to a progressive geometric change of the ventricle. Currently, our concepts of the remodeling process have evolved to include not only changes in ventricular size and shape, but cellular and molecular remodeling, particularly as the ventricle evolves towards failure. In recent years, much attention has focused on the role of cell-extracellular matrix (ECM) connections in this process. In this review, we will specifically delineate how cell membrane-linked molecules of three classes: integrins, membrane-type matrix metalloproteinases, and ADAMs (A Disintegrin And Metalloproteinase) might play crucial roles in myocardial remodeling. These molecules are essential for cell-ECM adhesion, cell signaling, matrix modification, and proteolysis of surface receptors. Our goal is to put forth concepts on how they might interrelate to modulate the remodeling process in the heart.

Dietary copper (Cu) restriction leads to cardiac hypertrophy and failure in mice, and Cu repletion (CuR) reverses the hypertrophy and prevents the transition to heart failure. The present study was undertaken to determine changes in myocardial gene expression involved in Cu deficient (CuD) cardiomyopathy and its reversal by CuR. Analysis was performed on three groups of mice: 4-week-old CuD mice that exhibited signs of cardiac failure, their age-matched copper-adequate (CuA) controls, and the CuD mice that were re-fed adequate Cu for 2 weeks. Total RNA was isolated from hearts and subjected to cDNA microarray and real-time reverse transcription-polymerase chain reaction analysis. Dietary CuD caused a decrease in cardiac mRNA of β-MHC, L-type Ca2+ channel, K-dependent NCX, MMP-2, -8, and -13, NF-κB, and VEGF. The mRNA levels of ET-1, TGF-β, TNF-α, and procollagen-l-α1 and III-α1 were increased in the CuD cardiac tissue. Copper repletion resulted in cardiac mRNA levels of most of the genes examined returning to control levels, although the K-dependent NCX and MMP-2 values did not reach those of the CuA control. In addition, CuR caused an increase in β-MHC, L-type Ca2+channel, MMP-13 to levels surpassing those of CuA control, and a decrease in ET-1, and TNF-at mRNA levels. In summary, changes in gene expression of elements involved in contractility, Ca2+ cycling, and inflammation and fibrosis may account for the altered cardiac function found in CuD mice. The return to normal cardiac function by CuR may be a result of the favorable regression in gene expression of these critical components in myocardial tissue.

Copper Deficiency (CuD) leads to hypertrophic cardiomyopathy in various experimental models. The morphological, electrophysiological, and molecular aspects of this hypertrophy have been under investigation for a long time. However the transition from compensated hypertrophy to decompensated heart failure has not been investigated in the study of CuD. We set out to investigate the contractile and hemodynamic parameters of the CuD mouse heart and to determine whether heart failure follows hypertrophy in the CuD heart. Dams of FVB mice were fed CuD or copper-adequate (CuA) diet starting from the third day post delivery and the weanling pups were fed the same diet for a total period of 5 weeks (pre- and postweanling). At week 4, the functional parameters of the heart were analyzed using a surgical technique for catheterizing the left ventricle. A significant decrease in left ventricle systolic pressure was observed with no significant change in heart rate, and more importantly contractility as measured by the maximal rate of left ventricular pressure rise (+dP/dt) and decline (�dP/dt) were significantly depressed in the CuD mice. However, left ventricle end diastolic pressure was elevated, and relaxation was impaired in the CuD animals; the duration of relaxation was prolonged. In addition to significant changes in the basal level of cardiac function, CuD hearts had a blunted response to the stimulation of the -adrenergic agonist isoproterenol. Furthermore, morphological analysis revealed increased collagen accumulation in the CuD hearts along with lipid deposition. This study shows that CuD leads to systolic and diastolic dysfunction in association with histopathological changes, which are indices commonly used to diagnose congestive heart failure. Exp Biol Med 228:811–817, 2003

Sickle cell nephropathy (SCN) is a prevalent complication among adults with sickle cell disease (SCD) and has been observed in younger populations, suggesting potential early renal involvement in pediatric patients. Initial hyperfiltration and albuminuria followed by frank proteinuria, leading to declining GFR and eventual end stage renal disease is assumed to be the typical progression of SCN; however, few clinical biomarkers exist to identify early-stage renal disease. We describe the renal profile in 119 children with SCD at Kamuzu Central Hospital in Lilongwe, Malawi and propose a novel urinary biomarker for the identification of children with early renal disease. Among children with confirmed HbSS disease (females 47.9%; median age 9.0 years, IQR: 5, 11), 21.6% were found to have a urinary albumin to creatinine ratio (UACR) 30mg/g or above. Baseline laboratory and clinical parameters stratified by UACR are shown in Tables 1 and 2. Patients with increased levels of UACR were found to be significantly older, and have significantly elevated plasma levels of lactate dehydrogenase (LDH), total bilirubin, and indirect bilirubin when compared to those without albuminuria (p= 30). Urine nephrin was significantly associated with albuminuria (regression coefficient estimate: 0.00188, SE: 0.000571, p = 0.0010). Additional analysis using a nephrin cut-point of 293 pg/mL, the median value in the cohort, revealed a 17.8 times greater odds of having albuminuria in children with nephrinuria above this value. These data taken together suggests that a significant proportion of children with SCD in Malawi exhibit renal involvement early in life and could be at risk for worsening nephropathy and end-stage renal disease as they grow older. Our data further indicates that urinary nephrin could be utilized as an early marker of glomerular disease in SCN and possibly prompt earlier intervention in these children. The discordant association of albuminuria with clinical markers of hemolysis suggests that hemolysis may not play a substantial role in the pathogenesis of albuminuria in this population. Increased surveillance of children with SCD for renal complications can ultimately inform management strategies to improve outcomes and increase life expectancy among children with SCD. Disclosures No relevant conflicts of interest to declare.

Suicidal NETosis (Neutrophil Extracellular Traps) is a mode of cell death involving de-condensation of chromatin and fusion with cytoplasmic granule proteins to create a net-like structure for entrapping pathogenic elements. Despite its name, NETosis does not occur exclusively in neutrophils but rather in all myeloid cells of the immune system except dendritic cells. A growing list of diseases and conditions implicate NETs in their etiology and the progression of symptoms. These conditions include sepsis, cystic fibrosis, lupus nephritis, acute respiratory distress syndrome (ARDS) and deep vein thrombosis. There are several different quantitative methods to assess NETosis making comparative analysis across the field of NETosis research extremely challenging. To further define NETosis and facilitate new discoveries in this field, we have developed a novel assay that combines high-content microscopy with Convolutional Neural Networks (CNN). CNN is the revolutionary image recognition technology used in popular social media facial features recognition systems to 1) quantify the percentage of NETotic and non-NETotic nuclei in a cell population, 2) classify NETotic, necrotic and normal nuclei in a mixed population of neutrophils and 3) differentiate between two different NETotic nuclei generated by two of the most commonly used agonists in NETosis research, the PKC activator PMA and A23187, a Ca+2 ionophore. Furthermore we have optimized neutrophil isolation and culture conditions to yield reproducible results with low inter-and intra-assay variability. Image acquisition was performed on adherent neutrophils using the automated BD Pathway Bio-imaging Systems with autofocusing function that allows imaging of approximately 10% of a well in a 96-well plate. An algorithm was created for image segmentation and recognition of NETotic nuclei based on size, DNA staining intensity and pattern. Data generated by this semi-automated analysis system were used to train a CNN to count NETotic and non-NETotic nuclei. These methods allowed us to calculate the EC50 for PMA (2.2 nM) and A23187 (2.5 uM) and to differentiate between their distinct mechanisms of NETosis. CNNs are able to classify NETotic phenotypes, without the use of expensive antibodies or reagents. Our assay represents a crucial first step for standardizing and quantitating NETosis, which will allow better data interpretation across different labs and provide a potential screening tool for known and as yet undiscovered NETosis agonists. Disclosures No relevant conflicts of interest to declare.

Mice expressing the tetracycline transactivator (tTA) transcription factor driven by the rat α-myosin heavy chain promoter (α-MHC-tTA) are widely used to dissect the molecular mechanisms involved in cardiac development and disease. However, these α-MHC-tTA mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury in both in vitro and in vivo models in the absence of associated cardiac hypertrophy or remodeling. Cardiac function, as assessed by echocardiography, did not differ between α-MHC-tTA and control animals, and there were no noticeable differences observed between the two groups in HW/TL ratio or LV end-diastolic and end-systolic dimensions. Protection against ischemia/reperfusion injury was assessed using isolated perfused hearts where α-MHC-tTA mice had robust protection against ischemia/reperfusion injury which was not blocked by pharmacological inhibition of PI3Ks with LY294002. Furthermore, α-MHC-tTA mice subjected to coronary artery ligation exhibited significantly reduced infarct size compared to control animals. Our findings reveal that α-MHC-tTA transgenic mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury similar to cardiac pre- and post-conditioning effects. However, in contrast to classical pre- and post-conditioning, the α-MHC-tTA phenotype is not inhibited by the classic preconditioning inhibitor LY294002 suggesting involvement of a non-PI3K-AKT signaling pathway in this phenotype. Thus, further study of the α-MHC-tTA model may reveal novel molecular targets for therapeutic intervention during ischemic injury.

Introduction: Sickle cell disease (SCD) may be associated with iron overload following repeated RBC transfusions. In beta thalassemia major, a disease characterized by hemolysis and ineffective erythropoiesis, iron overload occurs due to both repeated RBC transfusions and hyperabsorption of iron. Hyperabsorption of iron occurs due to ineffective erythropoiesis and subsequent downregulation of hepcidin, the main regulator of systemic iron homeostasis. With the presence of a thalassemia gene, HbSβ-thalassemia patients may have an increased tendency to hyperabsorb iron compared to HbSS patients. The purpose of this exploratory study was to compare hepcidin levels in HbSS and HbSβ-thalassemia as well as the levels of GDF-15 and any associations between hepcidin and ferritin, other measures of iron balance, erythropoietin and markers of inflammation. Methods: Eligible patients with HbSS or HbSβ-thalassemia (HbSβ0-thalassemia or HbSβ+-thalassemia) were evaluated during routine clinic visits. Hepcidin, GDF -15 and erythropoietin were analyzed using commercially available ELISA kits, while plasma cytokines were analyzed by Luminex® multi-analyte profiling technology. Complete blood counts, serum iron, transferrin, transferrin saturation and ferritin, high-sensitivity CRP and routine chemistries were performed at the clinical laboratories of UNC Hospitals. Patients' clinical data, including SCD-related clinical complications and estimates of the number of lifetime RBC transfusions, were obtained at the time of evaluation combined with reviews of their medical records. The levels of hepcidin and GDF-15 were compared in HbSS and HbSβ-thalassemia (and SCD vs. healthy controls) using Wilcoxon rank sum tests, and the association of hepcidin and laboratory covariates was evaluated using Spearman correlation coefficient. Reported p-values are for individual tests, unadjusted for multiple comparisons. Results: Baseline demographic variables were similar in HbSS and HbSβ-thalassemia patients, but platelet counts, lactate dehydrogenase, total bilirubin, direct bilirubin, indirect bilirubin, aspartate transaminase and alkaline phosphatase were significantly higher in HbSS patients (Table 1). No significant differences were observed when hepcidin levels were compared between control subjects (N = 15) and SCD patients (N = 40) (25.2 ± 14.3 ng/mL vs. 19.5 ± 17.3 ng/mL, p = 0.13). In addition, no difference was observed in hepcidin levels (16.1 ± 11.4 ng/mL vs. 23.7 ± 22.2 ng/mL, p = 0.53) or GDF-15 levels (1635.1 ± 1357.7 pg/mL vs. 2051 ± 2385.4 pg/mL, p = 0.9) when HbSS patients were compared with HbSβ-thalassemia. Hepcidin was associated with ferritin (r= 0.57, p = 0.0001) and transferrin (r = -0.46, p = 0.0029) in SCD patients. However, no significant correlations were observed between hepcidin and erythropoietin, high sensitivity CRP, IL-6, IL-8, IL-10 and TNFα. Conclusion: No differences in hepcidin levels were observed in SCD patients compared with healthy controls, and in HbSS patients compared with HbSβ-thalassemia patients. We confirm the association of hepcidin with ferritin, but surprisingly find no association of hepcidin with IL-6. Table 1. Baseline Variables Variable HbSS (N = 22)Mean ± SD (or %) HbSβ-thalassemia (N = 18)Mean ± SD (or %) 95% CI for Mean Difference Age (years) 37.96 ± 12.7 36.4 ± 11.4 (-6.2,9.2) Gender (females) 15 (68.2) 12 (66.7) Weight (kg) 70.8 ± 15.1 74.7 ± 14.8 (-13.7,5.9) Transfusions 0 - 5 6 - 10 11 - 20 21 - 50 9 (23.1) 5 (12.8) 5 (12.8) 2 (5.1) 11 (28.2) 3 (7.7) 3 (7.7) 1 (2.6) White Blood Cell Count (× 109/L) 9.5 ± 3.1 9.4 ± 3.1 (-1.9,2.04) Hemoglobin(g/dL) 9.4 ± 1.7 10 ± 1.99 (-1.7,0.6) Platelet Count (× 109/L) 402.6 ± 134.9 310.1 ± 151.6 (1.8,183.1) Ferritin (ng/mL) 346.3 ± 406.9 174 ± 257.7 (-50.3,395) Reticulocyte Count (× 109/L) 233.5 ± 139.9 215.7 ± 138.9 (-71.8,107.6) Lactate Dehydrogenase (U/L) 954.3 ± 322.95 720.4 ± 174.2 (73.8,394) Total Bilirubin (mg/dL) 3.9 ± 3.1 1.4 ± 0.9 (1.2,3.9) Direct Bilirubin (mg/dL) 0.56 ± 0.5 0.3 ± 0.1 (0.04,0.48) Indirect Bilirubin (mg/dL) 3.3 ± 2.8 1.06 ± 0.8 (1.03,3.5) Aspartate Transaminase (U/L) 46.5 ± 18.8 32.8 ± 11.9 (3.4,23.9) Alanine Transaminase (U/L) 30.7 ± 13.5 28.7 ± 9.7 (-5.6,9.6) Alkaline Phosphatase (U/L) 110.6 ± 45.6 78.1 ± 24.7 (9.7,55.3) Creatinine (mg/dL) 0.67 ± 0.17 1.1 ± 1.25 (-1.07,0.18) Disclosures No relevant conflicts of interest to declare.

The dystrophin–glycoprotein complex is a large complex of membrane-associated proteins linking the cytoskeleton to the extracellular matrix in muscle. Transmembrane heterodimeric (αβ) integrins serve also as cellular adhesion molecules and mechanotransducers. In the animal model for Duchenne muscular dystrophy, the mdx mouse, loss of dystrophin causes more severe abnormalities in skeletal than in cardiac muscle. We hypothesized that ablation of cardiac myocyte integrins in the mdx background would lead to a severe cardiomyopathic phenotype. Mdx mice were crossed to ones with cardiac myocyte-specific deletion of β1 integrin (β1KO) to generate β1KO mdx . Unstressed β1KO mdx mice were viable and had normal cardiac function; however, high mortality was seen in peri- and postpartum females by 6 months of age, when severe myocardial necrosis and fibrosis and extensive dystrophic calcification was seen. Decreased ventricular function and blunted adrenergic responsiveness was found in the β1KO mdx mice compared with control (Lox/Lox, no Cre), β1KO, and mdx . Similarly, adult β1KO mdx males were more prone to isoproterenol-induced heart failure and death compared with control groups. Given the extensive calcification, we analyzed transcript levels of genes linked to fibrosis and calcification and found matrix γ-carboxyglutamic acid protein, decorin, periostin, and the osteoblast transcription factor Runx2/Cbfa1 significantly increased in β1KO mdx cardiac muscle. Our data show that combined deficiency of dystrophin and integrins in murine cardiac myocytes results in more severe cardiomyopathic changes in the stressed myocardium than reduction of either dystrophin or integrins alone and predisposes to myocardial calcification.

Background— Our previous studies showed that transgenic mice that overexpress cardiac-specific metallothionein (MT) are highly resistant to diabetes-induced cardiomyopathy. Zinc is the major metal that binds to MT under physiological conditions and is a potent inducer of MT. The present study therefore explored whether zinc supplementation can protect against diabetic cardiomyopathy through cardiac MT induction. Methods and Results— Diabetes was induced in mice (C57BL/6J strain) by a single injection of streptozotocin. Half were supplemented intraperitoneally with zinc sulfate (5 mg/kg) every other day for 3 months. After zinc supplementation, mice were maintained for 3 more months and then examined for cardiomyopathy by functional and morphological analysis. Significant increases in cardiac morphological impairment, fibrosis, and dysfunction were observed in diabetic mice but not in diabetic mice supplemented with zinc. Zinc supplementation also induced a significant increase in cardiac MT expression. The role of MT in cardiac protection by zinc supplementation was examined in cultured cardiac cells that were directly exposed to high levels of glucose (HG) and free fatty acid (FFA) (palmitate), treatment that mimics diabetic conditions. Cell survival rate was significantly decreased for cells exposed to HG/FFA but did not change for cells exposed to HG/FFA and pretreated with zinc or low-dose cadmium, each of which induces significant MT synthesis. When MT expression was silenced with the use of MT small-interfering RNA, the preventive effect of pretreatment with zinc or low-dose cadmium was abolished. Conclusions— These results suggest that the prevention of diabetic cardiomyopathy by zinc supplementation is predominantly mediated by an increase in cardiac MT.

PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on in vitro studies and its role in angiogenesis in vivo has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6-8 week old PAK1-/- and PAK1+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1-/- mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1-/- mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1-/- but not PAK1+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1-/- mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1-/- and PAK1+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.

Dietary copper (Cu) restriction leads to cardiac hypertrophy and failure in mice, and Cu repletion (CuR) reverses the hypertrophy and prevents the transition to heart failure. The present study was undertaken to determine changes in myocardial gene expression involved in Cu deficient (CuD) cardiomyopathy and its reversal by CuR. Analysis was performed on three groups of mice: 4-week-old CuD mice that exhibited signs of cardiac failure, their age-matched copper-adequate (CuA) controls, and the CuD mice that were re-fed adequate Cu for 2 weeks. Total RNA was isolated from hearts and subjected to cDNA micro-array and real-time reverse transcription-polymerase chain reaction analysis. Dietary CuD caused a decrease in cardiac mRNA of beta-MHC, L-type Ca(2+) channel, K-dependent NCX, MMP-2, -8, and -13, NF-kappaB, and VEGF. The mRNA levels of ET-1, TGF-beta, TNF-alpha, and procollagen-I-alpha1 and III-alpha1 were increased in the CuD cardiac tissue. Copper repletion resulted in cardiac mRNA levels of most of the genes examined returning to control levels, although the K-dependent NCX and MMP-2 values did not reach those of the CuA control. In addition, CuR caused an increase in beta-MHC, L-type Ca(2+)channel, MMP-13 to levels surpassing those of CuA control, and a decrease in ET-1, and TNF-alpha mRNA levels. In summary, changes in gene expression of elements involved in contractility, Ca(2+) cycling, and inflammation and fibrosis may account for the altered cardiac function found in CuD mice. The return to normal cardiac function by CuR may be a result of the favorable regression in gene expression of these critical components in myocardial tissue.

Dietary copper deficiency (CuD)(3) leads to cardiac hypertrophy in various animal models. We showed recently that heart failure develops after hypertrophy in FVB mice fed a CuD diet. The present study was undertaken to determine whether CuD-induced cardiac failure is reversible upon copper repletion (CuR). Dams of FVB mice were fed a CuD diet (0.3 mg/kg) starting from d 3 postdelivery; the weanling pups were fed the same diet until CuR with 6.0 mg/kg Cu in the diet at 4 or 5 wk of age. CuR at 4 wk of age prevented the body weight loss; at 5 wk of age, it resulted in the regaining of the lost weight caused by CuD. A significant regression of CuD-induced cardiac hypertrophy was observed in the CuR mice. Histopathological examination revealed that CuR eliminated CuD-caused lipid deposition in the myocardium, and electron microscopy demonstrated that CuD-induced ultrastructural changes such as mitochondrial swelling and organelle structural disarray were all reversed in the CuR mice. Hemodynamic analysis showed that the CuD-depressed systolic and diastolic parameters such as the maximal rate of left ventricular pressure rise (+dP/dt) and decline (-dP/dt), and the contraction and relaxation times were completely recovered in the CuR mice. Furthermore, the CuD-blunted myocardial responses to the beta-adrenergic agonist, isoproterenol, were also restored in the CuR mice. This study thus demonstrates for the first time that CuR results in the regression of heart failure induced by CuD as demonstrated by the reversal of depressed cardiac hemodynamic and contractile function and the restored responsiveness to beta-adrenergic stimulation.