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Supplemental Digital Content is available in the text., Objective— To establish the cellular source of plasma factor (F)XIII-A. Approach and Results— A novel mouse floxed for the F13a1 gene, FXIII-Aflox/flox (Flox), was crossed with myeloid- and platelet-cre–expressing mice, and cellular FXIII-A mRNA expression and plasma and platelet FXIII-A levels were measured. The platelet factor 4-cre.Flox cross abolished platelet FXIII-A and reduced plasma FXIII-A to 23±3% (P

Background Endothelial cell injury, induced by dyslipidemia, is the initiation of atherosclerosis, resulting in an imbalance in endothelial fatty acid ( FA ) transport. Pigment epithelial‐derived factor ( PEDF ) is an important regulator in lipid metabolism. We hypothesized that PEDF is involved in endothelium‐mediated FA uptake under hyperlipidemic conditions. Methods and Results Circulating PEDF levels were higher in patients with atherosclerotic cardiovascular disease than in normal individuals. However, decreasing trends of serum PEDF levels were confirmed in both wild‐type and apolipoprotein E–deficient mice fed a long‐term high‐fat diet. Apolipoprotein E–deficient/PEDF‐deficient mice were generated by crossing PEDF‐deficient mice with apolipoprotein E–deficient mice, and then mice were fed with 24, 36, or 48 weeks of high‐fat diet. Greater increases in body fat and plasma lipids were displayed in PEDF ‐deficient mice. In addition, PEDF deficiency in mice accelerated atherosclerosis, as evidenced by increased atherosclerotic plaques, pronounced vascular dysfunction, and increased lipid accumulation in peripheral tissues, whereas injection of adeno‐associated virus encoding PEDF exerted opposite effects. Mechanistically, PEDF inhibited the vascular endothelial growth factor B paracrine signaling by reducing secretion of protein vascular endothelial growth factor B in peripheral tissue cells and decreasing expression of its downstream targets in endothelial cells, including its receptors (namely, vascular endothelial growth factor receptor‐1 and neuropilin‐1), and FA transport proteins 3 and 4, to suppress endothelial FA uptake, whereas PEDF deletion in mice activated the vascular endothelial growth factor B signaling pathway, thus causing markedly increased lipid accumulation. Conclusions Decreasing expression of PEDF aggravates atherosclerosis by significantly impaired vascular function and enhanced endothelial FA uptake, thus exacerbating ectopic lipid deposition in peripheral tissues.

Background Muscular dystrophy ( MD ) causes a progressive cardiomyopathy characterized by diffuse fibrosis, arrhythmia, heart failure, and early death. Activation of the thromboxane‐prostanoid receptor ( TP r) increases calcium transients in cardiomyocytes and is proarrhythmic and profibrotic. We hypothesized that TP r activation contributes to the cardiac phenotype of MD , and that TP r antagonism would improve cardiac fibrosis and function in preclinical models of MD . Methods and Results Three different mouse models of MD (mdx/utrn double knockout, second generation mdx/ mTR double knockout, and delta‐sarcoglycan knockout) were given normal drinking water or water containing 25 mg/kg per day of the TP r antagonist ifetroban, beginning at weaning. After 6 months (10 weeks for mdx/utrn double knockout), mice were evaluated for cardiac and skeletal muscle function before euthanization. There was a 100% survival rate of ifetroban‐treated mice to the predetermined end point, compared with 60%, 43%, and 90% for mdx/utrn double knockout, mdx/mTR double knockout, and delta‐sarcoglycan knockout mice, respectively. TP r antagonism improved cardiac output in mdx/utrn double knockout and mdx/ mTR mice, and normalized fractional shortening, ejection fraction, and other parameters in delta‐sarcoglycan knockout mice. Cardiac fibrosis in delta‐sarcoglycan knockout was reduced with TP r antagonism, which also normalized cardiac expression of claudin‐5 and neuronal nitric oxide synthase proteins and multiple signature genes of Duchenne MD. Conclusions TP r antagonism reduced cardiomyopathy and spontaneous death in mouse models of Duchenne and limb‐girdle MD . Based on these studies, ifetroban and other TP r antagonists could be novel therapeutics for treatment of the cardiac phenotype in patients with MD .

Background Herein, we report an in vivo study of a biodegradable flow diverter ( BDFD ) for aneurysm occlusion. Conceptually, BDFD s induce a temporal flow‐diverting effect and provide a vascular scaffold for neointimal formation at the neck of the aneurysm until occlusion. This offers several potential advantages, including a reduced risk of remote ischemic complications and more treatment options in case of device failure to occlude the aneurysm. Methods and Results A BDFD consisting of 48 poly‐ l ‐lactic acid wires with radiopaque markers at both ends was prepared. An in vitro degradation test of the BDFD was performed. Thirty‐six BDFD s were implanted in a rabbit aneurysm model. Digital angiography, optical coherence tomography, histopathology, and scanning electron microscopy were performed after 1, 3, and 6 months, and 1 year. The in vitro degradation test showed that the BDFD was almost degraded in 1.5 years. In the in vivo experiment, aneurysm occlusion rates were 0% at 1 month, 20% at 3 months, 50% at 6 months, and 33% at 1 year. Optical coherence tomography showed that luminal area stenosis was the highest at 3 months (16%) and decreased afterward. Immunohistochemical analysis showed that more than half of the luminal surface area was covered by endothelial cells at 1 month. Device fragmentation was not observed in any lesions. Conclusions This first in vivo study of a BDFD shows the feasibility of using BDFDs for treating aneurysms; however, a longer follow‐up is required for comprehensive evaluation of the biological and mechanical behavior peculiar to biodegradable devices.

Background Previous studies have demonstrated that functional autoantibodies to adrenergic receptors may be involved in the pathogenesis of postural tachycardia syndrome. The objective of this study was to examine the impact of these autoantibodies on cardiovascular responses to postural changes and adrenergic orthosteric ligand infusions in immunized rabbits. Methods and Results Eight New Zealand white rabbits were coimmunized with peptides from the α1‐adrenergic receptor and β1‐adrenergic receptor (β1AR). Tilt test and separate adrenergic agonist infusion studies were performed on conscious animals before and after immunization and subsequent treatment with epitope‐mimetic peptide inhibitors. At 6 weeks after immunization, there was a greater percent increase in heart rate upon tilting compared with preimmune baseline. No significant difference in blood pressure response to tilting was observed. The heart rate response to infusion of the β‐adrenoceptor agonist isoproterenol was significantly enhanced in immunized animals, suggesting a positive allosteric effect of β1AR antibodies. In contrast, the blood pressure response to infusion of the α1‐adrenergic receptor agonist phenylephrine was attenuated in immunized animals, indicating a negative allosteric effect of α1‐adrenergic receptor antibodies. Injections of antibody‐neutralizing peptides suppressed the postural tachycardia and reversed the altered heart rate and blood pressure responses to orthosteric ligand infusions in immunized animals at 6 and 30 weeks. Antibody production and suppression were confirmed with in vitro bioassays. Conclusions The differential allosteric effect of α1‐adrenergic receptor and β1AR autoantibodies would lead to a hyperadrenergic state and overstimulation of cardiac β1AR. These data support evidence for an autoimmune basis for postural tachycardia syndrome., See Editorial Miller and Doherty

Background Few reports have addressed the mechanism by which microRNA miR‐10b‐5p regulates post–myocardial infarction (post‐ MI ) cardiomyocyte apoptosis under hypoxic conditions. Methods and Results C57 BL /6 mice underwent surgical ligation of the left anterior descending artery to create an MI or ischemia/reperfusion animal model. The expression of miR‐10b‐5p, PTEN (phosphatase and tensin homolog), and HIF‐1α (hypoxia‐inducible factor 1α) was detected in infarct border zone tissues at various time points. After precordial injections of the negative control or miR‐10b‐5p, overexpression lentiviruses were made in the areas surrounding the MI sites at 1 week, and myocardial infarct size, cardiac function, and cardiomyocyte apoptosis were examined. A miR‐10b‐5p mimic was transfected into primary mouse cardiomyocytes to analyze its effects on cardiomyocyte apoptosis and PTEN expression. Meanwhile, PTEN as a target of miR‐10b‐5p was verified via luciferase reporter gene assays. Cotransfection of miR‐10b‐5 and PTEN verified the relationship between miR‐10b‐5 and PTEN . Under hypoxic stress, the expression of HIF ‐1α and miR‐10b‐5p was examined. The results showed that miR‐10b‐5p expression was markedly reduced in the infarct border zone. Overexpression of miR‐10b‐5p in the murine model of MI significantly reduced MI size, improved cardiac function, and inhibited apoptosis. Overexpression of miR‐10b‐5p in vitro antagonized hypoxia‐induced cardiomyocyte apoptosis and specifically inhibited the expression of the apoptosis‐related gene PTEN , but overexpression of PTEN weakened these effects. We also found that hypoxia‐induced accumulation of HIF ‐1α resulted in decreased expression of miR‐10b‐5p. Interfering with the activation of the HIF ‐1α signaling pathway promoted Pri‐miR‐10b and miR‐10b‐5p expression and inhibited PTEN expression. Conclusions MicroRNA miR‐10b‐5p antagonizes hypoxia‐induced cardiomyocyte apoptosis, indicating that miR‐10b‐5p may serve as a potential future clinical target for the treatment of MI .

Background Arterial bypass and interposition grafts are used routinely across multiple surgical subspecialties. Current options include both autologous and synthetic materials; however, each graft presents specific limitations. Engineering artificial small‐diameter arteries with vascular cells derived from induced pluripotent stem cells could provide a useful therapeutic solution. Banking induced pluripotent stem cells from rare individuals who are homozygous for human leukocyte antigen alleles has been proposed as a strategy to facilitate economy of scale while reducing the potential for rejection of induced pluripotent stem cell–derived transplanted tissues. Currently, there is no standardized model to study transplantation of small‐diameter arteries in major histocompatibility complex–defined backgrounds. Methods and Results In this study, we developed a limb‐sparing nonhuman primate model to study arterial allotransplantation in the absence of immunosuppression. Our model was used to compare degrees of major histocompatibility complex matching between arterial grafts and recipient animals with long‐term maintenance of patency and function. Unexpectedly, we (1) found that major histocompatibility complex partial haplomatched allografts perform as well as autologous control grafts; (2) detected little long‐term immune response in even completely major histocompatibility complex mismatched allografts; and (3) observed that arterial grafts become almost completely replaced over time with recipient cells. Conclusions Given these findings, induced pluripotent stem cell–derived tissue‐engineered blood vessels may prove to be promising and customizable grafts for future use by cardiac, vascular, and plastic surgeons.

Background Reduced oxygen delivery in congenital heart disease causes delayed brain maturation and white matter abnormalities in utero. No treatment currently exists. Tetrahydrobiopterin ( BH 4) is a cofactor for neuronal nitric oxide synthase. BH 4 availability is reduced upon NOS activation, such as during hypoxic conditions, and leads to toxin production. We hypothesize that BH 4 levels are depleted in the hypoxic brain and that BH4 replacement therapy mitigates the toxic effects of hypoxia on white matter. Methods and Results Transgenic mice were used to visualize oligodendrocytes. Hypoxia was introduced during a period of white matter development equivalent to the human third trimester. BH 4 was administered during hypoxia. BH 4 levels were depleted in the hypoxic brain by direct quantification (n=7–12). The proliferation (n=3–6), apoptosis (n=3–6), and developmental stage (n=5–8) of oligodendrocytes were determined immunohistologically. Total oligodendrocytes increased after hypoxia, consistent with hypoxia‐induced proliferation seen previously; however, mature oligodendrocytes were less prevalent in hypoxia, and there was accumulation of immature oligodendrocytes. BH 4 treatment improved the mature oligodendrocyte number such that it did not differ from normoxia, and accumulation of immature oligodendrocytes was not observed. These results persisted beyond the initial period of hypoxia (n=3–4). Apoptosis increased with hypoxia but decreased with BH 4 treatment to normoxic levels. White matter myelin levels decreased following hypoxia by western blot. BH 4 treatment normalized myelination (n=6–10). Hypoxia worsened sensory‐motor coordination on balance beam tasks, and BH 4 therapy normalized performance (n=5–9). Conclusions Suboptimal BH 4 levels influence hypoxic white matter abnormalities. Repurposing BH 4 for use during fetal brain development may limit white matter dysmaturation in congenital heart disease.

Background In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH 7b, is regulated by alternative splicing, and while the protein is restricted to specialized muscles such as extraocular muscles or muscle spindles, RNA that cannot encode protein is expressed in most skeletal muscles and in the heart. Remarkably, birds and snakes express MYH 7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH 7b may only be needed during development since its expression is prevented in adult tissue, possibly because it could promote disease by unbalancing myocardial contractility. Methods and Results We have analyzed MYH 7b null mice to determine the potential role of MYH 7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH 7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH 7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH 7b protein expression induced early cardiac dilation in males with significantly increased left ventricular mass in both sexes. Cardiac dilation is progressive, leading to early cardiac dysfunction in males, but later dysfunction in females. Conclusions The data presented show that the expression of MYH 7b protein in the mammalian heart has been inhibited during the evolution of mammals most likely to prevent the development of a severe cardiomyopathy that is sexually dimorphic.

Background Duchenne muscular dystrophy ( DMD ) is an X‐linked disease that causes progressive muscle weakness. Affected boys typically die from respiratory or cardiac failure. Golden retriever muscular dystrophy ( GRMD ) is genetically homologous with DMD and causes analogous skeletal and cardiac muscle disease. Previous studies have detailed features of GRMD cardiomyopathy in mostly young dogs. Cardiac disease is not well characterized in adult GRMD dogs, and cardiac magnetic resonance ( CMR ) imaging studies have not been completed. Methods and Results We evaluated echocardiography and CMR in 24 adult GRMD dogs at different ages. Left ventricular systolic and diastolic functions, wall thickness, and myocardial strain were assessed with echocardiography. Features evaluated with CMR included left ventricular function, chamber size, myocardial mass, and late gadolinium enhancement. Our results largely paralleled those of DMD cardiomyopathy. Ejection fraction and fractional shortening correlated well with age, with systolic dysfunction occurring at ≈30 to 45 months. Circumferential strain was more sensitive than ejection fraction in early disease detection. Evidence of left ventricular chamber dilatation provided proof of dilated cardiomyopathy. Late gadolinium enhancement imaging showed DMD ‐like left ventricular lateral wall lesions and earlier involvement of the anterior septum. Multiple functional indexes were graded objectively and added, with and without late gadolinium enhancement, to give cardiac and cardiomyopathy scores of disease severity. Consistent with DMD , there was parallel skeletal muscle involvement, as tibiotarsal joint flexion torque declined in tandem with cardiac function. Conclusions This study established parallels of progressive cardiomyopathy between dystrophic dogs and boys, further validating GRMD as a model of DMD cardiac disease.

Background Obesity‐associated chronic inflammation has been known to contribute to atherosclerosis development, but the underlying mechanisms remain elusive. Recent studies have revealed novel functions of IKK β (inhibitor of NF ‐κB [nuclear factor κB] kinase β), a key coordinator of inflammation through activation of NF ‐κB, in atherosclerosis and adipose tissue development. However, it is not clear whether IKK β signaling in adipocytes can also affect atherogenesis. This study aims to investigate the impact of adipocyte IKK β expression on atherosclerosis development in lean and obese LDLR (low‐density lipoprotein receptor)–deficient ( LDLR −/− ) mice. Methods and Results To define the role of adipocyte IKK β in atherogenesis, we generated adipocyte‐specific IKK β‐deficient LDLR −/− ( IKK β ΔAd LDLR −/− ) mice. Targeted deletion of IKK β in adipocytes did not affect adiposity and atherosclerosis in lean LDLR −/− mice when fed a low‐fat diet. In response to high‐fat feeding, however, IKK β ΔAd LDLR −/− mice had defective adipose remodeling and increased adipose tissue and systemic inflammation. Deficiency of adipocyte IKK β did not affect atherosclerotic lesion sizes but resulted in enhanced lesional inflammation and increased plaque vulnerability in obese IKK β ΔAd LDLR −/− mice. Conclusions These data demonstrate that adipocyte IKK β signaling affects the evolution of atherosclerosis plaque vulnerability in obese LDLR −/− mice. This study suggests that the functions of IKK β signaling in atherogenesis are complex, and IKK β in different cell types or tissues may have different effects on atherosclerosis development.

Background Sepsis is the overwhelming host response to infection leading to shock and multiple organ dysfunction. Cardiovascular complications greatly increase sepsis‐associated mortality. Although murine models are routinely used for preclinical studies, the benefit of using genetically engineered mice in sepsis is countered by discrepancies between human and mouse sepsis pathophysiology. Therefore, recent guidelines have called for standardization of preclinical methods to document organ dysfunction. We investigated the course of cardiac dysfunction and myocardial load in different mouse models of sepsis to identify the optimal measurements for early systolic and diastolic dysfunction. Methods and Results We performed speckle‐tracking echocardiography and assessed blood pressure, plasma inflammatory cytokines, lactate, B‐type natriuretic peptide, and survival in mouse models of endotoxemia or polymicrobial infection (cecal ligation and puncture, [ CLP ]) of moderate and high severity. We observed that myocardial strain and cardiac output were consistently impaired early in both sepsis models. Suppression of cardiac output was associated with systolic dysfunction in endotoxemia or combined systolic dysfunction and reduced preload in the CLP model. We found that cardiac output at 2 hours post‐ CLP is a negative prognostic indicator with high sensitivity and specificity that predicts mortality at 48 hours. Using a known antibiotic (ertapenem) treatment, we confirmed that this approach can document recovery. Conclusions We propose a non‐invasive approach for assessment of cardiac function in sepsis and myocardial strain and strain rate as preferable measures for monitoring cardiovascular function in sepsis mouse models. We further show that the magnitude of cardiac output suppression 2 hours post‐ CLP can be used to predict mortality.

Background Protein posttranslational modifications by O ‐linked β‐N‐acetylglucosamine (O‐GlcNAc) increase with cardiac hypertrophy, yet the functional effects of these changes are incompletely understood. In other organs, O‐GlcNAc promotes adaptation to acute physiological stressors; however, prolonged O‐GlcNAc elevations are believed to be detrimental. We hypothesize that early O‐GlcNAcylation improves cardiac function during initial response to pressure overload hypertrophy, but that sustained elevations during established pathological hypertrophy negatively impact cardiac function by adversely affecting calcium handling proteins. Methods and Results Transverse aortic constriction or sham surgeries were performed on littermate controls or cardiac‐specific, inducible O‐GlcNAc transferase knockout (OGTKO) mice to reduce O‐GlcNAc levels. O‐GlcNAc transferase deficiency was induced at different times. To evaluate the initial response to pressure overload, OGTKO was completed preoperatively and mice were followed for 2 weeks post‐surgery. To assess prolonged O‐GlcNAcylation during established hypertrophy, OGTKO was performed starting 18 days after surgery and mice were followed until 6 weeks post‐surgery. In both groups, OGTKO with transverse aortic constriction caused significant left ventricular dysfunction. OGTKO did not affect levels of the calcium handling protein SERCA2a. OGTKO reduced phosphorylation of phospholamban and cardiac troponin I, which would negatively impact cardiac function. O‐GlcNAcylation of protein kinase A catalytic subunit, a kinase for phospholamban, decreased with OGTKO. Conclusions O‐GlcNAcylation promotes compensated cardiac function in both early and established pathological hypertrophy. We identified a novel O‐GlcNAcylation of protein kinase A catalytic subunit, which may regulate calcium handling and cardiac function.

Background Because of the failure of numerous clinical trials, various recommendations have been made to improve the usefulness of preclinical studies. Specifically, the STAIR (Stroke Therapy Academic Industry Roundtable) recommendations highlighted functional outcome as a critical measure. Recent reviews of experimental subarachnoid hemorrhage ( SAH ) studies have brought to light the numerous neurobehavioral scoring systems that are used in preclinical SAH studies. To gain insight into the utility of these scoring systems, as well as to identify a scoring system that best captures the deficits caused by SAH in mice, we designed the current study. Methods and Results Adult male C57 BL /6J mice were used. One cohort of mice was randomly allocated to either sham or SAH and had functional testing performed on days 1 to 3 post‐ SAH using the modified Bederson Score, Katz Score, Garcia Neuroscore, and Parra Neuroscore, as well as 21 individual subtests. A new composite neuroscore was developed using the 8 most diagnostically accurate subtests. To validate the use of the developed composite neuroscore, another cohort of mice was randomly assigned to either the sham or SAH group and neurobehavior was evaluated on days 1 to 3, 5, and 7 after injury. Receiver operating characteristic curves were used to analyze the diagnostic accuracy of each scoring system, as well as the subtests. Of the 4 published scoring systems, the Parra Neuroscore was diagnostically accurate for SAH injury in mice versus the modified Bederson and Katz Scores, but not the Garcia Neuroscore. However, the newly developed composite neuroscore was found to be statistically more diagnostically accurate than even the Parra Neuroscore. Conclusions The findings of this study promote use of the newly developed composite neuroscore for experimental SAH studies in mice.

Background Systemic innate immune priming is a recognized sequela of post‐ischemic neuroinflammation and contributor to delayed neurodegeneration. Given mounting evidence linking acute stroke with reactive lung inflammation, we asked whether enhanced expression of the endogenous antioxidant extracellular superoxide dismutase 3 (SOD3) produced by alveolar type II pneumocytes would protect the lung from transient global cerebral ischemia and the brain from the delayed effects of ischemia‐reperfusion. Methods and Results Following 15 minutes of global cerebral ischemia or sham conditions, transgenic SOD3 and wild‐type mice were followed daily for changes in weight, core temperature, and neurological function. Three days after reperfusion, arterial and venous samples were collected for complete blood counts, flow cytometry, and SOD3 protein blotting, and immunohistochemistry was performed on lung and brain tissue to assess tissue injury, blood‐brain barrier permeability, and neutrophil transmigration. Relative to ischemic controls, transgenic SOD3 mice performed better on functional testing and exhibited reduced peripheral neutrophil activation, lung inflammation, and blood‐brain barrier leak. Once released from the lung, SOD3 was predominantly not cell associated and depleted in the venous phase of circulation. Conclusions In addition to reducing the local inflammatory response to cerebral ischemia, targeted enrichment of SOD3 within the lung confers distal neuroprotection against ischemia‐reperfusion injury. These data suggest that therapies geared toward enhancing adaptive lung‐neurovascular coupling may improve outcomes following acute stroke and cardiac arrest.

Background We occasionally noticed that native collateral blood flow showed a recessive trend in the early stages of acute myocardial infarction in rats, which greatly interferes with the accurate assessment of native collateral circulation levels. Here, we sought to recognize the coronary collateral circulation system in depth, especially the microcirculation part, on this basis. Methods and Results In this study, we detected native collateral flow with positron emission tomography perfusion imaging in rats and found that the native flow is relatively abundant when it is initially recruited. However, this flow is extremely unstable in the early stage of acute myocardial infarction and quickly fails. We used tracers to mark the collateral in an ischemic area and a massive preformed collateral network was labeled. The ultrastructures of these collateral microvessels are flawed, which contributes to extensive leakage and consequent interstitial edema in the ischemic region. Conclusions An unrecognized short‐lived native coronary collateral microcirculation reserve is widely distributed in rat hearts. Recession of collateral blood flow transported by coronary collateral microcirculation reserve contributes to instability of native collateral blood flow in the early stage of acute myocardial infarction. The immature structure determines that these microvessels are short‐lived and provide conditions for the development of early interstitial edema in acute myocardial infarction.

Background Mutations in αB‐crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting (IF) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy‐lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy. Methods and Results Mice overexpressing the R120G mutant of αB‐crystallin in cardiomyocytes (Myh6‐CryABR120G) were subjected to IF or ad‐lib feeding, or transduced with adeno‐associated virus–TFEB or adeno‐associated virus—green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno‐associated virus–short hairpin RNA–mediated knockdown of TFEB and HSPB8 was performed simultaneously with IF. Myh6‐CryABR120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno‐associated virus–TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6‐CryABR120G mice. CryABR120G‐expressing hearts demonstrated increased interaction of desmin with αB‐crystallin and reduced interaction with chaperone protein, HSPB8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB8, to restore normal desmin localization in CryABR120G‐expressing cardiomyocytes. Short hairpin RNA–mediated knockdown of TFEB and HSPB8 abrogated IF effects, in vivo. Conclusions IF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G αB‐crystallin mutant‐induced cardiomyopathy by normalizing desmin localization via autophagy‐dependent and autophagy‐independent mechanisms., See Editorial by Mukai et al

Background Chronic intermittent hypoxia ( CIH ) is a distinct pathological mechanism of obstructive sleep apnea ( OSA ), which is recognized as an independent risk factor for cardiovascular diseases. The aims of this study were to ascertain whether CIH induces atrial fibrillation ( AF ), to determine whether cardiac sympathetic denervation ( CSD ) can prevent it and suppress blood pressure, and to explore the potential molecular mechanisms involved. Methods and Results Sixty Sprague‐Dawley male rats were randomly divided into 4 groups: sham, CSD , CIH , CIH + CSD . The rats were exposed either to CIH 8 hours daily or normoxia for 6 weeks. Cardiac pathology and structure were analyzed by hematoxylin and eosin staining and echocardiogram. ECG, blood pressure, body weight, and blood gas were recorded. Connexin 43 and tyrosine hydroxylase were detected by western blot, immunohistochemistry, and immunofluorescence. CIH induced atrial remodeling, and increased AF inducibility. CSD treatment reduced postapneic blood pressure rises and AF susceptibility, which could attenuate CIH ‐associated structural atrial arrhythmogenic remodeling. In addition, CIH ‐induced sympathetic nerve hyperinnervation and CSD treatment reduced sympathetic innervation, which may affect CIH ‐induced AF ‐associated sympathovagal imbalance. Connexin 43 was specifically downregulated in CIH , whereas CSD treatment increased its expression. Conclusions These results suggested CIH induces atrial remodeling, increases AF inducibility, results in sympathetic nerve hyperinnervation, and decreases connexin 43 expression, but CSD treatment reduces AF susceptibility, postapneic blood pressure increase, sympathetic innervation, and the alteration of Cx43, which may be a key point in the genesis of CIH ‐induced AF .

Background In pregnancy, a high plasma volume maintains uteroplacental perfusion and prevents placental ischemia, a condition linked to elevated maternal blood pressure ( BP ). Reducing BP by increasing Na + intake via plasma volume expansion appears contra‐intuitive. We hypothesize that an appropriate Na + intake in pregnancy reduces maternal BP and adapts the renin‐angiotensin system in a pregnancy‐specific manner. Methods and Results BP was measured by implanted telemetry in Sprague‐Dawley rats before and throughout pregnancy. Pregnant and nonpregnant animals received either a normal‐salt (0.4%; NS ), high‐salt (8%; HS ), or low‐salt (0.01%; LS ) diet, or HS (days 1–14) followed by LS (days 14–20) diet ( HS / LS ). Before delivery (day 20), animals were euthanized and organs collected. Food, water, and Na + intake were monitored in metabolic cages, and urinary creatinine and Na + were analyzed. Na + intake and retention increased in pregnancy ( NS , LS ), leading to a positive Na + balance ( NS , LS ). BP was stable during LS , but reduced in HS conditions in pregnancy. The renin‐angiotensin system was adapted as expected. Activating cleavage of α‐ and γ‐subunits of the renal epithelial Na + channel and expression of‐full length medullary β‐subunits, accentuated further in all LS conditions, were upregulated in pregnancy. Conclusions Pregnancy led to Na + retention adapted to dietary changes. HS exposure paradoxically reduced BP . Na + uptake while only modestly linked to the renin‐angiotensin system is enhanced in the presence of posttranslational renal epithelial Na + channel modifications. This suggests (1) storage of Na + in pregnancy upon HS exposure, bridging periods of LS availability; and (2) that potentially non–renin‐angiotensin–related mechanisms participate in EN aC activation and consecutive Na + retention.

Background Vascular endothelial cell (EC) alignment in the direction of flow is an adaptive response that protects against aortic diseases, such as atherosclerosis. The Rho GTP ases are known to regulate this alignment. Herein, we analyze the effect of ARHGAP 18 on the regulation of EC alignment and examine the effect of ARHGAP 18 deficiency on the development of atherosclerosis in mice. Methods and Results We used in vitro analysis of ECs under flow conditions together with apolipoprotein E −/− Arhgap 18 −/− double‐mutant mice to study the function of ARHGAP 18 in a high‐fat diet–induced model of atherosclerosis. Depletion of ARHGAP 18 inhibited the alignment of ECs in the direction of flow and promoted inflammatory phenotype, as evidenced by disrupted junctions and increased expression of nuclear factor‐κB and intercellular adhesion molecule‐1 and decreased endothelial nitric oxide synthase. Mice with double deletion in ARHGAP 18 and apolipoprotein E and fed a high‐fat diet show early onset of atherosclerosis, with lesions developing in atheroprotective regions. Conclusions ARHGAP 18 is a protective gene that maintains EC alignments in the direction of flow. Deletion of ARHGAP 18 led to loss of EC ability to align and promoted atherosclerosis development.

Background The impairment of endothelium‐dependent vasodilation, increased endothelial permeability, and glycocalyx degradation are all important pathophysiological components of endothelial dysfunction. However, it is still not clear whether in atherosclerosis, glycocalyx injury precedes other features of endothelial dysfunction or these events coincide. Methods and Results Herein, we demonstrate that in 4‐ to 8‐week‐old apolipoprotein E/low‐density lipoprotein receptor‐deficient mice, at the stage before development of atherosclerotic plaques, impaired acetylcholine‐induced vasodilation, reduced NO production in aorta, and increased endothelial permeability were all observed; however, flow‐mediated dilation in the femoral artery was fully preserved. In 4‐week‐old mice, glycocalyx coverage was reduced and endothelial stiffness was increased, whereas glycocalyx length was significantly decreased at 8 weeks of age. Early changes in endothelial function were also featured by increased plasma concentration of biomarkers of glycocalyx disruption (endocan), biomarkers of endothelial inflammation (soluble vascular cell adhesion molecule 1), increased vascular permeability (angiopoietin 2), and alterations in hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). In 28‐week‐old mice, at the stage of advanced atherosclerotic plaque development, impaired NO production and nearly all other features of endothelial dysfunction were changed to a similar extent, compared with the preatherosclerotic plaque phase. The exceptions were the occurrence of acetylcholine‐induced vasoconstriction in the aorta and brachiocephalic artery, impaired flow‐mediated vasodilation in the femoral artery, and further reduction of glycocalyx length and coverage with a concomitant further increase in endothelial permeability. Conclusions In conclusion, even at the early stage before the development of atherosclerotic plaques, endothelial dysfunction is a complex multifactorial response that has not been previously appreciated.

Background In the presence of arterial stenosis, collateral artery growth (arteriogenesis) can alleviate ischemia and preserve tissue function. In patients with poorly developed collateral arteries, Gal‐2 (galectin 2) expression is increased. In vivo administration of Gal‐2 inhibits arteriogenesis. Blocking of Gal‐2 potentially stimulates arteriogenesis. This study aims to investigate the effect of Gal‐2 inhibition on arteriogenesis and macrophage polarization using specific single‐domain antibodies. Methods and Results Llamas were immunized with Gal‐2 to develop anti–Gal‐2 antibodies. Binding of Gal‐2 to monocytes and binding inhibition of antibodies were quantified. To test arteriogenesis in vivo, Western diet‐fed LDLR .(low‐density lipoprotein receptor)–null Leiden mice underwent femoral artery ligation and received treatment with llama antibodies 2H8 or 2C10 or with vehicle. Perfusion restoration was measured with laser Doppler imaging. In the hind limb, arterioles and macrophage subtypes were characterized by histology, together with aortic atherosclerosis. Llama‐derived antibodies 2H8 and 2C10 strongly inhibited the binding of Gal‐2 to monocytes (93% and 99%, respectively). Treatment with these antibodies significantly increased perfusion restoration at 14 days (relative to sham, vehicle: 41.3±2.7%; 2H8: 53.1±3.4%, P =0.016; 2C10: 52.0±3.8%, P =0.049). In mice treated with 2H8 or 2C10, the mean arteriolar diameter was larger compared with control (vehicle: 17.25±4.97 μm; 2H8: 17.71±5.01 μm; 2C10: 17.84±4.98 μm; P P =0.007; 2C10: 0.75±0.18, P =0.006). In vitro antibody treatment decreased the expression of M1‐associated cytokines compared with control ( P P =0.59). Conclusions Inhibition of Gal‐2 induces a proarteriogenic M2 phenotype in macrophages, improves collateral artery growth, and increases perfusion restoration in a murine hind limb model.

Background Myocarditis is an important cause of acute and chronic heart failure. Men with myocarditis have worse recovery and an increased need for transplantation compared with women, but the reason for the sex difference remains unclear. Elevated sera soluble (s) ST 2 predicts mortality from acute and chronic heart failure, but has not been studied in myocarditis patients. Methods and Results Adults with a diagnosis of clinically suspected myocarditis (n=303, 78% male) were identified according to the 2013 European Society of Cardiology position statement. Sera sST 2 levels were examined by ELISA in humans and mice and correlated with heart function according to sex and age. Sera sST 2 levels were higher in healthy men ( P =8×10 −6 ) and men with myocarditis ( P =0.004) compared with women. sST 2 levels were elevated in patients with myocarditis and New York Heart Association class III ‐ IV heart failure ( P =0.002), predominantly in men ( P =0.0003). Sera sST 2 levels were associated with New York Heart Association class in men with myocarditis who were ≤50 years old ( r =0.231, P =0.0006), but not in women ( r =0.172, P =0.57). Sera sST 2 levels were also significantly higher in male mice with myocarditis ( P =0.005) where levels were associated with cardiac inflammation. Gonadectomy with hormone replacement showed that testosterone ( P P =0.32), increased sera sST 2 levels in male mice with myocarditis. Conclusions We show in a well‐characterized subset of heart failure patients with clinically suspected and biopsy‐confirmed myocarditis that elevated sera sST 2 is associated with an increased risk of heart failure based on New York Heart Association class in men ≤50 years old.

Background: It is well known that after menopause women are exposed to a greater cardiovascular risk, but the intracellular modifications are not properly described. The sodium/proton exchanger (NHE) and the sodium/bicarbonate cotransporter (NBC) regulate the intracellular pH and, indirectly, the intracellular sodium concentration ([Na+]). There are 2 isoforms of NBC in the heart: the electrogenic (1Na+/2HCO 3 ; NBCe1) and the electroneutral (1Na+/1HCO 3 ; NBCn1). Because NHE and NBCn1 hyperactivity as well as the NBCe1 decreased activity have been associated with several cardiovascular pathologies, the aim of this study was to investigate the potential alterations of the alkalinizing transporters during the postmenopausal period. Methods and Results: Three-month ovariectomized rats (OVX) were used. The NHE activity and protein expression are significantly increased in OVX. The NBCe1 activity is diminished, and the NBCn1 activity becomes predominant in OVX rats. p-Akt levels showed a significant diminution in OVX. Finally, NHE activity in platelets from OVX rats is also higher in comparison to sham rats, resulting in a potential biomarker of cardiovascular diseases. Conclusions: Our results demonstrated for the first time that in the cardiac ventricular myocytes of OVX rats NHE and NBC isoforms are altered, probably because of the decreased level of p-Akt, compromising the ionic intracellular homeostasis. (J Am Heart Assoc. 2019;8:e011066. DOI: 10.1161/JAHA.118.011066.), Centro de Investigaciones Cardiovasculares

BACKGROUND: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with important vascular and hemostatic alterations that should be taken into account during diagnosis and treatment. OBJECTIVES: This study evaluates whether anticoagulation with dabigatran, a clinically approved oral direct thrombin inhibitor with a low risk of intracerebral hemorrhage, ameliorates AD pathogenesis in a transgenic mouse model of AD. METHODS: TgCRND8 AD mice and their wild-type littermates were treated for 1 year with dabigatran etexilate or placebo. Cognition was evaluated using the Barnes maze, and cerebral perfusion was examined by arterial spin labeling. At the molecular level, Western blot and histochemical analyses were performed to analyze fibrin content, amyloid burden, neuroinflammatory activity, and blood-brain barrier (BBB) integrity. RESULTS: Anticoagulation with dabigatran prevented memory decline, cerebral hypoperfusion, and toxic fibrin deposition in the AD mouse brain. In addition, long-term dabigatran treatment significantly reduced the extent of amyloid plaques, oligomers, phagocytic microglia, and infiltrated T cells by 23.7%, 51.8%, 31.3%, and 32.2%, respectively. Dabigatran anticoagulation also prevented AD-related astrogliosis and pericyte alterations, and maintained expression of the water channel aquaporin-4 at astrocytic perivascular endfeet of the BBB. CONCLUSIONS: Long-term anticoagulation with dabigatran inhibited thrombin and the formation of occlusive thrombi in AD; preserved cognition, cerebral perfusion, and BBB function; and ameliorated neuroinflammation and amyloid deposition in AD mice. Our results open a field for future investigation on whether the use of direct oral anticoagulants might be of therapeutic value in AD. This work was funded by a Proof-of-Concept Award from the Robertson Therapeutic Development Fund (Dr. Cortes-Canteli), The Rockefeller University; NINDS/NIH grant NIS106668 (Drs. Norris and Strickland); European Union’s Seventh Framework Programme (FP7-PEOPLE-2013-IIF), grant agreement n PIIF-GA-2013-624811 (Drs. Cortes-Canteli and Fuster), CNIC, Madrid, Spain; Miguel Servet type I research contract (CP16/00174 and MS16/00174 [Dr. Cortes-Canteli]), Instituto de Salud Carlos III (ISCIII), CNIC; Iniciativa de Empleo Juvenil (PEJ16/MED/TL-1231 [A. Marcos-Diaz] and PEJ-2018-AI/BMD-11477 [C. Ceron]) from Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid; European Regional Development Funds (FEDER “Una manera de hacer Europa”) and European Social Funds (FSE “El FSE invierte en tu futuro”); and with the support of the Marie Curie Alumni Association (Dr. Cortes-Canteli). The CNIC is supported by the ISCIII, the Spanish Ministerio de Ciencia, Innovación y Universidades (MCNU), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). CIC biomaGUNE is a Maria de Maeztu Unit of Excellence (MDM-2017-0720). Dr. Sanchez-Gonzalez is an employee of Philips Healthcare. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Sí

Background Particulate matter (particles < 2.5 μm [PM 2.5]) exposure during the in utero and postnatal developmental periods causes cardiac dysfunction during adulthood. Here, we investigated the potential priming effects of preconception exposure of PM 2.5 on cardiac function in adult offspring. Methods and Results Male and female friend leukemia virus b (FVB) mice were exposed to either filtered air (FA) or PM 2.5 at an average concentration of 38.58 μg/m3 for 6 hours/day, 5 days/week for 3 months. Mice were then crossbred into 2 groups: (1) FA male×FA female (both parents were exposed to FA preconception) and, (2) PM 2.5male×PM 2.5female (both parents were exposed to PM 2.5 preconception). Male offspring were divided: (1) preconception FA (offspring born to FA exposed parents) and, (2) preconception PM 2.5 (offspring born to PM 2.5 exposed parents) and analyzed at 3 months of age. Echocardiography identified increased left ventricular end systolic volume and reduced posterior wall thickness, reduced %fractional shortening and %ejection fraction in preconception PM 2.5 offspring. Cardiomyocytes isolated from preconception PM 2.5 offspring showed reduced %peak shortening, −dL/dT, TPS90 and slower calcium reuptake (tau). Gene and protein expression revealed modifications in markers of inflammation (IL‐6, IL‐15, TNFα, NFқB, CRP, CD26E, CD26P, intercellular adhesion molecule 1, and monocyte chemoattractant protein‐1) profibrosis (collagen type III alpha 1 chain), oxidative stress (NOS2), antioxidants (Nrf2, SOD, catalase), Ca2+ regulatory proteins (SERCA2a, p‐PLN, NCX), and epigenetic regulators (Dnmt1, Dnmt3a, Dnmt3b, Sirt1, and Sirt2) in preconception PM 2.5 offspring. Conclusions Preconception exposure to PM 2.5 results in global cardiac dysfunction in adult offspring, suggesting that abnormalities during development are not limited to the prenatal or postnatal periods but can also be determined before conception., See Editorial by Stapleton

Background Immune cells are key regulators of the vascular inflammatory response characteristic of hypertension. In hypertensive rodents, regulatory T lymphocytes (Treg, CD 4 + CD 25 + ) prevented vascular injury, cardiac damage, and endothelial dysfunction of mesenteric arteries. Whether Treg modulate the cerebrovascular damage induced by hypertension is unknown. Methods and Results C57 BL /6 mice were perfused with angiotensin II (Ang II ; 1000 ng/kg per minute) for 14 days and adoptive transfer of 3×10 5 CD 4 + CD 25 + T cells was performed via 2 intravenous injections. Control mice received a sham surgery and PBS . Treg prevented Ang II ‐induced neurovascular uncoupling ( P P II ‐induced neurovascular uncoupling ( P II ( P P P Conclusions Treg prevent impaired cerebrovascular responses in Ang II ‐induced hypertension. The neuroprotective effects of Treg involve the modulation of inflammation in the brain and periphery.

Background Left ventricular ( LV ) diastolic dysfunction often precedes heart failure with preserved ejection fraction, the dominant form of heart failure in postmenopausal women. The objective of this study was to determine the effect of oral estradiol treatment initiated early after ovariectomy on LV function and myocardial gene expression in female cynomolgus macaques. Methods and Results Monkeys were ovariectomized and randomized to receive placebo (control) or oral estradiol at a human‐equivalent dose of 1 mg/day for 8 months. Monkeys then underwent conventional and tissue Doppler imaging to assess cardiac function, followed by transcriptomic and histomorphometric analyses of LV myocardium. Age, body weight, blood pressure, and heart rate were similar between groups. Echocardiographic mitral early and late inflow velocities, mitral annular velocities, and mitral E deceleration slope were higher in estradiol monkeys (all P LV filling pressure. MCP1 (monocyte chemoattractant protein 1) and LV collagen staining were lower in estradiol animals ( P 1.2‐fold change; false discovery rate, P P Conclusions Estradiol treatment initiated soon after ovariectomy resulted in enhanced LV diastolic function, and altered myocardial gene expression towards decreased extracellular matrix deposition, improved myocardial contraction, and calcium homeostasis, suggesting that estradiol directly or indirectly modulates the myocardial transcriptome to preserve cardiovascular function.

Background Statins reduce aneurysm growth in mouse models of Marfan syndrome, although the mechanism is unknown. In addition to reducing cholesterol, statins block farnesylation and geranylgeranylation, which participate in membrane‐bound G‐protein signaling, including Ras. We dissected the prenylation pathway to define the effect of statins on aneurysm reduction. Methods and Results Fbn1 C1039G/+ mice were treated with (1) pravastatin (HMG‐CoA [3‐hydroxy‐3‐methylglutaryl coenzyme A] reductase inhibitor), (2) manumycin A ( MA ; FPT inhibitor), (3) perillyl alcohol ( GGPT 1 and ‐2 inhibitor), or (4) vehicle control from age 4 to 8 weeks and euthanized at 12 weeks. Histological characterization was performed. Protein analysis was completed on aortic specimens to measure ERK (extracellular signal‐regulated kinase) signaling. In vitro Fbn1 C1039G/+ aortic smooth muscle cells were utilized to measure Ras‐dependent ERK signaling and MMP (matrix metalloproteinase) activity. Pravastatin and MA significantly reduced aneurysm growth compared with vehicle control (n=8 per group). In contrast, PA did not significantly decrease aneurysm size. Histology illustrated reduced elastin breakdown in MA ‐treated mice compared with vehicle control (n=5 per group). Although elevated in control Marfan mice, both phosphorylated c‐Raf and phosphorylated ERK 1/2 were significantly reduced in MA ‐treated mice (4–5 per group). In vitro smooth muscle cell studies confirmed phosphorylated cR af and phosphorylated ERK 1/2 signaling was elevated in Fbn1 C1039G/+ smooth muscle cells (n=5 per group). Fbn1 C1039G/+ smooth muscle cell Ras‐dependent ERK signaling and MMP activity were reduced following MA treatment (n=5 per group). Corroborating in vitro findings, MMP activity was also decreased in pravastatin‐treated mice. Conclusions Aneurysm reduction in Fbn1 C1039G/+ mice following pravastatin and MA treatment was associated with a decrease in Ras‐dependent ERK signaling. MMP activity can be reduced by diminishing Ras signaling.

Background Hereditary hemorrhagic telangiectasia ( HHT ) is a rare genetic vascular disorder caused by mutations in endoglin ( ENG ), activin receptor‐like kinase 1 ( ACVRL 1 ; ALK 1 ), or SMAD 4 . Major clinical symptoms of HHT are arteriovenous malformations ( AVM s) found in the brain, lungs, visceral organs, and mucosal surface. Animal models harboring mutations in Eng or Alk1 recapitulate all of these HHT clinical symptoms and have been useful resources for studying mechanisms and testing potential drugs. However, animal models representing SMAD 4 mutations have been lacking. The goal of this study is to evaluate Smad4 ‐inducible knockout ( iKO ) mice as an animal model of HHT and compare the phenotypes with other established HHT animal models. Methods and Results Global Smad4 deletion was induced at neonatal and adult stages, and hemoglobin levels, gastrointestinal hemorrhage, and presence of aberrant arteriovenous connections were examined. Neonatal Smad4 ‐ iKO mice exhibited signs of gastrointestinal bleeding and AVM s in the brain, intestine, nose, and retina. The radial expansion was decreased, and AVM s were detected on both distal and proximal retinal vasculature of Smad4 ‐ iKO s. Aberrant smooth muscle actin staining was observed in the initial stage AVM s and their connecting veins, indicating abnormal arterial flow to veins. In adult mice, Smad4 deficiency caused gastrointestinal bleeding and AVM s along the gastrointestinal tract and wounded skin. HHT ‐related phenotypes of Smad4 ‐ iKO s appeared to be comparable with those found in Alk1 ‐ iKO and Eng ‐ iKO mice. Conclusions These data further confirm that SMAD signaling is crucial for normal arteriovenous network formation, and Smad4 ‐ iKO will be an alternative animal model of AVM research associated with HHT .

Background Neurological deficits in hypoxic‐ischemic encephalopathy, even with therapeutic hypothermia, are partially attributed to white matter injury. We theorized that proteasome insufficiency contributes to white matter injury. Methods and Results Neonatal piglets received hypoxia‐ischemia ( HI ) or sham procedure with normothermia, hypothermia, or hypothermia+rewarming. Some received a proteasome activator drug (oleuropein) or white matter–targeted, virus‐mediated proteasome knockdown. We measured myelin oligodendrocyte glycoprotein, proteasome subunit 20S (P20S), proteasome activity, and carbonylated and ubiquitinated protein levels in white matter and cerebral cortex. HI reduced myelin oligodendrocyte glycoprotein levels regardless of temperature, and myelin oligodendrocyte glycoprotein loss was associated with increased ubiquitinated and carbonylated protein levels. Ubiquitinated and carbonyl‐damaged proteins increased in white matter 29 hours after HI during hypothermia to exceed levels at 6 to 20 hours. In cortex, ubiquitinated proteins decreased. Ubiquitinated and carbonylated protein accumulation coincided with lower P20S levels in white matter; P20S levels also decreased in cerebral cortex. However, proteasome activity in white matter lagged behind that in cortex 29 hours after HI during hypothermia. Systemic oleuropein enhanced white matter P20S and protected the myelin, whereas proteasome knockdown exacerbated myelin oligodendrocyte glycoprotein loss and ubiquitinated protein accumulation after HI . At the cellular level, temperature and HI interactively affected macroglial P20S enrichment in subcortical white matter. Rewarming alone increased macroglial P20S immunoreactivity, but this increase was blocked by HI . Conclusions Oxidized and ubiquitinated proteins accumulate with HI ‐induced white matter injury. Proteasome insufficiency may drive this injury. Hypothermia did not prevent myelin damage, protect the proteasome, or preserve oxidized and ubiquitinated protein clearance after HI .

Background Tetrahydrobiopterin is a cofactor of endothelial NO synthase ( eNOS ), which is critical to embryonic heart development. We aimed to study the effects of sapropterin (Kuvan), an orally active synthetic form of tetrahydrobiopterin on eNOS uncoupling and congenital heart defects ( CHD s) induced by pregestational diabetes mellitus in mice. Methods and Results Adult female mice were induced to pregestational diabetes mellitus by streptozotocin and bred with normal male mice to produce offspring. Pregnant mice were treated with sapropterin or vehicle during gestation. CHD s were identified by histological analysis. Cell proliferation, eNOS dimerization, and reactive oxygen species production were assessed in the fetal heart. Pregestational diabetes mellitus results in a spectrum of CHD s in their offspring. Oral treatment with sapropterin in the diabetic dams significantly decreased the incidence of CHD s from 59% to 27%, and major abnormalities, such as atrioventricular septal defect and double‐outlet right ventricle, were absent in the sapropterin‐treated group. Lineage tracing reveals that pregestational diabetes mellitus results in decreased commitment of second heart field progenitors to the outflow tract, endocardial cushions, and ventricular myocardium of the fetal heart. Notably, decreased cell proliferation and cardiac transcription factor expression induced by maternal diabetes mellitus were normalized with sapropterin treatment. Furthermore, sapropterin administration in the diabetic dams increased eNOS dimerization and lowered reactive oxygen species levels in the fetal heart. Conclusions Sapropterin treatment in the diabetic mothers improves eNOS coupling, increases cell proliferation, and prevents the development of CHD s in the offspring. Thus, sapropterin may have therapeutic potential in preventing CHD s in pregestational diabetes mellitus.

Background The sodium channel, Na v 1.5, encoded by SCN 5A , undergoes developmentally regulated splicing from inclusion of exon 6A in the fetal heart to exon 6B in adults. These mutually exclusive exons differ in 7 amino acids altering the electrophysiological properties of the Na v 1.5 channel. In myotonic dystrophy type 1, SCN 5A is mis‐spliced such that the fetal pattern of exon 6A inclusion is detected in adult hearts. Cardiac manifestations of myotonic dystrophy type 1 include conduction defects and arrhythmias and are the second‐leading cause of death. Methods and Results This work aimed to determine the impact of SCN 5A mis‐splicing on cardiac function. We used clustered regularly interspaced short palindromic repeat ( CRISPR) /CRISPR‐associated protein 9 (Cas9) to delete Scn5a exon 6B in mice, thereby redirecting splicing toward exon 6A. These mice exhibit prolonged PR and QRS intervals, slowed conduction velocity, extended action potential duration, and are highly susceptible to arrhythmias. Conclusions Our findings highlight a nonmutational pathological mechanism of arrhythmias and conduction defects as a result of mis‐splicing of the predominant cardiac sodium channel. Animals homozygous for the deleted exon express only the fetal isoform and have more‐severe phenotypes than heterozygotes that also express the adult isoform. This observation is directly relevant to myotonic dystrophy type 1, and possibly pathological arrhythmias, in which individuals differ with regard to the ratios of the isoforms expressed.

Background The American Heart Association recommends use of physiologic feedback when available to optimize chest compression delivery. We compared hemodynamic parameters during cardiopulmonary resuscitation in which either end‐tidal carbon dioxide ( ETCO 2 ) or diastolic blood pressure ( DBP ) levels were used to guide chest compression delivery after asphyxial cardiac arrest. Methods and Results One‐ to 2‐week‐old swine underwent a 17‐minute asphyxial‐fibrillatory cardiac arrest followed by alternating 2‐minute periods of ETCO 2 ‐guided and DBP ‐guided chest compressions during 10 minutes of basic life support and 10 minutes of advanced life support. Ten animals underwent resuscitation. We found significant changes to ETCO 2 and DBP levels within 30 s of switching chest compression delivery methods. The overall mean ETCO 2 level was greater during ETCO 2 ‐guided cardiopulmonary resuscitation (26.4±5.6 versus 22.5±5.2 mm Hg; P =0.003), whereas the overall mean DBP was greater during DBP ‐guided cardiopulmonary resuscitation (13.9±2.3 versus 9.4±2.6 mm Hg; P =0.003). ETCO 2 ‐guided chest compressions resulted in a faster compression rate (149±3 versus 120±5 compressions/min; P =0.0001) and a higher intracranial pressure (21.7±2.3 versus 16.0±1.1 mm Hg; P =0.002). DBP ‐guided chest compressions were associated with a higher myocardial perfusion pressure (6.0±2.8 versus 2.4±3.2; P =0.02) and cerebral perfusion pressure (9.0±3.0 versus 5.5±4.3; P =0.047). Conclusions Using the ETCO 2 or DBP level to optimize chest compression delivery results in physiologic changes that are method‐specific and occur within 30 s. Additional studies are needed to develop protocols for the use of these potentially conflicting physiologic targets to improve outcomes of prolonged cardiopulmonary resuscitation.

Background Dysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease ( CKD ). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD , not only by direct effects on the endothelium but also by an increment of α‐Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia‐induced endothelial damage and the extent to which this was dependent on increased α‐Klotho concentrations. Methods and Results In a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial‐gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α‐Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real‐time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro‐permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial‐cadherin–based cell‐cell junctions and diminished F‐actin stress fiber organization, preventing the formation of endothelial intracellular gaps. Conclusions Our results demonstrate that paricalcitol attenuates the CKD ‐induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell‐cell interactions.

Background Hypertension is a complex condition and a common cardiovascular risk factor. Dietary docosahexaenoic acid ( DHA ) modulates atherosclerosis and hypertension, possibly via an inflammatory mechanism. IL‐1 (interleukin 1) has an established role in atherosclerosis and inflammation, although whether IL ‐1 inhibition modulates blood pressure is unclear. Methods and Results Male apoE −/− (apolipoprotein E–null) mice were fed either a high fat diet or a high fat diet plus DHA (300 mg/kg per day) for 12 weeks. Blood pressure and cardiac function were assessed, and effects of DHA on wall shear stress and atherosclerosis were determined. DHA supplementation improved left ventricular function, reduced wall shear stress and oscillatory shear at ostia in the descending aorta, and significantly lowered blood pressure compared with controls (119.5±7 versus 159.7±3 mm Hg, P DHA feeding in mice demonstrated a 4‐fold reduction in lesion burden in distal aortas and in brachiocephalic arteries ( P DHA treatment selectively decreased plaque endothelial IL ‐1β ( P Conclusions Our findings revealed that raised blood pressure can be reduced by inhibiting IL ‐1 indirectly by administration of DHA in the diet through a mechanism that involves a reduction in wall shear stress and local expression of the proinflammatory cytokine IL ‐1β.