Nature Communications 8: Article number: 14095 (2017); Published: 24 January 2017; Updated: 30 August 2017. The authors inadvertently omitted Eltyeb Abdelwahid, who contributed to the generation of ...animal models and their initial evaluation, from the author list. This has now been corrected in both the PDF and HTML versions of the Article.
Hypoxia-inducible factors (HIFs) are the master regulators of angiogenesis, a process that is impaired in patients with diabetes mellitus (DM). The transcription factor aryl hydrocarbon receptor ...nuclear translocator (ARNT, also known as HIF1β) has been implicated in the development and progression of diabetes. Angiogenesis is driven primarily by endothelial cells (ECs), but both global and EC-specific loss of ARNT-cause are associated with embryonic lethality. Thus, we conducted experiments in a line of mice carrying an inducible, EC-specific ARNT-knockout mutation (
) to determine whether aberrations in ARNT expression might contribute to the vascular deficiencies associated with diabetes. Mice were first fed with a high-fat diet to induce diabetes.
mice were then adminstrated with oral tamoxifen to disrupt
and peripheral angiogenesis was evaluated by using laser-Doppler perfusion imaging to monitor blood flow after hindlimb ischemia. The
mice had impaired blood flow recovery under both non-diabetic and diabetic conditions, but the degree of impairment was greater in diabetic animals. In addition, siRNA-mediated knockdown of ARNT activity reduced measurements of tube formation, and cell viability in human umbilical vein endothelial cells (HUVECs) cultured under high-glucose conditions. The
mutation also reduced measures of cell viability, while increasing the production of reactive oxygen species (ROS) in microvascular endothelial cells (MVECs) isolated from mouse skeletal muscle, and the viability of
MVECs under high-glucose concentrations increased when the cells were treated with an ROS inhibitor. Collectively, these observations suggest that declines in endothelial ARNT expression contribute to the suppressed angiogenic phenotype in diabetic mice, and that the cytoprotective effect of ARNT expression in ECs is at least partially mediated by declines in ROS production.
The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS mice or from pharmacological inhibition of nNOS are ...contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of H-l-arginine to H-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17±3% decrease of +dp/dtmax compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR CaATPase and additionally with L-type Ca- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, ICa,L density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca-channels and in turn reduces Ca-transients which accounts for the negative inotropic effect.
MicroRNA-210 (miR-210) increases in hypoxia and regulates mitochondrial respiration through modulation of iron-sulfur cluster assembly proteins (ISCU1/2), a protein that is involved in Fe/S cluster ...synthesis. However, it is not known how miR-210 affects cellular iron levels or production of heme, another iron containing molecule that is also needed for cellular and mitochondrial function.
To screen for micro-ribonucleic acids (miRNAs) regulated by iron, we performed a miRNA gene array in neonatal rat cardiomyocytes treated with iron chelators. Levels of miR-210 are significantly increased with iron chelation, however, this response was mediated entirely through the hypoxia-inducible factor (HIF) pathway. Furthermore, miR-210 reduced cellular heme levels and the activity of mitochondrial and cytosolic heme-containing proteins by modulating ferrochelatase (FECH), the last enzyme in heme biosynthesis. Mutation of the 2 miR-210 binding sites in the 3' untranslated region (UTR) of FECH reversed the miR-210 response, while mutation of either binding site in isolation did not exert any effects. Changes mediated by miR-210 in heme and FECH were independent of ISCU, as overexpression of an ISCU construct lacking the 3' UTR does not alter miR-210 regulation of heme and FECH. Finally, FECH levels increased in hypoxia, and this effect was not reversed by miR-210 knockdown, suggesting that the effects of miR-210 on heme are restricted to normoxic conditions, and that the pathway is overriden in hypoxia.
Our results identify a role for miR-210 in the regulation of heme production by targeting and inhibiting FECH under normoxic conditions.
The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the ...nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importinβ1 to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, H-leucine incorporation (159%±9 versus 111%±11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%±11 versus 133%±8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%±9 versus 270%±11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity.
Abstract only Rongxue Wu, Kusum Chawla, Yoshihiko Ichikawa, Mohsen Granefar Hossein Ardehali Background: Aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of basic helix-loop-helix ...Per/ARNT/Sim (bHLH-PAS) proteins and serves as a binding partner for a number of other family members. ARNT is also a required dimerization partner of HIF1a. Although HIF1a is known to be required for normal cardiac development, the role of ARNT in basal cardiac function in the adult heart is not known. We hypothesized that ARNT is required for normal cardiac physiology, and its deletion in adult heart results in cardiomyopathy. Methods and Results: The deletion of ARNT gene in the heart of 3 month old mice was achieved by crossing ARNT flox/flox mice with αMHC-MCM (tamoxifen-inducible heart specific Cre) transgenic mice followed by administration of tamoxifen chow. MHC-MCM/ARNT +/+ (WT) littermates were used as a control. The ARNT knock out (KO) mice exhibited enlarged left ventricle with a significant reduction in ejection fraction (KO vs. WT: 35.8 ± 3.6% vs. 61.2 ± 2.8%, n=12, p<0.01) and fractional shortening (KO vs. WT: 18.3 ± 1.5 % vs. 30.1 ± 1.1%, p<0.01), assessed by Echocardiography. Closed-chest catheterization also demonstrated reduced +dP/dt (KO vs. WT: 4355 ± 538 vs. 9426 ± 180 mmHg, p<0.01) and increased left ventricular end diastolic pressure (KO vs. WT: 3.12 ± 1.1 vs. 8.47± 1.2 mmHg, P<0.05) in KO mice but not in WT mice. The worsened cardiac function in the KO mouse heart was associated with increase in ANF and BNP expression, interstitial fibrosis, and apoptosis as determined by TUNEL staining. Furthermore, electron microscopy reveals a variety of degenerative changes and some lipid droplet in the KO hearts, and histological studies demonstrated intramyocardial lipid accumulation in the perivascular area in the KO mouse hearts. Analysis of gene expression in the KO heart revealed up-regulation of peroxisome proliferator-activated receptor alpha. Conclusion: ARNT is essential for the maintenance of structural and functional homeostasis in the adult heart, and its inactivation leads to cardiac contractile dysfunction. Our findings implicate a novel critical transcriptional requirement for ARNT in the maintenance of adult cardiac function.
The role of estrogens during myocardial ischemia has been extensively studied. However, effects of a standard hormone replacement therapy including 17β-estradiol (E2) combined with ...medroxyprogesterone acetate (MPA) have not been assessed, and this combination could have contributed to the negative outcomes of the clinical studies on hormone replacement. We hypothesized that adding MPA to an E2 treatment would aggravate chronic heart failure after experimental myocardial infarction (MI). To address this issue, we evaluated clinical signs of heart failure as well as left ventricular (LV) dysfunction and remodeling in ovariectomized rats subjected to chronic MI receiving E2 or E2 plus MPA. After eight weeks MI E2 showed no effects. Adding MPA to E2 aggravated LV remodeling and dysfunction as judged by increased heart weight, elevated myocyte cross-sectional areas, increased elevated left ventricle end diastolic pressure, and decreased LV fractional shortening. Impaired LV function in rats receiving MPA plus E2 was associated with increased cardiac reactive oxygen species generation and myocardial expression levels of NADPH oxidase subunits. These results support the interpretation that adding MPA to an E2 treatment complicates cardiovascular injury damage post-MI and therefore contributes to explain the adverse outcome of prospective clinical studies.
Abstract only
Introduction:
Mitochondrial injury occurs following cardiac arrest (CA) and contributes to myocardial stunning and anoxic brain injury. Hypoxia has been proposed as a therapeutic ...strategy for neurodegenerative diseases associated with mitochondrial respiration defects, but its benefits in the post-CA setting remain unexplored. Here, we hypothesized mild hypoxia as therapy for post-CA mitochondrial injury.
Methods and Results:
Mice underwent brief asystolic CA (12 min) followed by cardiopulmonary resuscitation (CPR). One hour following successful CPR, mice were randomized to receive a brief episode (6 hours) of hyperoxia (33% O
2
), normoxia (21% O
2
), or hypoxia (10% O
2
) in an environmental chamber. Post exposure, the mice were returned to room air (21% O
2
). Hypoxia improved myocardial fractional shortening compared to normoxia and hyperoxia (Hypoxia: 45.6±2.3 %, Normoxia: 40.0±0.8 %, Hyperoxia: 29.0±4.0 %, n=5, P<0.05, respectively). Hypoxia also elevated neurological scores (Hypoxia: 8.4±1.3, Hyperoxia: 5.6±1.2, n=5, P<0.05) 48 hours post CA compared to hyperoxia. Ten-day survival was prolonged by hypoxia (Hypoxia: 87.5 %, Normoxia: 62.5 %, Hyperoxia: 12.5 %, n=10, P<0.05, respectively). Hypoxia also improved mitochondrial function (28% increase in basic oxygen consumption and 49% increase in ATP-related oxygen consumption, n=3, P<0.05, respectively) and a 20% decrease in ROS production (n=6, P<0.05) compared to normoxia.
Conclusion:
This study has two important findings:
First
, we demonstrate that oxygen availability in the early post-CA period is a critical determinant of long-term post-CA outcomes.
Second
, we show that mild hypoxic therapy in the early post-CA period improves early mitochondrial function and long-term post-CA outcomes. These findings reveal an important oxygen-dependent therapeutic window following resuscitation from CA that has implications for post-CA critical care.
Abstract only Introduction: Cardiovascular dysfunction is a major complication of severe sepsis. Microvascular leakage and mitochondrial dysfunction during sepsis are critically involved in heart ...failure. However, the underlying mechanisms between cardiac microvascular barrier function and sepsis-induced heart failure are not well studied. Kinin B1 receptor (B1R) regulates inflammation and induces capillary leakage. Previous findings from our lab also revealed an increase in B1R serum level in both septic patients and animal models within 24 hours. Hypothesis: We hypothesized that treatment with B1R antagonist protects against sepsis-induced microvascular leakage and mitochondrial dysfunction. Methods and Results: To address the hypothesis, we pre-treated WT male mice with either vehicle (control) or a specific B1R antagonist B6929 via subcutaneous injection, then subjected them to 40% cecal ligation and puncture (CLP) to induce polymicrobial sepsis. Echocardiographic analysis revealed an early cardioprotective effect of B1R inhibition, as evidenced by the improvement of EF%, FS%, and cardiac output in mice pre-treated with B1R antagonist (n=6-8, p<0.01) at 4-6 hours post-CLP. Furthermore, mitochondrial H 2 O 2 measurement of the freshly isolated cardiac mitochondria from CLP-induced septic mice also showed that B1R inhibition partially abolished the production of H 2 O 2 . The opening of mPTP was also reduced with B1R antagonist treatment. In vivo cardiac vascular permeability assessed using the Miles assay further demonstrated that B1R inhibition prevented sepsis-induced cardiac microvascular leakage and edema. Similar improvements were also observed in the liver and kidney. Interestingly,B1R antagonist treatment abolished CLP-induced serum level of IL-6 by 70%. In in-vitro study, B1R antagonist treatment inhibited LPS-induced IL-6 expressions in human cardiac microvacuolar endothelial cells (HCMECs). Finally, ECIS based analysis demonstrated that IL-6 deletion using either siRNA or B1R antagonist treatment rescued LPS-induced endothelial barrier leakage. Conclusions: Our findings suggest that targeting B1R via IL-6 may be a novel therapeutic approach for the treatment of sepsis-associated cardiovascular disease.
Abstract only Introduction: Oxygen administration following cardiac arrest (CA) is associated with cardiac dysfunction, brain injury, and poor survival. These injuries are driven in part by ...mitochondrial injury, but the specific mechanisms underlying oxygen-induced mitochondrial injury are unclear. Here we hypothesized that oxygen-mediated injury following CA is influenced by the mitochondrial fission protein Dynamin Related Protein 1 (Drp1) and that targeting Drp1 could mitigate the effects of post-CA oxygen toxicity. Methods and Results: Mice underwent brief asystole CA (12 min) followed by cardiopulmonary resuscitation (CPR). One hour following successful CPR, mice were randomized to receive hyperoxia (33% O 2 ), normoxia (21% O 2 ), or hypoxia (10% O 2 ) in an environmental chamber for 6 hours. After exposure, the mice were returned to room air. Hyperoxia and normoxia groups exhibited lower 10-day survival rate compared to mice exposed to hypoxia (15% and 67% vs. 92%, n=13/group, P<0.05, respectively). The hyperoxia group also showed decreased myocardial fractional shortening (30.1±3.0% vs 45.0±1.4%) and neurological scores (5.8±0.8 vs 9.8±0.3) 48 hours post CA compared to the hypoxia group (n=13, P<0.05, respectively). Hyperoxia increased the expression of the activated form of Drp1 (Drp1-S616) to 134% in the heart (n=4, P<0.05) and decreased cardiomyocyte mitochondrial size to 75% (n=30, P<0.05) compared to hypoxia-treated CA mice. The hypoxia-dependent decrease in Drp1 activity was associated with a 46% increase in myocardial mitochondrial respiration (n=5, P<0.01) and a 32% reduction in ROS production vs hyperoxia-treated CA mice (n=6, P<0.05). Administration of the Drp1 GTPase inhibitor Drpitor1a to hyperoxia-exposed post-CA mice increased survival (40% vs 20%), myocardial function (31.5±4.4% vs 42.0±1.3%) and neurological scores (4.1±0.7 vs 9.0±0.0) compared to untreated hyperoxia-exposed post-CA mice (n=10, P<0.05, respectively). Conclusions: Oxygen dependent post-CA injury is partly dependent on the mitochondrial fission factor Drp1. Targeting Drp1 is a promising strategy to limit post-CA oxygen toxicity.
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