bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 837: Beta1 And Beta2 Adrenergic Receptor Transgenic Mice Display Distinct MicroRNA Expression Patterns That Converge On The Hypertrophic Signature
Authors: Sayed Danish , Rane Shweta , Chen Leng-Yi , He Minzhen , Lypowy Jacqueline , Engelhardt Stefan , Yan Lin , Vatner Dorothy , Sadoshima Junichi , Abdellatif Maha
Published: July 2021

MicroRNA (miRNA) are ~22 ribonucleotides-long, with a potential to recognize multiple mRNA targets guided by sequence complimentarity. This class of molecules is functionally versatile, with the capacity to specifically inhibit translation, as well as, induce mRNA degradation, through targeting the 3′-untranslated regions. The levels of individual miRNA vary under different developmental, biological, or pathological conditions, thus, implicating them in normal and pathological cellular attributes. We have previously reported a miRNA signature that distinguishes pressure-overload compensatory hypertrophy by recapitulating the neonatal pattern. We hypothesized that this ’signature’ might aid in discriminating the underlying molecular differences in genetic models of cardiac hypertrophy, as seen in the beta1 and 2 adrenergic receptor (B1AR and B2AR) transgenic (Tg) mice. To address this, we used microarray analysis of RNA isolated from the hearts of 3 months old B1AR and B2AR mice. In general, while both mice exhibited an overlap with the hypertrophy signature including, upregulation of miR-21 and downregulation of miR-133a, miR-133b, and miR-185, the B2-AR Tg exhibited a more extensive overlap with the hypertrophy pattern, which further included upregulation of miR-199a*, miR-214, and miR-15b. To understand the functional significance of these miRNA in myocyte hypertrophy, we cloned them and their anti-sense sequences into adenoviral vectors. Significantly, over-expression miR-21 resulted in a, dose-dependent, branching (sprouting) of the cells. Computational predictions by ’TargetScanS’ identified sprouty as potential target. Subsequently, we confirmed down-regulation of sprouty by over-expression of miR-21 and vice versa. Sprouty is a known inhibitor of the Ras-MAPK signaling pathway and is, concordantly, downregulated in many forms of cancer. In the heart, sprouty has been suggested to control myocyte size and vascularization during cardiac hypertrophy. Thus, we propose that B1AR and B2AR Tg models exhibit distinct miRNA profiles that converge on that of pressure-overload cardiac hypertrophy. Moreover, the commonly over-expressed miR-21 plays a role in downregulating sprouty, an antagonist of the Ras-MAPK pathway.
Abstract 842: Messenger RNA Processing Body Participates In Cardiac Protection Induced By Heat Shock Preconditioning In Mouse Hearts
Authors: Yin Chang , Kukreja Rakesh C
Published: July 2021

mRNA Processing Body (P-Body) is a specialized cytoplasmic structure that functions as a major site for post-transcriptional and trnanslational repression. Target mRNA is guided into P-Body through base-pairing with its micro-RNA (miRNA), where they further bind to P-Body components such as GW182 and AGO2; thus get retained away from translation machinery or degraded. Our previous study detected an increase of a key component of P-Body, miRNA, in heat-shock (HS) protected hearts. To gain further insights into the protective mechanism, we hypothesized that the miRNA-associated protection is mediated through P-Body formation. This hypothesis was tested by measuring three key components of P-Body, i.e., miRNA (target seeker), GW182 (marker of P-Body, also called GW-Body) and AGO2 (mRNA catalytic enzyme).

METHODS
: ICR mice were either HS-preconditioned (15 min, 42°C, anal temperature) or kept at room temperature (controls). miRNA and proteins were extracted 2 hour after HS. miRNA induction was verified by RT-PCR. P-Body formation was evaluated by measuring the binding of GW182 and AGO2, using a combination of immunopricipitation and Western blotting techniques. To study the role of miRNA in P-Body formation, identical experiments were also repeated in mice treated with miRNA-1 inhibitor (antisense miRNA-1 with 2′-O-methyl base at every nucleotide).

RESULTS
: Compared to the control, HS-preconditioning significantly induced miRNA-1 (150 ± 11%, mean ± SEM), miRNA-21 (71 ± 10%) and miRNA-24 (68 ± 14%). GW182 (109 ± 16%) and AGO2 (50 ± 16%) were also increased in the HS-group. More importantly, there was an increase (39 ± 11%) in co-immunopricipitation between GW182 and AGO2 in the HS-group than in the control, indicating more binding of the two key P-Body components. However, this co-immunoprecipitation was significantly reduced (−68 ± 8%) in the mice treated with the miRNA-1 inhibitor after HS.

CONCLUSION
: The formation of P-Body following HS-preconditioning represents a novel protective pathway against ischemic injury. The pharmaceutical potential of P-Body formation may offer a new strategy in cardiac preconditioning.
Abstract 851: The Cardioprotective Effect Of Glycogen Synthase Kinase-3β (gsk-3β) Inhibitors Involves Inhibition Of Mitochondrial Adenine Nucleotide Transport
Authors: Das Samarjit , Murphy Elizabeth , Steenbergen Charles
Published: July 2021

Inhibition of GSK-3β has been shown to reduce ischemia-reperfusion injury by acting on the mitochondria. We aimed to determine the mechanism by which inhibition of GSK-3β mediates cardioprotection. Rats were treated with specific GSK-3β inhibitors, SB216763 (3 μM) or SB415286 (10 μM) 15 min prior to ischemia. Both inhibitors significantly improved post-ischemic left ventricular function after 20 minutes of ischemia compared to control. Mitochondria were isolated from all three groups of hearts immediately after 15 min perfusion with/without GSK-3β inhibitors. Both the SB compounds significantly reduced state 3 respiration as well as ATP consumption during anoxia. Consumption of ATP during anoxia was measured by allowing mitochondria in an oxygraph chamber to consume oxygen until they became anoxic, and the rate of consumption of added ATP was measured at 20, 40, and 60 minutes of anoxia. GSK-3β inhibitors significantly slowed anoxic mitochondrial ATP consumption. Similarly, GSK-3β inhibitors significantly reduced ATP consumption when sodium cyanide was used to stop mitochondrial respiration and also when the mitochondria were deenergized by uncoupler addition. This reduction in ATP consumption could be due to inhibition of ATP entry into the mitochondria through Voltage dependent anion channel (VDAC) and/or the adenine nucleotide transporter (ANT), inhibition of some other unknown protein which may directly inhibit mitochondrial transport, or inhibition of the F1F0 ATPase. To identify proteins that might be involved in this reduced ATP consumption, western blot analysis with phospho-Akt substrate antibody and 1-D gel phosphorylation site analysis staining revealed the GSK-3β inhibitors significantly decrease the phosphorylation of a 32 kDa protein compared to control mitochondria. We then cut out the 32 kDa band and used mass spectrometry to identify proteins. Voltage dependent anion channel members 1, 2 and 3 were all identified along with the adenine nucleotide translocator and γ-subunit of the F1 ATPase. Taken together the data indicate that GSK-3β regulates mitochondrial energetics under anoxic conditions, which may play an important role in cardioprotection.
Abstract 852: Conditional Overexpression Of Neuronal Nitric Oxide Synthase Is Cardioprotective In Ischemia-reperfusion
Authors: Burkard Natalie , Rokita Adam , Kaufmann Susann , Hallhuber Matthias , Gebhardt Claudia , Hu Kai , Neyses Ludwig , Maier Lars , Schuh Kai , Ritter Oliver
Published: July 2021

Introduction: We previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nNOS, NOS1) inhibited L-type Ca
2+
-channels. We now hypothesize that nNOS overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion.

Methods and results: We assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nNOS overexpression. NOS-activity (22 ± 1.5 vs. 29 ± 1μM /sec, n=18, p<0.05) was significantly enhanced after nNOS overexpression. Co-immunoprecipitation experiments indicated interaction of nNOS with SR Ca
2+
ATPase and additionally with L-type Ca
2+
-channels in nNOS overexpressing animals. I
ca,L
(reduction of 40±6 rel. %, n=12, p<0.05) as well as intracellular Ca
2+
-transients and fractional shortening in cardiomyocytes were clearly impaired in nNOS overexpressing mice (3.0 ± 0.4F/F
0
vs. 2.2 ± 0.2F/F
0
, n=13, p<0.005 and 7.7 ± 1.3% vs. 3.8 ± 0.5%, n=13, p<0.05). In vivo examinations of the nNOS overexpressing mice showed a decrease of +dp/dt
max
(reduction for 52 ± 17%, n=12, p<0.05) as well as a reduced ejection fraction (43±5% vs. 63±9%, n=12, p<0.05). Ischemia-reperfusion experiments showed a cardio-protective effect of nNOS overexpression (30 min post-ischemia, LVDP 20±6 in non-induced animals vs. 60±11 mmHg in nNOS overexpressing animals, n=6, p<0.05).

Discussion: In summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nNOS resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the L-type Ca
2+
-channel. In contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nNOS overexpression acts cardioprotective. These effects might be caused by a reduction of myocardial Ca
2+
-overload after reperfusion.
Abstract 892: The Importance of Mitochondrial Oxidative Stress and Integrity of Mitochondrial DNA in Coronary Arteriogenesis
Authors: Rocic Petra , Wilson Glenn , Shokolenko Inna , Joseph Joy , Kalyanaraman Balaraman , Chilian William
Published: July 2021

The prediabetic metabolic syndrome (pdMS) leads to many pathological cardiac and vascular phenotypes including impaired arteriogenesis. Mitochondrial (mt) oxidative stress (mtOxStress) is postulated to contribute to the development of such phenotypes, but whether reduction of mtOxStress, or imposition of a maneuver to mimic its effects, would restore or inhibit coronary arteriogenesis (CA), respectively, has not been determined. We tested the hypothesis that in the pdMS, mtOxStress plays a key role in inhibition of coronary arteriogenesis. Lean (LN, n = 6) and obese (OB, n = 6) Zucker obese fatty rats (model of pdMS) were instrumented with a pneumatic snare around the left anterior descending artery (LAD), and subjected to a protocol of repetitive ischemia (RI: 24, 40 sec occlusions/day for 10 days) to stimulate growth. CA was determined from the ratio of flows (radioactive microspheres) measured during LAD occlusion to collateral-dependent and normal zones (CZ/NZ). Prior to RI, CZ/NZ in all groups was less than 0.2 indicating that the native collateral conductance could supply less than 20% of the normally occurring flow during LAD occlusion. After RI, CZ/NZ was 0.82Â ± 0.09 in LN, which was markedly higher (p < 0.05) than that in the OB (0.21Â ± 0.08) indicating impaired CA in the OB rats. To reduce mtOxStress, OB animals (n = 5) were given mito-Quinone (mt-directed free radical scavenger) in drinking water (750 mg/L); this treatment restored CA (CZ/NZ = 0.79Â ± 0.03) comparable to that in LN. Because one manifestation of mt-OxStress is mt-DNA fragmentation, which results in impaired expression of key mt-proteins and compromised mt-function, we determined if administration of a recombinant protein engineered with a TAT sequence to enable intracellular delivery, a mt-localization sequence, and exonuclease ExoIII (mt-tat-ExoIII) to fragment mtDNA would impair coronary arteriogenesis in LN (n = 5). Mt-tat-ExoIII (iv, 0.2 ug/g, every other day) in LN rats impaired CA in response to RI (CZ/NZ = 0.32Â ± 0.09, p < 0.05 vs. LN controls). We conclude that mitochondrial oxidative stress, and its effects on mtDNA integrity, are pivotal in the abrogation of CA in the pdMS.
Abstract 894: Leptin Potentiates the Angiogenic Properties of Endothelial Progenitor Cells In Vitro and In Vivo
Authors: Heida Nana-Maria , Schroeter Marco R , Cheng I-Fen , Deryugina Elena I , Korff Thomas , Quigley James P , Ruggeri Zaverio M , Mueller Jan-Peter , Hasenfuss Gerd , Konstantinides Stavros , Schaefer Katrin
Published: July 2021

Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.
Abstract 908:
In Vivo
Role of the Pharmacogenetics of Glucuronidation in the Disposition and Triglyceride Response to Fenofibrate
Authors: Straka Robert J , Li Na , Guillemette Chantal , Tsai Michael Y , Hanson Naomi Q , Ordovas Jose M , Arnett Donna K
Published: July 2021

In spite of optimal low-density lipoprotein (LDL-C) lowering with statins, cardiovascular risk remains high for patients with elevated triglyceride (TG)-low high-density cholesterol (HDL-C) profiles. Fenofibric acid, the active moiety of fenofibrate, acts on the peroxisome proliferator-activated nuclear receptor-alpha (PPAR-α) to lower TG and raise HDL-C. The extent of lipid response has been shown to be associated with serum concentrations (conc.) of fenofibric acid.
In vitro
work confirms fenofibric acid is primarily eliminated by UDP-glucuronosyltransferases (UGTs) including UGT2B7, and others (UGT1A1, UGT1A3 and UGT1A9). Therefore, we hypothesized genetic variations in UGT activity may be associated with fenofibric acid conc. and thus may influence lipid response.

Purpose:
To test if the A-327G SNP (rs7662029) for UGT2B7, which has been shown to modulate UGT2B7 expression
in vitro,
is associated with serum conc. of fenofibric acid and TG response
in vivo
.

Methods:
As part of the NHLBI sponsored Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study, participants providing consent and not currently taking lipid lowering medications, were analyzed for fasting lipid response pre and post 21 days of once daily 160mg fenofibrate. TG response was calculated by the log of the ratio of the average of the 2 pre- and post-fenofibrate exposure TG determinations. Associations between UGT2B7 A-327G genotype and steady-state trough fenofibric acid conc. as well as TG response were analyzed by linear regression.

Results
: 745 participants (51% male) with both genotype and phenotype data were included. The mean (SD) age was 49 (16) years and the median (25
th
,75
th
percentile) % change in TG concentrations was -32.3 (−45.2, −19.0). The G allele frequency for UGT2B7A>G was 0.49 and was in HWE (p = 0.8). After adjusting for age, age
2
, sex, alcohol use, height and serum creatinine, strong associations were observed between UGT2B7 A-327G genotypes and steady-state trough fenofibric acid conc. (log-transformed, p = 4 × 10
−8
) as well as TG response (p = 0.002, also adjusted for baseline TG).

Conclusion
: This study suggests UGT2B7 A-327G genotype may contribute to TG response by way of influencing glucuronidation activity of fenofibric acid.
Abstract 925: Rit GTPase Protects Cells from Oxidative Damage
Authors: Rudolph Jennifer L , Shi Geng-Xian , Harrison Susan M , Harrison Douglas A , Andres Douglas A
Published: July 2021

Signaling cascades that contribute to the regulation of cellular survival influence many cardiovascular diseases. Elucidating the mechanisms of these cascades is thus important for understanding basic biology and for therapeutic intervention. We have identified an evolution-arily conserved group of Ras-related GTPases, including two mammalian genes (Rit and Rin) and a single Drosophila ortholog (Ric), that we hypothesize plays a critical role in trophic factor-mediated anti-apoptotic signaling. Expression of activated Rit in PC6 cells induces potent activation of the MAP kinases ERK1/2 and p38, and promotes neurite outgrowth and cell survival. Furthermore, RNAi-mediated Rit silencing sensitized PC6 cells to a wide variety of stresses, in part by inhibiting NGF-mediated activation of ERK and p38 MAP kinases and CREB. Here we present studies from two genetic models designed to further assess our hypothesis. First, we generated a transgenic mouse over-expressing constitutively activated Rit (RitL79). To begin to assess the contribution of Rit signaling to cellular survival, we examined the ability of cortical neuronal cultures isolated from Rit-TG versus wild-type littermates to survive reactive-oxygen-species (ROS)-mediated cell death. Cell viability for wild-type cortical neurons following a 4 h exposure to H2O2 (60 μM) was reduced approximately 90%, while cortical neurons isolated from Rit-TG mice were largely unaffected by this treatment. Thus, RitL79-mediated signaling protects cortical neurons against ROS-mediated apoptosis. As further confirmation of the important role this protein family plays in survival from stress, we generated a Drosophila strain null for D-Ric. Mutants are homozygous viable and show no obvious developmental abnormalities. However, D-Ric mutants are susceptible to environmental stresses, including heat stress and dry starvation. Taken together, these studies suggest a conserved role for Rit/Ric in promoting cellular survival and suggest that Rit signaling is a potential target for pharmaceutical intervention. We look forward to assessing Rit’s role in other systems including cardiac and vascular cell types.

This work was supported by a Predoctoral Fellowship from the AHA.
Abstract 932: Temporal Changes In The Contribution Of Protein Kinase C Isoforms To Myogenic Constriction In Rat Posterior Cerebral Arteries
Authors: Kashihara Toshihide , Nakayama Koichi , Ishikawa Tomohisa
Published: July 2021

Background

Cerebral arteries respond to an increase in intraluminal pressure with vasoconstriction, being referred to as myogenic tone. A number of studies have postulated the mechanisms involved in the development of myogenic tone; however, less information is available about how myogenic tone is maintained. The present study thus investigated the mechanisms underlying sustained myogenic constriction in isolated rat posterior cerebral arteries.

Methods and Results

Long-term elevation of intraluminal pressure from 5 to 60 mmHg for 1 hr caused sustained constriction and [Ca
2+
]
i
elevation. In the presence of rottlerin, a PKCδ inhibitor, the pressure-induced constriction and [Ca
2+
]
i
elevation were gradually declined, and nearly abolished at the end of the 1-hr stimulation (sustained phase). In contrast, Gö 6976, a cPKC inhibitor, significantly inhibited the constriction for up to 5 min after the start of stimulation (initial phase), but had no effects on the [Ca
2+
]
i
elevation. The pressure stimulation induced small [Ca
2+
]
i
elevation even in the presence of nicaripine. Ruthenium red (RuR), a TRPV inhibitor, significantly inhibited the nicardipine-resistant [Ca
2+
]
i
elevation in the initial phase. However, slowly developing [Ca
2+
]
i
elevation was still observed in the sustained phase. In contrast, rottlerin had little effect on the initial phase, but significantly inhibited the sustained one. Moreover, the combination of RuR and rottlerin nearly abolished the nicardipine-resistant [Ca
2+
]
i
elevation. PKCα, γ, δ, and ε, but not PKCβ, were detected by immunohistochemisty in smooth muscle cells of rat posterior cerebral arteries.

Conclusions

PKC isoforms have different roles in the development and maintain of the myogenic constriction in rat posterior cerebral arteries: cPKC (α and/or γ) mediates Ca
2+
sensitization in the initial phase, whereas PKCδ mediates [Ca
2+
]
i
elevation via the activation of RuR-resistant cation channels in the sustained phase.

Table.

Effects of PKC inhibitors on pressure-induced constriction and [Ca
2+
]
i
elevation
Abstract 939: Galpha-i Signaling in Neural Crest Cells is Required for Normal Migration of Cardiac Neural Crest Cells, Cardiac Development, and Survival
Authors: Ruppel Kathleen M , Kataoka Hiroshi , Iwaki Michelle , Cornelissen Ivo , Coughlin Shaun R
Published: July 2021

G protein coupled receptors (GPCRs) have long been known to play crucial roles in transducing environmental signals to the adult cardiovascular system. In recent years, the roles of G protein-mediated signaling pathways in orchestrating the interactions of different tissues during cardiovascular development have become increasingly evident. To analyze the role of G protein signaling pathways in vivo we have generated mice where the function of the heterotrimeric G alpha subunit Gai can be ablated in a cell type specific manner utilizing the Cre-loxP system. We have mated these mice to two different neural crest-specific Cre lines in order to probe the effects of loss of Gai mediated signaling on the ability of neural crest cells (NCC) to contribute to the developing outflow tract and aortic arch arteries.

METHODS: We have generated mice that express the Gai-inhibiting pertussis toxin S1 subunit (PTX) from the ROSA26 locus in a Cre recombination dependent manner (ROSA-PTX mice). These were mated to mice expressing either the Wnt1 Cre or P0 Cre transgene. Wnt1Cre is active in both premigratory and migratory NCC, whereas P0Cre is active only in migratory NCC and their derivatives.

RESULTS: P0Cre-ROSA-PTX mice were normal at birth and demonstrated no structural heart defects. In contrast, Wnt1Cre-ROSA-PTX mice were present in normal numbers at late gestation but died perinatally due in part to cardiac outflow tract defects. Excision reporter and in situ hybridization studies suggest this is secondary to a delay/blockage of cardiac NCC migration into the developing outflow tract. NCC migration into the pharyngeal arches was unaffected in these mice and no craniofacial, thymic, or aortic arch abnormalities were observed.

CONCLUSIONS: These results indicate that Gai-mediated signaling is required in premigratory or early migratory cardiac NCC for normal development of the outflow tract. In contrast, endothelin A receptor knockout mice (currently the only GPCR knock out with a neural crest phenotype) are thought to exhibit defects of postmigratory NCC function. RNA profiling of NCC for GPCRs involved in this Gai-dependent pathway has revealed several potential candidate receptors, including orphan receptors. Further analysis of these receptors is underway.
Abstract 958: Cardiac MyBP-C Modulates Actomyosin Interactions: Evidence From Cardiac MyBP-c−/ − Mice
Authors: Lecarpentier Yves , Vignier Nicolas , Oliviero Patricia , Cortes-Morichetti Miguel , Carrier Lucie , Coirault Catherine
Published: July 2021

The precise role of cardiac myosin binding protein C (cMyBP-C) on actomyosin interaction (AMI) remains unknown. We hypothesized that the lack of cMyBP-C impaired cardiac AMI. Experiments were performed on 16 weeks old cMyBP-C
−/−
(KO) and age-matched wild-type (WT) mice (n=20/group).
In vitro
mechanical and energetics properties were performed on left ventricular (LV) papillary muscles and Huxley’s equations were used to characterize AMI.
In vitro
motility assays were performed using myosin purified from LV. Myosin-based sliding velocities of actin filaments were analyzed at baseline, after pretreatment of the myosin solution with 10 umol of the catalytic subunit of PKA and/or in the presence of increasing amount of α-actinin, an actin-binding protein that acts as an internal load thereby providing an index of relative isometric force. Western-blot analysis was used to quantify cMyBP-C and phosphorylated cMyBP-C in myosin solutions. Compared to WT, both total tension and maximum shortening velocity were lower in KO (p<0.001). The probability for myosin to be weakly bound to actin was higher in KO than in WT (8.6±0.3 vs. 5.4±0.2%, p<0.05), whereas the number of strongly bound, high-force generated state cross-bridges was lower in KO (6.4±0.9 vs. 11.6±1.0 10
9
/mm
2
, p<0.001). The unitary force per AMI was lower in KO than in WT (p<0.01). At baseline, myosin-based velocities of actin were slower in KO than in WT (1.65±0.01 vs. 1.98±0.01 um/s, p<0.01). The minimum amount of α-actinin needed to completely arrest the thin filament motility was significantly higher in WT than in KO (73.3±1.1 vs 29.1±0.1 ug/l, p<0.001). As expected, cMyBP-C was present in WT myosin solution whereas cMyBP-C was not detected in KO. In WT, PKA induced a 1.6-fold increased in cMyBP-C phosphorylation (p<0.01) associated with a 53±1% increase in the amount of α-actinin required to arrest thin filament motility (p<0.001). PKA did not modify sliding velocity in WT. In KO, PKA had no effect on myosin sliding. We conclude that cMyBP-C regulates AMI by limiting inefficient cross-bridge formation and by enhancing the power stroke step. Phosphorylation status of cMyBP-C appears to play a critical role on cardiac contractility through a direct effect on the myosin molecular motor.
Abstract 967: Parathyroidectomy Reduces Cardiovascular Events And All-cause Mortality In Renal Hyperparathyroidism
Authors: Lima Jose , Costa-Hong Valeria , Jorgetti Vanda , Gowdak Luis Henrique W , Moyses Rosa Maria A , Krieger Eduardo M
Published: July 2021

Background:
Secondary hyperparathyroidism (SHPT) and its associated abnormalities in mineral metabolism increase the risk of cardiovascular morbidity and death in chronic kidney disease (CKD). The effect of parathyroidectomy (PTX) on the incidence of major cardiovascular events in CKD patients with SHPT is unknown. We tested the hypothesis that PTX reduces the incidence of cardiovascular complications and death in CKD patients with severe SHPT scheduled for PTX, comparing the outcome of patients treated or not treated by surgery.

Methods:
The study comprised 118 CKD patients with SHPT on maintenance hemodialysis, unresponsive to medical treatment, and scheduled for PTX. Patients underwent comprehensive cardiovascular evaluations at baseline. They were followed up until death, occurrence of major cardiovascular events, or kidney transplantation.

Results:
No deaths related to surgery occurred. After a median follow-up of 30 months, 50 patients (42.4%) had undergone PTX while 68 (57.9%) had not. The groups were comparable in terms of age, sex, race, parathyroid hormone (PTH), calcium, phosphate, calcium x phosphate product, and all major cardiovascular variables, except diastolic blood pressure. PTX was associated with a reduced incidence of major cardiovascular events (log-rank= 0.02) and overall mortality (log-rank= 0.001). Cox proportional multivariate analysis showed that variables significantly and independently associated with events were PTX (RR=2.36, CI 1.11–6.32, p=0.02) and age (RR=1.07, IC 1.02–1.14, p=0.009). All-cause mortality was related to PTX (RR=2.34, CI 1.25–5.14, p=0.007) and hematocrit (RR=1.15, CI 1.03–1.29, p=0.01).

Conclusion:
PTX confers protection against future major cardiovascular events and death in select CKD patients with severe refractory SHPT.
Abstract 971: Inhibition Of Vascular Bk Channel Activity By Caveolae-mediated Angiotensin II Type I Receptor Signaling In Diabetic Vessels
Authors: Lu Tong , Wang Xiaoli , Lee Hon-Chi
Published: July 2021

Angiotensin II (Ang II) type I receptor (ATR
1
) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca
2+
actviated K
+
(BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR
1
, and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR
1
inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β
1
subunit, ATR
1
, cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR
1
activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR
1
, Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR
1
and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.
Abstract 987: Functional Inhibition Of p300 Leads To Mitochondrial Dysfunction And Autophagic Myocardial Cell Death In The Adult Heart
Authors: Nakagawa Yasuaki , Kuwahara Koichiro , Takemura Genzo , Akao Masaharu , Arai Yuji , Takano Makoto , Harada Masaki , Murakami Masao , Nakanishi Michio , Usami Satoru , Yasuno Shinji , Kinoshita Hideyuki , Fujiwara Masataka , Ueshima Kenji , Nakao Kazuwa
Published: July 2021

p300 is one of transcriptional co-activators fundamentally important in various signal-modulated transcriptional events. It was shown that the cardiac specific overexpression of p300 developed heart failure in adult mice. On the other hand, in embryonic stage p300 has been shown to be essential for cardiac development. In this study, to evaluate the physiological role of p300 in the adult heart, we analyzed the mice carrying cardiac specific overexpression of dominant-negative mutant of p300 (C/H3 domain deletion mutant of p300; DN-p300). DN-p300 transgenic mice(TG) showed the significantly reduced survival rate compared to non-transgenic mice(NTG) and mostly died by 20 weeks of age At 12 weeks, TG showed the significant increase in the heart or lung weight-to-body weight ratio (P < 0.01). In echocardiography, TG revealed the depressed ejection fraction (NTG [n = 8],65.5Â ± 1.5%; TG [n = 11], 33.1Â ± 2.6%; P < 0.01) and the increased LV end-systolic diameter (NTG [n = 8], 4.3Â ± 0.12 mm; TG [n = 11], 5.26Â ± 0.11 mm; P < 0.01), but no significant difference in the wall thickness of the left ventricles. Histological analysis demonstrated the increased interstitial fibrosis and myofibrillar disarray in the ventricles of TG. In electron microscopic (EM) analysis, we found the markedly increased number and reduced size of mitochondria in TG hearts. Tetramethylrhodamine ethylesters (TMRE) staining revealed the diminished mitochondrial membranous potential in TG. RT-PCR analysis showed that the expression of genes encoding multiple mitochondrial genes and PGC-1alpha, a pivotal master regulator of mitochondrial genes, was significantly decreased in TG. In vitro, DN-p300 suppressed the activity of PGC-1alpha promoter. As mitochondrial dysfunction was known to lead to cell death including apoptosis and autophagy, in TG heart we examined the cell death and found the increase in autophagic degeneration of cardiac myocytes in EM analysis. Consistent with this finding, protein expression of cathepsin D and LC3 was increased in TG. Collectively, all these data indicate the critical role of p300 in the maintanance of mitochondrial gene expression and function, and the myocardial cell survival in post-natal heart.
Abstract 992: Novel Role of Copper-dependent Transcription Factor Antioxidant-1 in Cell Proliferation and Wound Healing
Authors: Kim Ha Won , Ozumi Kiyoshi , Ronald McKinney D , Itoh Shinichi , Yang Jay , Nakagawa Osamu , Lessner Susan , Fukai Masuko Ushio , Fukai Tohru
Published: July 2021

Copper plays a fundamental role in regulating cell proliferation involved in wound healing and angiogenesis. Recently, we found that antioxidant-1 (Atox1), previously appreciated as a copper chaperone at the transGolgi network (TGN), stimulates cyclin D1 transcription and cell proliferation in a copper dependent manner. However, functional role of Atox1 in wound healing in vivo remains unknown. Here we show that Atox1 was highly expressed at the BrdU positive nuclei in the wounding tissue, which was associated with an increase in copper content (2.1-fold), as assessed by ICP-mass spectrometry, as compared to non-wounded skin. In Atox1 knockout (KO) mice, the rate of wound closure was markedly delayed as compared to wild-type (WT) mice (7.1 day vs. 11.2 day for complete wound closure in WT vs. Atox1 KO mice, p < 0.001), and BrdU-positive proliferating cells and cyclin D1 protein expression were significantly decreased. Capillary density in healing wound tissue was also reduced in Atox1 KO mice (27.8% decrease). To segregate a copper-dependent transcription factor and a copper chaperone function of Atox1, we have generated lentivirus expressing nuclear targeted Atox1 (Atox1-NLS), TGN-targeted Atox1 (Atox1-TGN) or Atox1-WT. and examine their effect on cell proliferation and wound healing in vitro and in vivo. In cultured Atox1 KO mouse fibroblasts, copper-induced cell proliferation and cyclin D1 expression were abolished, which was rescued by re-expression of both Atox1-NLS and Atox1-WT, but not Atox1-TGN. Promoter deletion analysis identified Atox1 responsive element (Atox1-RE) at cyclin D1 promoter and EMSA and ChIP assays demonstrated that Atox1 binds to Atox1-RE at the cyclin D1 promoter in a copper dependent manner. The impairment of wound healing in Atox1 KO mice was rescued by re-expression with lenti-Atox1-NLS and Atox1-WT, but not Atox1-TGN, which was associated with the increase in cyclin D1 expression as well as number of BrdU positive proliferating nuclei. In summary, transcription factor function of Atox1, but not copper-chaperone function of Atox1, plays a critical role in copper-induced cell proliferation, which may contribute to wound repair process in response to injury in vivo.
Abstract 995: Blockade Of Electron Transport Preserves The Contents Of Bcl-2 And Cytochrome
c
In Subsarcolemmal Mitochondria During Ischemia
Authors: Chen Qun , Lesnefsky Edward J
Published: July 2021

Cardiac ischemia decreases the rate of oxidative phosphorylation (OXPHOS) and the contents of cardiolipin and cytochrome
c
(CYTc) in subsarcolemmal mitochondria (SSM) in the isolated rabbit heart. CYTc release first requires damage to the inner mitochondrial membrane to delocalize CYTc to the intermembrane space, followed by breach of the outer membrane. The decrease in cardiolipin content allows CYTc detachment from the inner membrane. It is still unclear how CYTc passes the outer membrane for release into cytosol. We propose that ischemia increases outer-membrane leakage by depletion of bcl-2 content, and that oxidants generated by the electron transport chain (ETC) during ischemia favor bcl-2 depletion. We used blockade of the proximal ETC at complex I during ischemia with amobarbital (AMO) to test the role of ETC during ischemia. Langendorff perfused rabbit hearts were treated with AMO (2.5 mM for 1 min) or vehicle immediately before 30 min global ischemia (37°C). Time controls were perfused for 45 min. SSM were isolated at the end of ischemia. CYTc content (reduced minus oxidized spectra), OXPHOS and bcl-2 (western blotting) were measured. Ischemia decreased OXPHOS with TMPD-ascorbate as substrate (electron donor to complex IV via CYTc) and the contents of CYTc and bcl-2. In contrast, AMO preserves OXPHOS, CYTc and bcl-2. Thus, blockade of electron transport preserves bcl-2 content during ischemia with enhanced CYTc retention by SSM. The ETC contributes to mitochondrial damage during ischemia, depleting cardiolipin in the inner membrane and bcl-2 in the outer membrane favoring the two steps required for release of CYTc from mitochondria during ischemia and reperfusion.
Abstract 1000: Dichotomous Roles Of TNF-α Receptors p55 And p75 In Acute Ischemia/reperfusion Injury And Late Ischemic Preconditioning
Authors: Flaherty Michael P , Guo Yiru , Tang Xian-Liang , Tiwari Sumit , Hunt Greg , Rezazadeh Arash , Bolli Roberto , Dawn Buddhadeb
Published: July 2021

We have previously demonstrated that TNF-alpha signaling is critical for the development of protection afforded by the late phase of ischemic preconditioning (PC). In the current study, we investigated the roles of p55 (TNFR-I) and p75 (TNFR-II) in acute myocardial ischemia/reperfusion injury as well as late PC. Wild-type (WT, B6 and B6,129 strains), TNF-a−/−, p55−/−, p75−/−, and p55−/−/p75−/− double-knockout mice underwent a 30-min coronary occlusion followed by 4 h of reperfusion with or without six cycles of coronary occlusion/reperfusion (O/R) 24 h earlier. Six cycles of O/R reduced infarct size 24 h later in B6 as well as B6,129 WT mice, indicating a rob ust late PC effect (Figure

). This infarct-sparing effect of late PC was abolished in the absence of TNF-a, p55, p75, and both p55/p75, indicating

that TNF-a signaling is critical for the development of late PC protection; and

that signaling via both p55 and p75 is necessary for the development of protection.

In nonpreconditioned TNF-a−/− and p75−/− mice, infarct size was similar to that observed in strain-matched WT mice (Figure

). However, infarct size in nonpreconditioned p55−/− mice was reduced compared with nonpreconditioned WT mice (46.8 ± 2.8% vs. 63.4 ± 3.2%, P < 0.05, Figure

). These observations were confirmed via linear regression analysis of myocardial risk region and infarct size. We conclude that nonredundant TNF-a signaling via both p55 and p75 is crucial for the development of late PC protection. However, the reduction in infarct size in naïve p55−/− mice indicates a deleterious role of this receptor during acute myocardial ischemia/reperfusion injury.
Abstract 999: Gated Micro Computed Tomography Scanning: An Emerging Tool for Longitudinal Assessment of Murine Cardiac Remodeling.
Authors: Sheikh Ahmad Y , Doyle Timothy C , Sheikh Maryam K , Cao Feng , Ransohoff Katherine J , Yang Phillip C , Robbins Robert C , Fischbein Michael P , Wu Joseph C
Published: July 2021

Background:

Recent advances in high energy X-ray source computed tomography (CT) technology have made it possible to accurately image murine cardiac structure and function. We describe the use of a gated Micro CT system to assess cardiac remodeling in a murine model of myocardial infarction (MI).

Methods:

Adult FVB mice (n = 10) were randomized to surgically induced MI by left coronary artery ligation or sham procedure. Dual gated cardiac and respiratory Micro CT scans were performed pre-operatively and at weeks 4, 8 and 12 post-procedure. End-diastolic and end-systolic images were acquired by gating on the ECG P-and S’-waves, respectively. Post-acquisition analysis was performed using image analysis software and the following parameters were quantified: left ventricular (LV) volume and mass, right ventricular volume and mass, right and left atrial volumes, and ventricular ejection fraction (EF).

Results:

Images were successfully acquired with a resolution of 100 microns allowing for identification and quantification of key cardiac structures (Fig 1A

). Following MI, animals exhibited left ventricular failure with significantly increased* end systolic and diastolic volumes by week 4. Ventricular dilation continued through week 8, plateauing by week 12. Left ventricular mass increased steadily over 12 weeks*, with a significantly decreased* LVEF of 28.0 ± .05% by week 12 (pre-MI: 66.7 ± .06%,*p < 0.01). Post-MI left ventricular change is detailed in Fig 1B

.

Conclusions:

MicroCT scanning can be successfully used to characterize murine myocardial structure and function, making it a useful tool to assess cardiac phenotypes and models of cardiovascular disease.
Abstract 208: Deletion of the Circadian Rhythm Gene mPer2 is Cardioprotective
Authors: Dries Jessica L , Easton Peter , Friesland Amy , DeAntonio Jon , Chintalgattu Vishnu , Ding Jian , Lust Robert , Virag Jitka
Published: July 2021

Variations in circadian patterns are evident in the incidence of cardiovascular disease and derangements in normal circadian patterns are demonstrated to precipitate the onset of chronic diseases. “Clock genes” such as
Per-1 and -2, Cry-1 and -2, and clock/bmal
, are found in all peripheral tissues, including the heart. It is clear that they are involved in synchronization of vascular function and cardiac metabolism however, the downstream effector genes have yet to be identified. To evaluate the function of mPer2 in post-infarct myocardial remodeling, we used an mPer2 mutant mouse which lacks functional mPer2 protein (mPer2-KO). In anesthetized, mechanically ventilated wild-type (WT) and mPer2-KO mice, Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery. Our data reveal that, although there is no difference in the initial area at risk between the WT and mPer2-KO mouse hearts, at 4 days post-MI there is a 50% reduction of infarct size in mPer2-KO mice compared to WT. This is coincident with a 40% increase in vascular density, 25% less macrophage infiltration, and 34% less apoptosis in the infarct zone. Cytokine array analysis showed 3-fold increase in IGFBP-3 and -5 and a 90% increase in SDF-1α, IL12p70, and lymphotactin in mPer2-KO relative to WT at 4 days post-MI. Conversely, in mPer2-KO the levels of L-selectin, sTNFRII, and IL-6 were approx. 70% less than the WT. These data suggest that mice lacking mPer2 protein have enhanced activation of anti-apoptotic and hypertrophic pathways, augmented chemotaxis for reparative cells, and blunted production of adverse inflammatory mediators post-MI. We conclude that deletion of the mPER2 gene is cardioprotective. Further mechanistic studies are being conducted to understand the signaling pathways that afford this protection. Long-term studies are also in progress to determine the duration of these protective effects and whether cardiac function is altered. Understanding the complex interactions between circadian rhythms and cardiovascular disease may provide insights into potential preventative and therapeutics for susceptible populations.
Abstract 1011: SUR2 Short Form-Based Channels Confer A Glibenclamide Insensitive, ATP-Sensitive Potassium Current In Cardiac Myocytes
Authors: Pu Jie-Li , Ye Bin , Kroboth Stacie , McNally Elizabeth M , Makielski Jonathan C , Shi Nian-Qing
Published: July 2021

K
ATP
channels are widely distributed in various tissues and play important physiological roles in regulating insulin secretion in pancreatic beta-cells, providing ischemic protections to the heart and modulating vascular tone in smooth muscles. It is generally agreed that the sarcolemmal K
ATP
is a hetero-octamer consisting of a Kir6.0 pore and a sulfonylurea receptor (SUR). A SUR2 mutant mouse was generated by disrupting the first nucleotide-binding domain (NBD1), where a previously reported N-end component for the glibenclamide action site was located. In ventricular myocytes isolated from relatively young mutant mice, a novel ATP-sensitive current (
I
KATPn
) was detected in 33% single-channel recordings with an average amplitude of 12.3 ± 5.4 pA per patch (n = 15), which was 1/4 of the conventional
I
KATP
(55.4 ± 10.0 pA, n = 19). The IC
50
(ATP) for K
ATPn
was 10 μM compared to 39.3 μM for the conventional K
ATP
suggesting that K
ATPn
could respond faster to low-ATP conditions. Mean burst duration for K
ATPn
(20.6 ± 1.8 ms, n=4) was half of that for the conventional K
ATP
(44.2 ± 9.5 ms, n=4). K
ATPn
was insensitive to 10 μM glibenclamide and could not be re-activated by 0.1– 0.5 mM pinacidil in the presence of ATP. Newly designed SUR2-isoform or variant-specific antibodies identified several novel SUR2 short forms in the sizes of 28 – 68 kDa in addition to a 150-kDa long form in a purified WT cardiac membrane fraction. The 150-kDa long form was absent in the mutant as expected but the SUR2 short forms remained intact. We hypothesized that channels constituted by these short forms, presumably lacking NBD1, confer
I
KATPn
. The SUR2 short forms co-immunoprecipitated with Kir6.0 suggesting that the short forms may function like hemi-transporters reported in other ATP-binding cassette transporter families. Our results show that different K
ATP
compositions based on the novel SUR2 short forms may exist in cardiac sarcolemmal membrane.