bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P089: Estradiol Impairs Cardiac Contractile Function in Male Rodents
Authors: Kararigas Georgios , Bito Virginie , Tinel Hanna , Albrecht Barbara , Sipido Karin R , Regitz-Zagrosek Vera
Published: July 2021

Several clinical and animal studies have challenged the notion that 17beta-estradiol (E2) is cardioprotective. Recently, we identified that in E2-treated male human cardiac tissues there was an increased expression level of the myosin regulatory light chain (Mrlc) interacting protein (Mylip) gene compared to controls. Mylip has been shown to bind and target Mrlc for proteasomal degradation in neuronal cells. Modulation of contraction by Mrlc in the heart has been well documented. Mutant mice for Mrlc phosphorylation show defects in heart contraction. Here, we tested the hypothesis that the induction of Mylip by E2 is associated with reduced Mrlc protein levels and impaired contractile function. Eleven-month old male C57BL/6J mice were injected intraperitonealy with E2 (n = 6) or vehicle (Ctrl; n = 4). Five hours after injection, cardiomyocytes (CMs) were isolated and were either frozen in TRIzol reagent for RNA isolation or used for cell shortening measurements. Quantitative real-time PCR revealed that E2-treated CMs had higher Mylip levels than Ctrl CMs (49% induction, P < 0.05). In addition, there was a decreased abundance of Mrlc protein in E2-treated CMs compared to Ctrl CMs (74% reduction, P < 0.05). Recordings of unloaded cell shortening at 1, 2 and 4 Hz demonstrated that the treatment with E2 impaired CM contractile function compared to Ctrl CMs (1 Hz: 31%, adjusted P < 0.001; 2 Hz: 30%, adjusted P < 0.01; 4 Hz: 25%, adjusted P < 0.01). Similarly, there was a significant decrease in the rate of contraction of E2-treated CMs compared to controls. Next, we assessed the effect of E2 on cardiac contractility ex vivo using Langendorff-perfused rat hearts. Although at baseline there were no significant changes, the response to isoprenaline was blunted in E2-treated hearts. E2 levels in elderly and/or obese men might increase considerably and they have been associated with an increased risk and incidence of cardiovascular disease. However, explanations for causal pathways and putative mechanisms for this association have not been identified. Based on our present findings, we suggest that MYLIP could contribute to this association and we propose that MYLIP could become a pharmacological target in this high-risk group.
Abstract P100: Gene Targeting of
Npr1
Reveals Its Repressive Role in Cardiac Hypertrophy and Proinflammatory Cytokine Gene Expression
Authors: Pandey Kailash N , Vellaichamy Elangovan
Published: July 2021

The objective of the present study was to examine whether genetically determined differences in the
Npr1
gene (coding for guanylyl cyclase/natriuretic peptide receptor-A; GC-A/NPRA) affect the expression of cardiac pro-inflammatory cytokines and nuclear factor kappa-B (NF-kB) levels in
Npr1
gene-targeted mice. The
Npr1
gene-disrupted null mutant (
Npr1
−/−
; 0-copy) mice had 32-38 mmHg higher systolic blood pressure (SBP) and 60% greater heart weight to body weight (HW:BW) ratio, however,
Npr1
gene-duplicated mice (
Npr1
++/++
; 4-copy) exhibited 10-15 mmHg lower SBP with no change in the HW:BW ratio compared with wild-type (

Npr1
+/+
;

2-copy) mice. Significant up-regulation of interleukin-6 (IL-6; 2-fold tumor necrosis factor-alpha), TNF-α; 4-fold), and transforming growth factor-beta (TGF-β1; 4-fold), along with increases in NF-κB and activating protein-1 (AP-1) binding activities (170% and 130%, p < 0.01, respectively), were increased in the

Npr1
−/−

mice hearts. Conversely, a significant decrease in cytokine gene expression and NF-κB and AP-1 binding activities were found in gene-duplicated mice hearts compared with wild-type mice. The ventricular guanylyl cyclase (GC) activity and cGMP levels were reduced by almost 10-fold and 5-fold, respectively, in

Npr1
-/-

mice. However, GC activity and cGMP levels were increased, respectively, by almost 9-fold and 6-fold, respectively, in gene-duplicated mice. The present results provide direct evidence that duplication of
Npr1
gene in mice represses the inflammatory cytokine gene expression via inhibition NF-κB- and AP-1-mediated signaling mechanisms, and is associated with cardiac protection.
Abstract P132: Diabetes-Induced Cardiac Dysfunction Is Rescued by Endothelial Cationic Amino Acid Transporter 1 Overexpression in Mice
Authors: Ritchie Rebecca H , Khammy Makhala M , Bowden Marissa A , Irvine Jennifer , Andrews Karen , Huynh Karina , Kiriazis Helen , Kaye David , Venardos Kylie
Published: July 2021

Nitric oxide (NO) bioavailability is markedly impaired in diabetes, contributing to endothelial and myocardial dysfunction. Our aim was to determine if endothelial-specific overexpression of Cationic Amino Acid Transporter 1 (CAT-1, to enhance L-arginine uptake and NO generation) prevents diabetes-induced endothelial and cardiac dysfunction in diabetic mice. Male CAT-1 transgenic and non-transgenic (Ntg) 6 week old mice were administered streptozotocin (STZ, 55mg/kg ip daily for 5 days). Sham mice received citrate buffer vehicle. Myocardial function was assessed in anaesthetised mice in vivo 12 weeks later, via echocardiography. Vascular reactivity was then assessed in vitro. Compared to their non-diabetic littermates (n=6), Ntg STZ mice (n=6) exhibited reduced early-to-late blood flow velocity ratios (E/A, 1.7±0.2 vs 2.4±0.1, p<0.01) and prolonged deceleration time (DT, 40±1 vs 31±2msec, p<0.002), indicative of diastolic dysfunction. Decreased E-wave velocity (109±4 vs 76±12msec, p<0.01) and increased A-wave velocity (45±3 vs 55±4msec, p<0.05) were also observed. Systolic dysfunction was observed in Ntg STZ mice compared to shams, on reduced fractional shortening (FS, 30±1 vs 39±1%, p<0.002). In CAT-1 mice, diabetes did not impair E/A ratio (2.1±0.2), FS (40±3%) or DT (33±1msec, n=8). Thoracic aortic responses to the endothelium-dependent vasodilator acetylcholine (ACh) were unchanged by diabetes in Ntg and CAT-1 Tg mice compared to sham Ntg mice (pEC50 7.0±0.1 vs 7.2±0.1 and 7.1±0.1). There was a non-significant trend for sham CAT-1 Tg mice to show greater sensitivity to ACh than sham Ntg mice (pEC50 7.5±0.1 vs 7.1±0.1). Responses to the endothelium-independent vasodilator sodium nitroprusside and the vasoconstrictor noradrenaline were unaffected by diabetes and/or genotype. In conclusion, our results suggest that endothelial-specific CAT-1 overexpression protects against cardiac dysfunction via a mechanism independent of the systemic vasculature. The involvement of the coronary vasculature remains to be determined. Endothelial CAT-1 up-regulation as a potential therapeutic target for diabetic cardiomyopathy is an exciting prospect.
Abstract P134: Synoviolin Is a Stress-Inducible Endoplasmic/Sarcoplasmic Reticulum E3 Ubiquitin Ligase that Preserves Cardiac Function
Authors: Doroudgar Shirin , Thuerauf Donna J , Khan Mohsin , Mohsin Sadia , Völkers Mirko , Gude Natalie , Múller Oliver J , Sussman Mark A , Glembotski Christopher C
Published: July 2021

We recently reported that synoviolin1 (syvn1) is a novel endoplasmic reticulum stress response (ERSR) protein that is up-regulated in the mouse heart by ATF6, a cardioprotective, nodal transcription factor of the ERSR. Syvn1 is a unique E3 ubiquitin ligase that retrotranslocates misfolded proteins from endoplasmic/sarcoplasmic reticulum (ER/SR) to the cytosol and subsequently polyubiquitinates them, targeting them for degradation. We now report that syvn1 expression is induced with tunicamycin, thapsigargin, and dithiothreitol, ER stressors that activate ATF6. Moreover, adenovirus-mediated syvn1 overexpression in neonatal rat ventricular cardiac myocytes (NRVCMs) increases contractility. Consistent with this finding, syvn1 overexpression increases calcium transient amplitude as well as diastolic calcium. We also find that syvn1 overexpression decreases secretion of MANF, a protective, anti-hypertrophic, ER/SR protein which we find is conditionally secreted when ER/SR calcium is depleted. We also report MANF as the first example of a is protein whose secretion from ventricular myocytes is conditionally dependent on ER/SR calcium. Furthermore, syvn1 reduces the growth of NRVCMs treated with the α-adrenergic agonist, phenylephrine. Moreover, while knockdown of syvn1 in NRVCMs using syvn1-targeted siRNA increases cell death, overexpression of syvn1 promotes cell survival. To investigate the roles of syvn1,
in vivo
, we examined the effects of syvn1 overexpression in cardiac myocytes in a mouse model of trans-aortic banding. Syvn1 overexpression,
in vivo,
was achieved by intravenous delivery of recombinant AAV serotype 9 (AAV9; cardiac specific serotype) with MLC2v promoter driving syvn1 expression. Baseline cardiac function, as measured by echocardiography six weeks after gene delivery, shows no difference between AAV9-control and AAV9-syvn1 treated mice. Trans-aortic banding decreases cardiac function in mice injected with AAV9-control. In contrast, cardiac function is preserved in mice injected with AAV9-syvn1. The findings in this study suggest that syvn1 is a novel stress-inducible cardiac E3 ligase with unique functions in regulating protein secretion, maintaining cell viability, and preserving cardiac function.
Abstract P136: E2F2/4 Regulated by Serine 21 Phosphorylation of GSK-3α Play Compensatory Roles During Pressure Overload
Authors: Yamamoto Takanobu , Maejima Yasuhiro , Zhai Peiyong , Matsuda Takahisa , Sadoshima Junichi
Published: July 2021

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with two isoforms, alpha and beta, which have distinct functions in cardiomyocytes (CMs). GSK-3alpha is phosphorylated at S21 during pressure overload (PO), and inhibition of S21 phosphorylation in GSK-3alpha S21A knock-in (alpha−KI) mice promotes hypertrophy and heart failure in response to PO, accompanied by decreases in the total number of CMs in the heart. Since GSK-3alpha downregulates cyclinD1 in the nucleus, GSK-3alpha may negatively regulate E2F-mediated transcription. Reporter gene assays showed that the transcriptional activity of E2F was increased by GSK-3alpha knockdown (1.75 fold, p<0.05). To evaluate the role of E2F isoforms in regulating cardiac hypertrophy and function during PO, E2F1−/−, E2F2−/+, E2F4−/+, and wild type (WT) mice were subjected to transverse aortic constriction (TAC). Left ventricular (LV) weight/ tibial length (LVW/TL) was significantly greater and LV ejection fraction (LVEF) was significantly decreased in both E2F2−/+ and E2F4−/+ after 2 weeks of TAC (LVW/TL: E2F2−/+=7.1±0.3, E2F4−/+=7.0±0.4, WT=5.9±0.3, p<0.05 vs. WT; LVEF: E2F2−/+=53±1%, E2F4−/+=61±2%, WT=75±1%, p<0.05 vs. WT). Thus, downregulation of either E2F2 or E2F4 induced a phenotype similar to that of alpha−KI in response to TAC. To examine the causative role of E2F2/E2F4 downregulation in mediating the cardiac phenotype in alpha-KI mice, adenovirus (Ad) harboring either E2F2 or E2F4 was injected into alpha-KI hearts. Rescue with E2F2 or E2F4 attenuated cardiac hypertrophy (LVW/TL: alpha−KI+E2F2=7.1±0.4, alpha−KI+E2F4=7.3±0.3, alpha−KI+LacZ=8.7±0.4, p<0.05 vs. alpha−KI+LacZ) and improved LV dysfunction (LVEF: alpha−KI+E2F2=66±3%, alpha−KI+E2F4=60±2%, alpha−KI+LacZ=39±2%, p<0.05 vs. alpha−KI+LacZ) in alpha−KI mice under PO conditions. Injection of either Ad-E2F2 or Ad-E2F4, but not of Ad-LacZ, significantly increased the number of Ki67-positive myocytes in the alpha-KI mice (alpha-KI+LacZ =0.7±0.3%, alpha-KI+E2F2=10.4±2.3%, alpha-KI+E2F4=9.2±1.5%, p<0.05 vs. alpha-KI+LacZ). These results suggest that maintaining the activity of E2F2 and E2F4 through S21 phosphorylation of GSK-3alpha plays an essential role in preserving cardiac function during PO.
Abstract P145: Cathelicidin Antimicrobial Peptide Is Upregulated After Myocarditis and Inhibits Fibroblast Migration via P2x7 Receptor Signaling
Authors: Nakayama Hiroyuki , Matsui Kazuki , Yamashita Tomomi , Kawaguchi Haruyo , Fujio Yasushi
Published: July 2021

Rationale

_ Experimental autoimmune myocarditis in BALB/c mice is a model of inflammatory heart disease. It is well known that the process of this model exhibits the acute inflammation phase followed by the healing phase. However, the precise mechanisms how the inflammation terminates and the healing proceeds remain to be elucidated.

Objective

_ To identify the molecule which functions in the healing phase of EAM and investigate its pathophysiological roles on cardiac cells.

Methods and Results

_ To induce EAM, BALB/c mice were immunized twice with α-myosin heavy chain peptide. The severity of inflammation peaked at 21days after immunization and mitigated at 28days. To analyze the alterations of gene expression profile during healing phase, we performed microarray analyses using mRNA from hearts of 21day and 28day after immunization. Among up-regulated genes, we focused on cathelicidin antimicrobial peptide (CAMP) which has been reported to be involved in anti-inflammatory response and epithelial tissue repair. Histochemical staining using anti-CAMP antibody revealed that CAMP expresses in inflammatory cells in EAM hearts. Treatment with LL37, a human homologue of CAMP, mediated activation of ERK, JNK and p38 in fibroblasts from neonatal rat hearts, whereas it failed to activate those MAP kinases in rat neonatal cardiomyocytes, suggesting CAMP is engaged in healing process by regulating function of fibroblasts. In addition, LL–37 treatment mediated morphological changes of fibroblasts and decreased migration distances assessed by wound healing assay as well (283±43 mm in treated cells vs 183±54 mm in cells treated with 10 mg/ml of LL37, p<0.01). The expression of focal adhesion kinase is decreased in LL37 treated cardiac fibroblasts compared with non-treated cells. Moreover, blockade of P2X7 receptor which is known as a receptor for LL37 restored the migration distances after LL37 treatment, suggesting that LL37 suppresses cardiac fibroblast motility via P2X7 receptor signaling.

Conclusion

_ Our results indicate that CAMP is up-regulated after myocarditis and inhibits fibroblasts migration via P2X7 receptor signaling. Those results suggest that CAMP could be a useful tool to inhibit fibrotic changes in inflammatory heart disease.
Abstract P150: Estrogen Protects Against and Reverses Adverse Ventricular Remodeling in Pulmonary Hypertension-Induced Right Ventricular Failure
Authors: Nadadur Rangarajan D , Umar Soban , Iorga Andrea , Matori Humann , Partow-Navid Rod , Eghbali Mansoureh
Published: July 2021

Pulmonary hypertension (PH) leads to right-ventricular failure (RVF). RVF is characterized by adverse RV remodeling including hypertrophy and changes in the cardiac Extracellular Matrix (ECM) such as fibrosis and re-expression of cardiac fetal genes. Among the potentially re-expressed genes are the novel ECM interacting proteins Osteopontin (OPN) and Osteocalcin (OCN).

Gender differences found in experimental PH are attributed to protective effects of Estrogen (E2). We hypothesize that gender differences observed in experimental PH are partially due to the effects of E2 on the cardiac ECM, and that exogenous E2 may be able to reverse adverse RV remodeling.

Male and female rats received single monocrotaline (MCT, 60mg/kg) injection. Some rats were given E2 (42.5μg/kg/day) from day 21–30. Saline treated rats were controls. Cardiopulmonary hemodynamics were serially monitored and RV pressures (RVP) were recorded terminally. RV fibrosis was assessed by trichrome staining. Gene expression was determined by RT PCR and plasma OPN by ELISA.

All rats developed PH by day 21 and RVF by day 30. Male rats developed more severe PH-induced RVF than females (RVP=70 vs. 41.5±5 mmHg; RV/(LV+IVS)= 0.69±0.07 vs. 0.47±0.04; RVEF = 30.4±1.8 vs. 42.8±2% resp., all p<0.05). Males also revealed more severe RV fibrosis and greater re-expression of OPN (4.5 fold vs. females, p<0.05) and OCN (2-fold vs. females, p<0.05). Plasma OPN was also elevated in RVF males (1.00±0.11 in control to 1.47±0.18 pg/ml, p<0.05) but not RVF females (0.848±0.18 in control to 0.859±0.294 pg/ml, p=ns).

Since females experienced less severe RV remodeling than males, MCT injected males were treated with exogenous E2 from day 21–30. Some E2 treated male rats were sacrificed at day 30, and some were kept an additional 12 days after E2-withdrawal (E2-W group). E2 reversed PH-induced RVF (RVP=38mmHg; RV/(LV+IVS) = 0.28±0.03; RVEF=61.5±0.8%, all p<0.05 vs. male RVF) and fibrosis. OPN and OCN were fully restored following E2 withdrawal by day 42. E2 therapy also restored circulating OPN levels (p<0.05 vs. RVF) showing that OPN has potential value as a plasma marker for PH-induced RV failure.

These results suggest that E2 protects against adverse RV remodeling in females, and reverses adverse RV remodeling in males.
Abstract P161: Conversion of Secreted High-Molecular-Weight FGF-2 to an 18 kDa Low-Molecular-Weight FGF-2 by Limited Proteolysis Suppresses the Paracrine Induction of Cardiomyocyte Hypertrophy
Authors: Santiago Jon-Jon R , Bestvater Brian P , Fandrich Robert R , Kardami Elissavet
Published: July 2021

Background:
High molecular weight, 23 kDa rat FGF-2 (Hi-FGF-2) is composed of an N-terminal extension as well as the 18 kDa core sequence present in low molecular weight (Lo-) FGF-2. Hi- and Lo-FGF-2 have distinct biological activities: unlike Lo-FGF-2, Hi-FGF-2, which is secreted by cardiac non-myocytes, promotes cardiomyocytes hypertrophy
in vivo
and
in vitro
. We tested if the paracrine pro-hypertrophic effects of secreted Hi-FGF-2 can be blunted by limited proteolysis of its N-terminal extension.

Experimental:
Heparin-sepharose-bound Hi-FGF-2 present in rat tissue extracts converted to an approximately 18 kDa protein (*Lo-FGF-2) through truncation of its N-terminal extension, in the absence of protease inhibitors. Serine protease inhibitors such as PMSF fully prevented this conversion. Similarly, purified recombinant rat Hi-FGF-2 was converted to *Lo-FGF-2 by thrombin, a serine protease, and the effect was prevented by hirudin, a thrombin specific inhibitor. Conditioned media from unstimulated or angiotensin II-stimulated rat cardiac non-myocytes (fibroblasts) were used to treat neonatal cardiomyocytes in culture, in the absence or presence of thrombin. Conditioned medium from stimulated (but not unstimulated) cells contained Hi-FGF-2, which promoted cardiomyocyte hypertrophy (increase in cell surface area), and the effect was due to secreted FGF-2 because it was prevented by neutralizing anti-FGF-2 antibodies. Thrombin itself had no effect on cell size but prevented the pro-hypertrophic effect of the conditioned media; while converting the secreted Hi-FGF-2 to *Lo-FGF-2.

Conclusions:
The truncation of the N-terminal extension of secreted Hi-FGF-2 by serine protease(s) can provide a strategy to prevent undesirable paracrine effects of Hi-FGF-2 (such as cardiac hypertrophy), while maintaining potentially beneficial (cytoprotective, angiogenic) effects associated with Lo-FGF-2.
Abstract P169: A Novel Mitochondrial Permeability Transition Pore Inhibitor, Bendavia, Reduces Microvascular Obstruction (No-Reflow) due to Myocardial Ischemia/Reperfusion Injury in the Rabbit
Authors: Hale Sharon L , Dai Wangde , Kloner Robert A
Published: July 2021

Background:
The no-reflow phenomenon is an important detrimental component of reperfusion injury with clinical implications, as it is an independent predictor of outcome after myocardial infarction. Whether the mitochondrial permeability transition pore (MPTP) is involved in vascular integrity and no-reflow post reperfusion is unknown. Therefore, we tested Bendavia, a compound that is known to inhibit the MPTP, for its effects on no-reflow.

Methods:
Anesthetized rabbits were subjected to 30 min coronary artery occlusion and 3 hrs reperfusion. Treated animals (n=49) received Bendavia (0.05 mg/kg/hr) after the onset of ischemia, 1, 10 or 20 min before reperfusion. Control animals (n=15) received saline. Risk zone was measured by Unisperse blue staining, anatomic no-reflow area using thioflavin S and necrosis by triphenyltetrazolium chloride.

Results:
The ischemic insult was similar in both groups (31% of left ventricle (LV) in Bendavia and 30% in control). Bendavia treatment resulted in a non-significant 11% reduction in necrosis, expressed as a percentage of the risk zone. However for any given risk zone size, no-reflow was significantly reduced by Bendavia, as there was a significant group effect on the relationship between the extent of the no-reflow zone and that of the risk zone (ANCOVA, p = 0.0085). Overall, the no-reflow zone, expressed as a % of the risk zone was 22 ± 2% in treated hearts vs. 28 ± 3% in controls (p=0.07). In hearts with less severe risk zones (below the mean value of 31% of LV), Bendavia treatment significantly reduced the no-reflow zone by 35% (17±2 vs. 26±4% of risk zone, p <0.05); in hearts with large risk zone, Bendavia non-significantly reduced no-reflow by 17%. Bendavia treatment had no effect on hemodynamic parameters or regional myocardial blood flow during the ischemic period.

Conclusion:
These data suggest that the MPTP may play a role in the pathophysiology of no-reflow after myocardial ischemia/reperfusion. Bendavia reduced the extent of the no-reflow zone for any given risk zone size, suggesting that it helped prevent microvascular obstruction.
Abstract P185: Cardiac Characterization of Phlpp1 Knockout Mice: A Novel Phosphatase to Terminate Akt Signaling
Authors: Purcell Nicole H , Huang Katherine , Zhang Xiaoxue , Gude Natalie , Sussman Mark , Gustafsson Asa B , Miyamoto Shigeki , Heller Brown Joan
Published: July 2021

The serine/threonine kinase Akt, also known as protein kinase B (PKB), regulates a wide variety of cellular processes including metabolism, cell growth, survival, protein synthesis, and gene transcription. Akt is activated by upstream kinases through sequential phosphorylation on its activation loop (Thr308) followed by phosphorylation on the hydrophobic motif (Ser473). Studies using overexpression of Akt
in vitro
and
in vivo
have implicated the kinase in either attenuating or increasing cardiac hypertrophy, depending on signal intensity and localization of Akt in the cell. Recently the protein phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) was shown to dephosphorylate Akt on its hydrophobic motif (Ser473), thereby decreasing kinase activity. We hypothesized that PHLPP could serve as an important regulator of Akt signaling in the heart and that loss of PHLPP would accentuate Akt activation under physiological conditions. We generated
phlpp1
null mice to investigate the cardiac phenotype induced by modulating the kinetics of Akt activation
in vivo
. Myocytes were isolated from PHLPP1 knock-out and wild-type mice to demonstrate loss of PHLPP1 protein and increased Akt phosphorylation following stimulation with the cytokine LIF. Histological examination of hearts revealed that PHLPP1 KO mice have an increased capillary to myocyte ratio compared to age matched wild-type mice at baseline. Increased capillary networks were also detected in the PHLPP1 KO mice relative to wild-type mice using corrosion casting. To determine the role of PHLPP removal on cardiac hypertrophy, PHLPP1 null mice and wild-type mice were subjected to two weeks pressure overload by transverse aortic constriction (TAC). Cardiac hypertrophy was unexpectedly attenuated in PHLPP1 null mice (26% increase in HW/BW ratio vs. 48% in wild-type mice). In contrast, PHLPP1 null mice display an accentuated response to physiological hypertrophy induced by swimming compared to wild-type controls (46% vs. 38% respectively). Our data suggests that there is enhanced Akt activation following PHLPP removal and this attenuates pathological hypertrophy due to changes in capillary density.
Abstract P191: Calpain-Generated Free PKCα Catalytic Domains Induce HDAC5 Nuclear Export and Regulate Cardiac Gene Transcription
Authors: Zhang Yan , Matkovich Scot , Diwan Abhinav , Kang Min-Young , Dorn Gerald W
Published: July 2021

Receptor-mediated activation of protein kinase (PK) C is a central pathway regulating cell growth, homeostasis, and programmed death. Recently, we showed that calpain-mediated proteolytic processing of PKC in ischemic myocardium activates PKC signaling in a receptor-independent manner by releasing a persistent and constitutively active free catalytic C-terminal fragment, PKCα-CT. This unregulated kinase provokes cardiomyopathy, but the mechanisms remain unclear. We examined hypothesis that PKCα-CT has transcriptional activity. Using immunoblot analysis and confocal microscopy, we found that PKCα-CT localized in part to nuclei and spontaneously induced cytosolic relocalization HDAC5 of the transcriptional regulator. Co- expression of calpain 1 with full length PKCα can generate PKCα-CT and produced the same HDAC5 cytosolic relocalization, whereas full length PKCα alone had no such effect. HDAC5 cytosolic relocalization induced by PKCα-CT was abolished by the protein kinase inhibitor GO6976, but not by PKD inhibitor CID 755673. The in vivo relevance of these findings was examined in transgenic mice expressing PKCα and PKCα-CT. To assess the consequence on gene expression, we performed global transcriptome profiling by Affymetrix microarrays and mRNA sequencing. The two techniques substantially agreed. Compared to control hearts, 621 mRNAs were regulated at least 1.3 fold in PKCα-CT hearts (P< 0.001), only 59 in full-length PKCα hearts. MEF2-dependent inflammatory pathway genes which are putative HDAC targets were upregulated in PKCα-CT heart: 15 MEF2 target mRNAs were upregulated in PKCα-CT hearts (p<0.001), only one in PKCα hearts. These results reveal that PKCα-CT is a potent regulator of pathological cardiac gene expression by localizing to nuclei and directly promoting nuclei-cytoplasmic shuttling of HDAC5. Receptor-independent effect of PKCα-CT and HDAC phosphorylation in ischemic hearts has broad ramifications for understanding and preventing the pathological transcriptional stress response.
Abstract P194: CCAAT/Enhancer-Binding Protein Beta Negatively Regulates Hemodynamic Stress-Induced Cardiac Hypertrophy
Authors: Oyabu Jota , Yamaguchi Osamu , Oka Takafumi , Hikoso Shungo , Nishida Kazuhiko , Komuro Issei , Otsu Kinya
Published: July 2021

The CCAAT/enhancer-binding proteins (C/EBP) beta is a member of family of leucine-zipper transcription factors that regulate gene expression to control cellular proliferation, differentiation, inflammation and metabolism. Previous reports showed that the protein and mRNA levels of C/EBP beta were increased in rat ischemia-reperfused or human failing hearts. Recent study indicates an important role of C/EBP beta in physiological cardiac hypertrophic responses. In the present study, we generated cardiac-specific C/EBP beta-deficient mice (CKO) to elucidate its in vivo function in pathological cardiac hypertrophy. We crossed floxed C/EBP beta mice with mice expressing Cre recombinase in cardiac-specific manner. Echocardiographic and physiological analyses revealed that CKO showed no cardiac phenotypes under basal conditions at 10 weeks old, indicating that the C/EBP beta is not essential for mouse heart development. Then, we subjected CKO and control mice (CTL) to pressure overload by means of transverse aortic constriction (TAC). In wild-type mouse hearts, the expression level of C/EBP beta was increased after TAC. One week after TAC, CKO showed left ventricle (LV) hypertrophy (LV/body weight, CKO 4.64 ± 0.08 mg/g versus CTL 4.14 ± 0.06 mg/g, p<0.01, LV mass index, CKO 106.1 ± 1.7 versus CTL 98.5 ± 1.8, p<0.05), without showing contractile dysfunction. Cross-sectional area of cardiomyocytes was increased in CKO after TAC (CKO 373.6 ± 2.1 μm
2
versus CTL 300.5 ± 3.6 μm
2
, p<0.01). The increase in the atrial natriuretic factor (ANF) or alpha skeletal actin mRNA expression, molecular markers for cardiac remodeling, was observed in CKO hearts. Furthermore, overexpression of C/EBP beta in isolated neonatal rat cardiomyocytes inhibited an increase in
3
H-leucine uptake induced by phenylephrine stimulation. C/EBP beta is expressed as three distinct protein isoforms, namely FL, LAP, and LIP, which are encoded by a single gene, but transcribed from different initiation sites. Luciferase reporter gene assay showed that overexpression of LAP isoform attenuated the transcriptional activity of ANF induced by phenylephrine, but not LIP. Thus, we conclude that C/EBP beta attenuates the pathological cardiac hypertrophy induced by hemodynamic stresses.
Abstract P198: Aberrant Activation of γ2-AMPK Causes Cardiac Hypertrophy Independent of Glycogen Storage
Authors: Kim Maengjo , Hunter Roger , Sakamoto Kei , Seidman Christine E , Seidman Jonathan G , Garcia Menendez Lorena , Tian Rong
Published: July 2021

AMP-activated protein kinase (AMPK) is an energy sensor and a key regulator of cell metabolism, hence a promising drug target. The cardiac functions of AMPK have not been understood. Point mutations in the regulatory γ2-subunit (encoded by
PRKAG2
gene) have been shown to cause a unique form of cardiomyopathy in humans characterized by cardiac hypertrophy, arrhythmias and glycogen storage. We have previously shown that
PRKAG2
mutation caused aberrant activation of AMPK in the absence of energy deficit and subsequently triggered re-routing of excessive glucose into glycogen pool. In this study, we addressed two questions: 1) whether cardiac hypertrophy in PRKAG2 cardiomyopathy was secondary of glycogen storage; 2) which hypertrophic signaling pathways are involved. We sought to reduce glycogen storage in transgenic mice expressing a mutant
PRKAG2
(N488I) in the heart (TGγ2
N488I
) by crossing them to knock-in mice harboring a mutation in the muscle form of glycogen synthase (GYS1
KI
) that greatly reduced GYS activity in response to glucose-6-phosphate. Compared to TGγ2
N488I
, TGγ2
N488I
-GYS1-KI (double mutant) hearts showed lower GYS activity (0.7 ± 0.07 vs. 6.9 ± 0.49 nmol/min/mg, p<0.0001) and reduced glycogen content (35 ± 4.5 vs. 169 ± 40 umol/g, p<0.0001). Nonetheless, cardiac hypertrophy remained in the double mutant. The heart weight to body weight ratios were 6.8 ± 0.7 mg/g for TGγ2
N488I
, 6.7 ± 0.5 mg/g for the double mutant compared to 4.0 ± 0.2 mg/g in the wild type. Furthermore, we have observed significant changes in FOXO (forkhead-O transcription factor) and mTOR (mammalian target of rapamycin) pathways in the TGγ2
N488I
hearts. Increased phosphorylation of FOXO3a (Ser321, Ser253) and FoxO1a (Ser256) led to nuclear exclusion and degradation of FOXO proteins. Increased mTOR activity was evidenced by enhanced phosphorylation of Ser2448 as well as its downstream targets S6 and 4E-BP. Taken together these data indicate that aberrant γ2-AMPK activation causes cardiac hypertrophy independent of excessive glycogen accumulation. We found that increased mTOR activity and decreased FOXO signaling contributes to cardiac hypertrophy in TGγ2
N488I
mice, suggesting novel mechanisms underlying cardiac hypertrophy caused by abnormal γ2-AMPK activity.
Abstract P209: MyD88 Mediated Inflammatory Signaling Involved in Post-MI CaMKII Activation and Cardiac Hypertrophy
Authors: Singh Madhu V , Swaminathan Paari D , He Beixin J , Luczak Elizabeth E , Kutschke William , Anderson Mark E
Published: July 2021

Toll-like receptors (TLRs) and calmodulin kinase II (CaMKII) participate in pathological responses to myocardial infarction (MI), including activation of nuclear factor kappa B (NF-κB) transcription and complement factor B expression (Singh JCI 2009). We tested the effect of MyD88 deficiency on the adverse cardiac outcome after MI. MyD88 knock out (MyD88-/-) hearts had significantly reduced hypertrophy, hypertrophic gene expression, and inflammatory gene expression compared to WT control hearts. Cultured cardiomyocytes from expressing MyD88 and a NF-κB reporter demonstrated robust induction of NF-κB upon TLR induction by bacterial lipopolysaccharides (LPS). In contrast, cardiomyocytes from MyD88-/- mice failed to induce NF-κB upon LPS treatment. An alternative MyD88-independent pathway for NF-κB remained functional in WT and MyD88-/- cells. Both LPS treatment and MI increased a Ca2+/calmodulin independet activated form of CaMKII (CaMKIIox) in cultured cardiomyocytes and mouse hearts, respectively. MyD88-/- hearts, however, did not display increased CaMKIIox upon MI. Taken together, we interpret these results to show that: (1) MyD88 participates in MI mediated hypertrophic and inflammatory gene expression, (2) CaMKII enhances NF-kB activation in cardiomyocytes by a MyD88-dependent mechanism, and (3) CaMKIIox plays a role in MyD88 dependent signaling to induce expression of proinflammatory genes in post-MI hearts.
Abstract P224: Connective Tissue Growth Factor Plays a Key Role in Myocardial Fibrosis After Angiotensin II Exposure
Authors: Rosin Nicole L , Légaré Jean-François , Lee Timothy D
Published: July 2021

Background:
AngiotensinII (AngII) exposure to rodents is a common model of fibrosis, characterized by hypertension, hypertrophy and eventual deposition of excess extracellular matrix (ECM) proteins resulting in organ dysfunction. We have previously shown that cellular infiltration of bone marrow derived progenitor cells (fibrocytes) occurs prior to ECM deposition and is associated with production of the connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-β). The objective of this project was to characterize the role of CTGF in promoting fibrocyte recruitment and subsequent fibrosis after AngII exposure.

Methods:
Mice were treated with AngII or saline using an osmotic mini-pump at 2.8mg/kg/day. After 6hr to 7d hearts were excised and embedded in paraffin or prepared for mRNA isolation. Immunohistochemistry was used to determine extent and localization of CTGF protein. Quantitative RT-PCR was used to determine relative CTGF and TGF-β mRNA levels. Primary cardiomyocyte and fibrocyte cultures were isolated from neonatal or 3d AngII exposed animals respectively. Primary cultures were stimulated to determine the source (qRT-PCR) and function (proliferation, migration and differentiation) of CTGF.

Results:
In animals exposed to AngII, CTGF mRNA peaked the earliest at 6hr (21-fold; p<0.01) when compared to TGFbeta, which peaked at 3d (5-fold; p<0.05). Concurrent CTGF protein expression was evident by 3d of AngII exposure and appeared localized to the cardiomyocytes. Findings were confirmed using isolated cardiomyocytes, which significantly increased expression of CTGF in response to AngII (2-fold; p<0.05). While CTGF did not promote fibrocyte migration in transwell chamber assay it promoted significant fibrocyte proliferation in vitro (2-fold; p<0.05).

Conclusion:
We provide strong evidence that AngII exposure first results in the production of CTGF by cardiomyocytes. Furthermore, we have shown that CTGF does not promote migration as a chemokine but instead contributes to proliferation of fibrocytes once recruited from the bone marrow into the myocardium.
Abstract P231: Dispensability of Dmitofusin in Calcium Signaling in Intact Drosophila Heart Tubes
Authors: Jowdy Casey C
Published: July 2021

Sarcoplasmic Reticulum (SR) drives cardiomyocyte excitation-contraction coupling through the release and storage of Ca2+. Phasic fluctuations of Ca2+ require ATP. Mitochondria occupy ∼30% of the volume of cardiomyocytes and provide cardiomyocytes with ATP. Perturbations of mitochondria therefore have a detrimental effect on the heart. Mitochondrial homeostasis in mammals is regulated in part through fusion and fission by outer mitochondrial membrane proteins mitofusin 1 and 2 (Mfn1 and Mfn2) and in Drosophila by the homolog dMfn. Mutations in Mfn2 show impaired contractile function in humans (hMfn), mice (mMfn) and RNAi suppression in Drosophila (dMfn). Additionally Mfn2, independently of Mfn1, tethers the SR to mitochondria allowing for efficient transfer of Ca2+ between the organelles. It is currently not known whether mitochondria-SR tethering is required for proper Ca2+ cycling in cardiomyocytes. We monitored calcium transients in heart tubes of live flies, using the genetically encoded calcium indicator (GECI) GCaMP3.0 to study dynamic Ca2+ oscillations. Intensity signals from GCaMP3.0 in fly heart tubes are comparable to isolated cardiomyocytes in their rate cycling amplitude, and their response to L-type calcium channel antagonist, nifedipine. Additionally, non-calcium responsive GFP does not produce a signal that mimics the Ca2+ oscillations observed with GCaMP3.0. Comparisons between Wt Drosophila hearts and cardiomyopathic RNAi dMfn hearts expressing GCaMP3.0 have normal amplitude and time to decay constant Ca2+ signals. This study shows that suppression of dMfn with RNAi in the fly heart tube leads to cardiomyopathy. Monitoring intercellular Ca2+ levels with the (GECI) GCaMP3.0 demonstrates that Ca2+ signaling is not affected when mitochondria-SR tethering is disrupted.
Abstract P239: Differential Proteomics of Subsarcolemmal and Intermyofibrillar Mitochondria in Aging Rat Cardiac Muscle
Authors: Gelfi Cecilia , Leone Roberta , Capitanio Daniele , Viganò Agnese , Fania Chiara , Anastasia Luigi , Leri Annarosa
Published: July 2021

Recent studies revealed a key role of mitochondria in the 'axis of aging'. Mitochondrial dysfunction induces ROS production, leading to genotoxic damage, increasing erosion of telomeres followed by the activation of p53, blocking the mitochondrial biogenesis and generating a vicious cycle which induces the age-related mitochondrial dysfunction. This study, based on proteomic analysis, aims to elucidate the role of different mitochondrial subpopulations in the aging of heart in a rat model. Subsarcolemmal (SS) mitochondria play a major role in the adaptation of cells to physiological stimuli, whereas intermyofibrillar (IMF) mitochondria, are “power plants” for cardiac muscle contraction. The two subpopulations were isolated according to their localization in young (6 months), old adult (22 months) and senescent (30 months) Sprague Dawley rats by differential centrifugation in a density gradient. The differences at proteomic level were monitored by 2D-DIGE coupled with Maldi Tof and ESI MS/MS. While only few significant differences were detected in whole cardiac muscle proteome among young, adult and old rats, we detected many differences in SS mitochondrial proteome, particularly related to cell metabolism, cytoskeletal organization, membrane permeability and chaperon proteins indicating that aging may affect the SS mitochondria to maintain heart tissue homeostasis. Proteome of IMF mitocondria was characterized by a different pattern as compared to SS mitochondria and the number of proteins changed in aging were lower, even though a progression was observed from 22 to 30 months. These preliminary results suggest that protein dysregulation in mitochondrial proteome could significantly contribute to age-dependent impairment of cardiac function. However, these changes are not appreciable when analyzing the heart muscle proteome as a whole, highlighting the relevance of sub-cellular fractionation to detect molecular events involved in the aging process.
Abstract P244: Transcriptional Regulation of Cardiac Gene Expression by Med13 Alters Global Energy Balance
Authors: Grueter Chad E , Johnson Brett A , Qi Xiaoxia , McAnally John , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Aberrant cardiac metabolism is associated with obesity, type 2 diabetes and heart failure. The heart requires highly efficient metabolism to maintain the levels of ATP needed for contractility and pump function, however little is known about the role of the heart as a metabolic organ. Nuclear hormone receptors, such as thyroid hormone receptor play an important role in cardiovascular disease by significantly altering expression of genes involved in maintaining metabolic activity. The Mediator, a large multiprotein complex functions as a hub to control gene expression through association with transcriptional activators and repressors. We tested the hypothesis that Med13, a component of the Mediator complex, regulates cardiac function in a gain-of-function mouse model. Trangsenic mice overexpressing Med13 in the heart are lean, have increased energy expenditure, are resistant to high fat diet-induced obesity and have enhanced cardiac contractility. Microarray analysis and biochemical assays show that in vivo and in vitro Med13 selectively inhibits nuclear hormone receptor target genes of energy metabolism. These results implicate the Mediator complex regulates energy balance and cardiac contractility and suggests that the heart may function as a key component of mammalian energy homeostasis.
Abstract P276: Cardiac-Specific Knockout NF-kB Mice Resist Dysfunction Induced by Global Ischemia-Reperfusion
Authors: Zhang Xiu Q , Li Ling , Deng Yubin , Scott Lensey , Selzman Craig
Published: July 2021

Subjective:
Nuclear factor Kappa B (NF-kB) is a pluripotent transcriptional factor that has been implicated as mediator of cardiac injury after ischemia-reperfusion (IR). Multiple approaches have been used to target inhibition of NF-kB, yet these strategies are often non-specific. P65-NF-kB exists as a heterodimer with its p50 subunit and is the dominant transcriptional subunit of NF-kB. In order to better understand its role in the heart, we have recently generated mice that have cardiomyocyte (CMC) specific deletion of p65 NF-kB and hypothesized that these animals would be protected from ischemic stress.

Methods:
Transgenic mice were generated with (WT) or without CMC p65 (KO). NF-kB p65 expression was assessed with Western Blots. We subjected the mice to global IR using Langendorf system with baseline 20 min, ischemia 45 min and reperfusion 60 min. Cardiac contractile functional recovery was analyzed using rate pressure product (RPP), contractility (+dp/dt), and relaxation (-dp/dt). Infarct size was assessed with TTC staining. LDH and CK were measured in reperfused solutions.

Results:
1.
No differences were found between groups of WT (n=7) and KO (n=8) in body weight and heart weight.
2.
KO mice express significant less cardiac-specific NF-kB p65.
3.
During reperfusion, RPP, +dp/dt, -dp/dt recovery to base line were all better in p65 KO mice (RPP: 0.841±0.074 in KO vs 0.635±0.080 in WT; +dp/dt: 0.997±0.096 in KO vs 0.731±0.108 in WT; -dp/dt: 0.869±0.096 in KO vs 0.630±0.090 in WT; p<0.05 respectively).
4.
After ischemia, both LDH and CK release were markedly less in solutions collected from p65 KO heart (LDH: 45.2±28.0 u/L; CK: 28.1±23.6 u/L) than those in p65 WT heart (LDH: 143.2±25.9 u/L; CK: 107.2±26.2 u/L; p,0.01 respectively).
5.
TTC staining shown significant less necrotic tissue in KO heart vs WT heart (0.314±0.052 vs 0.653±0.061, p<0.05).

Conclusion:
CMC-specific knockout NF-kB p65 mice have improved cardiac recovery function, less heart necrosis, and decreased CMC injury compared to WT after global heart ischemia. These studies confirm many of the previous studies suggesting a protective role for anti-NF-kB strategies for ischemia. It furthermore provides a template to further define the mechanisms of p65-NF-kB afforded cardioprotection.
Abstract P292: Possible Mechanism and Potential Markers for Ozone-Induced Cardiac Toxicity
Authors: Sethi Rajat , Perepu Rama S , Ramirez Magdalena , Bandla Ashoka , Manchanda Shubham , Garcia Carlos , Dostal David
Published: July 2021

A significant number of deaths each year in the US have been linked to environmental pollutants such as ozone (O
3
). Earlier studies from our laboratory have shown that myocardial dysfunction, subsequent to chronic O
3
exposure, in normal adult rats may be associated with a decrease in antioxidant reserve and with an increased activity of inflammatory mediators. The present study tested the hypothesis that O
3
induced cardiac dysfunction in healthy normal adult rats may be due to changes in caveolin-1 and caveolin-3 levels. Sprague Dawley rats were exposed 8 hr/day for 28 and 56 days to filtered air or 0.8 ppm O
3
. In order to assess the chronic effects to O
3
,
in-vivo
cardiac function was assessed by measuring left ventricular developed pressure (LVDP), 24 hr after termination of O
3
exposure. Compared to rats exposed to filtered air, LVDP values significantly decreased in all O
3
exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-alpha (TNF-α) levels and decreased myocardial activities of superoxidase dismutase (SOD). Progressive increases in the expression of myocardial TNF-α in 4 and 8 week O
3
exposed animals were followed by decreases in cardiac caveolin-1 levels. However, differential changes in the expression of caveolin-3 in hearts from 4 and 8 week O
3
exposed animals were independent of intra-cardiac TNF-α levels. These novel findings suggest the interesting possibility that a balance between caveolin-1 and caveolin-3 may be involved in O
3
- mediated cardiac toxicity. Furthermore, differential changes in caveolin-3 content may serve as a marker that predicts moderate and chronic stages of cardiac injury specific to exposure to O
3
in human populations residing in urban areas with unhealthy levels of O
3
. The study is timely and has clinical significance related to environmental causes of cardiovascular disease. This novel study will form a basis for future studies to understand and define the various components of the mechanistic cascade responsible for generation of cell death signaling subsequent to O
3
exposure. The long-term goal of this study is in guiding regulatory policies to the USEPA regarding air quality standards pertaining to O
3
levels.
Abstract P293: Endogenous Lats2 Plays an Important Role in Mediating Myocardial Injury Caused by Ischemia/Reperfusion Through Inhibition of FoxO3
Authors: Shao Dan , Zhai Peiyong , Sadoshima Junichi
Published: July 2021

Lats2 is a tumor suppressor and a serine/threonine kinase, acting downstream of mammalian sterile 20 like kinase1 (Mst1), which stimulates apoptosis and inhibits hypertrophy in cardiomyocytes (CM). We investigated the role of Lats2 in mediating myocardial injury after ischemia/reperfusion (IR). Phosphorylation of YAP, an in vivo substrate of Lats2, was increased after 45 minutes ischemia followed by 24 hours reperfusion in control mouse hearts compared with sham, but not in dominant negative (DN) Lats2 transgenic mouse (Tg) hearts, suggesting that Lats2 is activated by IR. The size of myocardial infarction (MI)/area at risk was significantly smaller in Tg mice than in NTg mice (19% and 49%, p<0.01). And there were fewer TUNEL positive cells in Tg than in NTg mice (0.04% and 0.11%, p<0.05). Following 30 min of global ischemia and 60 min of reperfusion in Langendorff perfused heart preparations, left ventricular (LV) systolic pressure (100 vs 71mmHg, p<0.05) and LV developed pressure (79 vs 47 mmHg, p<0.05) were significantly greater in Tg than in NTg mice, indicating that suppression of Lats2 induces better functional recovery after IR. Oxidative stress, as evaluated by 8-OHdG staining, was attenuated in Tg mice. In cultured CMs, DN-Lats2 significantly decreased H
2
O
2
-induced cell death. Overexpression of Lats2 significantly downregulated (51% and 75%, p<0.05), whereas that of DN-Last2 upregulated (100 and 70%, p<0.05), MnSOD and catalase, suggesting that Lats2 negatively regulates expression of antioxidants. Reporter gene assays showed that overexpression of Lats2 significantly inhibits (−70%), whereas knocking down Lats2 by sh-Lats2 increases (+60%), FoxO3-mediated transcriptional activity. Overexpression of Lats2 in CMs inhibited FoxO3 expression, whereas that of DN-Lats2 significantly inhibited FoxO3 downregulation after IR in vivo, suggesting that Lats2 negatively regulates FoxO3 protein expression, which may lead to the downregulation of MnSOD and catalase. Taken together, these results suggest that endogenous Lats2 plays an important role in mediating myocardial injury in response to IR, In part through downregulation of FoxO3 and consequent downregulation of antioxidants and increased oxidative stress in the heart.