bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P199: Placental Growth Factor Regulates Cardiac Adaptation and Hypertrophy Through a Paracrine Mechanism
Authors: Accornero Federica , Van Berlo Jop , Benard Matthew D , Carmeliet Peter , Molkentin Jeffery D
Published: March 2022

Objective. Paracrine growth factor-mediated crosstalk between cardiac myocytes and non-myocytes in the heart is critical for programming adaptive cardiac hypertrophy in which myocyte size, capillary density, and the extracellular matrix function coordinately. Here we examined the role that placental growth factor (PGF) plays in the heart as a paracrine regulator of myocyte to non-myocyte communication and its influence on cardiac adaptation to stress stimulation using overexpressing and PGF knockout mice.

Methods and results. We identified PGF as a secreted factor that is predominantly produced in the heart during pressure overload. We studied mice with conditional post-natal PGF overexpression (PGF DTG). While these mice did not have a baseline phenotype, except increased fibrosis with aging, they responded to pressure overload stimulation induced by transverse aortic constriction (TAC) by an increase in hypertrophy (VW/BW (mg/g): 6.6 ± 0.2 for PGF DTG versus 5.6 ± 0.2 for controls; n ≥ 6 per group; p<0.01), capillary density (vessels/myocyte: 1.6 ± 0.05 for PGF DTG versus 1.4 ± 0.03 for controls; n ≥ 7 per group; p<0.01) and fibrosis. Despite a mild increase in fibrosis, cardiac function remained intact even after 12 weeks of pressure overload. On the other hand, PGF knockout mice (Pgf-/-) succumbed to heart failure within a week of pressure overload (fractional shortening (%): 20.2 ± 2.3 for Pgf-/- versus 30.4 ± 1.1 for controls; n ≥ 7 per group; p<0.01). These hearts displayed dilation and capillary rarefaction (vessels/myocyte: 1 ± 0.05 for Pgf-/- versus 1.2 ± 0.04 for controls; n ≥ 7 per group; p<0.01). Mechanistically we show that PGF has no direct effect on the cardiomyocytes but works through its direct actions on endothelial cells and fibroblasts by inducing capillary growth and fibroblasts proliferation.

Conclusion. PGF is a secreted factor that supports hypertrophy and cardiac function during pressure overload by affecting endothelial cells and fibroblasts.
Abstract P202: Role of Lipid Peroxidation--Derived Carbonyls in Left Ventricular Hypertrophy
Authors: Baba Shahid P , Singh Mahavir , Bhatnagar Aruni
Published: March 2022

Pathological left ventricular hypertrophy (LVH) is a key factor in the development of heart failure and cardiomyopathy. It is characterized by increased oxidative stress however the mechanisms by which it affects pathological LVH remain unclear. Because the effects of reactive oxygen species are mediated by products of lipid peroxidation, we hypothesize that myocardial injury induced by LVH is mediated by lipid carbonyls, which form covalent adducts with proteins that induces autophagy. To test this hypothesis, we examined the role of aldose reductase (AKR1B3; AR) an enzyme that catalyzes the detoxification of lipid carbonyls in modulating the cardiac stress responses in WT and AR-null mice hearts that were subjected to transverse aortic constriction (TAC). Two weeks after the TAC cardiac enlargement in the AR-null TAC mice was more prominent compared with WT TAC mice. Hemodynamic analysis of the AR-null TAC mice demonstrated left ventricular dilatation and severe contractile dysfunction compared with WT TAC mice. Expression levels of molecular marker for hypertrophy (ANF) increased 2-3 fold in AR-null TAC compared with the WT-TAC. Western blot analysis of the heart homogenates using anti-4 hydroxy-trans-2-nonenal (HNE) antibody showed that the formation of HNE-protein adduct (300KDa) increased 100-fold and the bands corresponding to molecular weights of 75KDa, 40KDa increased 2 to 3-fold after the TAC in WT hearts and the intensity of these bands in AR-null TAC hearts was significantly higher compared with WT TAC. Immuno-blot analysis of the heart homogenates revealed an increase in the LC3-I to LC3-II conversion in banded hearts compared with sham-operated controls and the autophagic activity was more prominent in the AR-null compared to the WT banded hearts. Isolated adult cardiomyocytes from WT and AR-null hearts perfused with HNE resulted in an increase in autophagy and the increase in autophagic activity was robust in AR-null than in WT myocytes. These data establish that TAC-induced hypertrophy increases protein-HNE-adduct formation and autophagic activity and the deletion of
akr1b3
gene exacerbates these responses to pressure overload.
Abstract P213: Epidermal Growth Factor Receptor and c-Src Are Involved in Stretch-Induced Activation of JNK1/2 and P38 in Cardiac Myocytes and Cardiac Fibroblasts
Authors: Feng Hao , Gerilechaogetu Fnu , Golden Honey B , Cristalis Ricardo , Dasuri Kumar , Nizamutdinov Damir , Foster Donald M , Tong Carl , Dostal David E
Published: March 2022

Mechanical stretch is a major determinant that leads to heart failure. Heart failure is associated with a steady increase in myocardial angiotensinogen (Ao) expression and the biological peptide angiotensin II (Ang II) formation. We have previously identified key downstream effectors that couple to mechanical stretch-induced Ao gene expression. JNK1/2 and p38α were found to have opposing roles on stretch-induced Ao gene expression in neonatal rat cardiac myocytes (NRVM) and cardiac fibroblasts (NRFB). JNK negatively regulated Ao expression in NRVM and NRFB following acute stretch, whereas with prolonged stretch, p38α was responsible for upregulation of Ao expression. However, the mechanisms leading to activation of these kinases and cross-talk between these signal molecules remain to be determined. Epidermal growth factor receptor (EGFR) is expressed in both NRVM and NRFB, but its precise role in mechanosensing is poorly understood. In NRVM, stretch caused JNK1/2 activation (phosphorylation) within 2 min, with a peak response at 15 min (3.96 ± 0.71 fold, p<0.01). Stretch induced p38 activation within 5 min, which remained elevated (2.93 ± 0.49 fold, p<0.05) at 60 min (final time point). Pretreatment with EGFR blockers AG1478 (100 nM) and BIBX 1382 (200 nM) inhibited stretch-induced activation of JNK at early time points (5 and 15 min), whereas BIBX 1382 and the Ang II receptor type I inhibitor losartan (10 µM) inhibited p38 activation at 60 min. Stretch induced activation of c-Src (phosphorylation of tyrosine 416) at 60 min (1.56 ± 0.21 fold, p<0.01), which was attenuated by losartan. In NRFB, stretch-induce-JNK-phosphorylation can be inhibited by both AG1478 and Src inhibitor PP2 (100 nM) at 15 min. Stretch also increased phosphorylation levels of c-Src at tyrosine 416 and EGFR at tyrosine 1068 at 15 and 60 min, which were blocked in the presence of losartan. In a summary, these studies give strong evidence that both EGFR and c-Src are involved in stretch-induced signal cascade in NRVM and NRFB.
Abstract P220: Anthrax Receptor Regulation of FAK and JNK Activation in Cardiac Myocytes
Authors: Nizamutdinov Damir , Golden Honey B , Gerilechaogetu FNU , Feng Hao , Foster Donald M , Tong Carl , Dostal David E
Published: March 2022

We previously demonstrated that anthrax lethal toxin (LT, protective antigen [PA] + lethal factor [LF]) produces acute systolic heart failure, characterized by increased left ventricular (LV) end-diastolic volume, reduced ejection fraction and decreased contractility in male Sprague-Dawley (SD) rats. The molecular basis for the cardiac dysfunction observed in either an animal model or clinical setting of anthrax toxicity remains to be examined. Previously, we tested LT dose-response effects on isolated neonatal rat ventricular myocytes (NRVM) at a dose of 0.05 ng/mL PA + 0.025 ng/mL LF diminished PLB and JNK phosphorylation from 1h to 4 h. During the LT+PA time-course, Akt phosphorylation at both Thr
308
and Ser
473
were also significantly reduced at all time points from 30 min to 4 h, accompanied by elevated B56α total protein and intracellular Ca
2+
, and reduced PLB phosphorylation. This response is mediated by translocation of LF into the cell through the anthrax receptors 1 and 2 (ATR1/2), which are structurally similar to integrins. In this study, we tested whether stimulation of ATR1/2 by PA alone can induce activation of the integrin effectors, FAK and JNK. Cardiac myocytes were treated time dependently (5 sec - 30 min) by PA at 0.25 ng/mL, and activation of FAK protein at different residues was determined. PA induced phosphorylation of FAK-Tyr
861
at 15 sec (p<0.05), whereas there was no effect on FAK-Tyr
397
or FAK-Tyr
925
phosphorylation for any of the time points. Furthermore, we found that PA alone maximally increased JNK phosphorylation at 15 min of treatment (p<0.05). This response was not blocked with either Losartan (10 µM) or AG1479 (100 nM) indicating that neither angiotensin type I receptor (AT1) or epidermal growth factor receptor (EGFR) transactivation was involved. In summary, this study suggests a key role of ATR1/2 in mediating of signal transduction across membrane of cardiac myocytes in response to
B. anthracis
PA stimulation and involvement in acute responses in regulation of FAK and JNK activation in independent of AT1 and EGFR pathway.
Abstract P229: Nongenomic Cardioprotective Effect of Dehydroepiandrosterone on Cardiac Hypertrophy: Comparison with 17β-Estradiol-Induced Cardioprotection
Authors: Tagashira Hideaki , Bhuiyan Md Shenuarin , Shioda Norifumi , Fukunaga Kohji
Published: March 2022

Objective:
We recently reported a decreased σ
1
receptor expression in heart following abdominal aortic stenosis in bilateral ovariectomized (OVX) rats. Here, we demonstrated non-genemic cardioprotective effects of dehydroepiandrosterone (DHEA) though σ1 receptor in pressure overload (PO)-induced cardiac dysfunction.

Methods:
Bilateral ovariectomy was performed in female rats. Two weeks after the sham operation or ovariectomy, pressure-overload was initiated by abdominal aortic banding. 17β-estradiol (E2: 0.1 mg/kg) and DHEA (30 mg/kg) were administered to rats subcutaneously and orally, respectively, for 14 days starting 2 weeks after aortic banding. Hemodynamic parameters and cardiac hypertrophy were measured after 2 weeks of drug treatment. After dissection out of left ventricular, gene expression and hypertrophic signaling were analyzed by real-time RT-PCR and western blot, respectively.

Results:
Both E2 and DHEA treatments significantly inhibited pressure overload-induced increases both in heart weight/body weight (HW/BW) ratio and lung weight/body weight (LW/BW) ratios. Both E2 and DHEA treatments also ameliorated hypertrophy-induced impairment of left ventricular end diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), left ventricular contraction and relaxation (±dp/dt) rates, heart rate (HR) and mean arterial blood pressure (MABP). Notably, DHEA but not E2 administration rescued PO-induced σ
1
receptor downregulation in left ventricular. Co-administration with NE-100, a σ
1
receptor selective antagonist, inhibited DHEA-induced amelioration of heart dysfunction without effects on E2-induced cardioprotection. Mechanistically, both E2 and DHEA treatments significantly restored PO-induced decreases in Akt phosphorylation and Akt-mediated eNOS phosphorylation (Ser1179). NE-100 treatment totally abolished DHEA-induced Akt and eNOS phosphorylation without effects on E2-induced Akt/eNOS activation.

Conclusions:
Taken together, based on these results with OVX rat heart, DHEA but not E2 elicits cardioprotective action through σ
1
receptor activation. DHEA-induced Akt/eNOS activation through σ
1
receptors likely mediates the cardioprotective activity.
Abstract P233: PPARα-Sirt1 Complex Mediates Metabolic Adaptation in Response to Starvation in the Heart
Authors: Oka Shinichi , Zhai Peiyong , Yamamoto Takanobu , Shao Dan , Sadoshima Junichi
Published: March 2022

Minimizing nutritional usage is believed to be important for prolonging survival time in organisms under starvation conditions such as fasting and hibernation. The heart is a major organ that constantly consumes nutrition coupling with mitochondrial ATP production. However, it remains unknown how mitochondrial gene expression is regulated in the heart during starvation conditions. We have previously shown that a PPARα-Sirt1 complex mediates downregulation of mitochondrial gene expression in the failing heart. Since both PPARα and Sirt1 are crucial regulators in physiological fasting responses, we hypothesized that the same mechanism is involved in the physiological fasting response in the heart. To investigate this hypothesis, heterozygous knockout PPARα (PPARα
+/-
) and Sirt1 (Sirt1
+/-
) mice were subjected to 24 hours fasting. Several mitochondrial genes, such as Sdha and Atp5g1, were downregulated following fasting in wild type (WT) mice, but this downregulation was at least partly prevented in PPARα
+/-
and Sirt1
+/-
mice, suggesting that PPARα and Sirt1 play an important role in fasting-induced downregulation of mitochondrial gene expression in the heart. After fasting, cardiac systolic function was slightly reduced in WT mice, but this response was not induced in PPARα
+/-
and Sirt1
+/-
mice (ejection fraction (%): Fed WT 74, Fasted WT 68*, Fed PPARα
+/-
76, Fasted PPARα
+/-
75, Fed Sirt1
+/-
74, Fasted Sirt1
+/-
74, p<0.05 vs Fed WT). Weight loss after 24 hours fasting increased in PPARα
+/-
mice, compared to WT mice (Body weight reduction (%): WT 9, PPARα
+/-
14*, p<0.05 vs WT). Under an isolated working mouse heart model with nutrient free perfusion buffer, spontaneous beating terminated earlier in hearts isolated from PPARα
+/-
and Sirt1
+/-
mice than in hearts from WT mice (Beating time (minutes): WT 63, PPARα
+/-
37*, Sirt1
+/-
36*, p<0.05 vs WT). Glucose deprivation-induced cell death was cooperatively prevented by overexpression of PPARα and Sirt1 in primary cultured myocytes (Cell survival rate (%): control 54, PPARα 75*, Sirt1 60, PPARα with Sirt1 94*, PPARα with Sirt1 knockdown 52, p<0.05 vs control). These results suggest that PPARα and Sirt1 are crucial regulators of adaptation to starvation in the heart.
Abstract P238: Snf1-Related Kinase Is a Novel Regulator of Cardiomyocyte Metabolism
Authors: Rines Amy K , Burke Michael A , Nagpal Varun , Ardehali Hossein
Published: March 2022

Background:
Snf1-related Kinase (SNRK) is a serine/threonine kinase with sequence homology to AMP-activated kinase (AMPK). We have found that SNRK is upregulated in hearts from patients with ischemic cardiomyopathy, but the function of SNRK has not been studied in cardiomyocytes. Since previous gene array data show that SNRK alters expression of metabolic genes, we determined whether SNRK regulates cardiomyocyte metabolism.

Results:
SNRK downregulation reduced ATP levels by 23.34% in neonatal rat cardiomyocytes compared to nonsilencing siRNA, and SNRK overexpression increased ATP by 20.67% compared to GFP control. SNRK knockdown reduced baseline oxygen consumption rate (OCR) by 23.69%, and by 54.53% with the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP). SNRK overexpression increased OCR by 9.57% with CCCP. Mitochondrial content was unchanged as measured by mitochondrial DNA and cardiolipin staining. However, tetramethylrhodamine ethyl ester (TMRE) staining was increased by SNRK, suggesting that SNRK maintains mitochondrial membrane potential. Furthermore, the extracellular acidification rate was reduced 19.47% with SNRK knockdown and increased 12.57% with overexpression, signifying enhanced glycolytic flux. SNRK also increased lactate levels and mRNA of the glycolytic proteins hexokinase I and II. Glucose uptake increased 29.61% with SNRK overexpression, and expression of glucose transporters GLUT1 and GLUT4 was also increased, demonstrating an increase in cellular glucose import. Glycogen was also decreased 40.69% with SNRK knockdown and increased 46.03% with overexpression, demonstrating that SNRK reduces storage of glucose into glycogen. Additionally, OCR with palmitate over baseline was reduced 43.56% with SNRK downregulation and increased 285.6% with overexpression, suggesting enhanced fatty acid oxidation. Phosphorylation of acetyl-coA carboxylase was also increased by SNRK. Fatty acid uptake, however, was unchanged.

Conclusions:
Our results demonstrate that SNRK increases ATP and oxygen consumption through augmented mitochondrial membrane potential, glucose utilization, and fatty acid oxidation. These findings reveal SNRK as a major modulator of cardiomyocyte metabolism.
Abstract P248: The DCM-Causing D230N Tropomyosin Mutation Produces an Age-Dependent Phenotype in Mice that Is Influenced by Transgene Dosage
Authors: Tal Lauren , Tardiff Jil C
Published: March 2022

A recent study in two large multigenerational families demonstrated a novel mutation in tropomyosin (Tm), Asp230Asn (D230N) that caused DCM with a unique natural history resulting in a striking bimodal disease distribution. Infants and young children with the D230N Tm mutation presented with a severe, often fatal DCM while adults developed a mild to moderate clinical phenotype. Position 230 in tropomyosin is proximal to the C-terminal unwinding of tropomyosin that is essential for the Tm-Tm overlap and for the Tm-cardiac troponin T (cTnT) interaction. We hypothesized that the age-dependent remodeling is a result of temporal isoform switches in the closely linked Tm-binding partner cTnT. To directly address this hypothesis, we used the regulated in vitro motility assay to determine the biophysical effects of the D230N Tm mutation as well as the effects of cTnT isoform changes on the regulatory function of thin filaments. We have found that both wild type and D230N Tm filaments inhibit filament sliding at low calcium indicating that the D230N mutation does not completely disrupt regulation. However in the presence of high calcium the D230N Tm mutation significantly decreases the maximal velocity of filament sliding (3.790 ± 0.1333 microns/sec, n=98) as compared to the wild type filaments (4.900 ± 0.1044, microns/sec n=76). These findings support an altered Tm-cTnT interaction because at maximal calcium concentrations, the myofilament activation at the level of cardiac troponin is not properly transmitted to tropomyosin thus decreasing the activity of myosin-ATPase. In order to extend our biophysical observations and to provide translational insight, we generated a D230N Tm transgenic mouse model. In preliminary studies we demonstrate that at two months the D230N transgenic mice have evidence for early dilatation. In addition, the differences in ventricular remodeling were highly dependent on transgene dose. At six months, we observed dilatation of the ventricle and thinning of the walls compared to non-transgenic siblings. However, the differences were not as marked as those at the two-month time point. Collectively, these findings support an age-dependent DCM phenotype that is modulated by transgene dosage.
Abstract P250: Reversal of Excessive
O
-GlcNAcylation Restores Myofilament Function in Diabetic Cardiomyopathy: Potential Role of OGT/OGA Enzymes Localization
Authors: Ramirez-Correa Genaro A , Slawson Chad , Ding Wengang , Shen Xiaoxu , Gao Wei D , Hart Gerald W , Murphy Anne M
Published: March 2022

Diabetes Mellitus impacts the heart contractile apparatus, as well as cellular ion homeostasis, and energy production systems. Diabetic cardiomyopathy is accompanied by extensive changes in protein post-translational modifications (PTMs), including phosphoylation and
O-
GlcNAcylation. The cellular and molecular mechanisms of diabetic cardiomyopathy are only partially understood. We previously identified 32 total
O-
GlcNAcylation sites on MHC, Actin, MLC 1, MLC2, and TnI from normal hearts, and showed that exposure of skinned muscles to GlcNAc induces myofilament Ca
2+
desensitization. In addition, Actin
O-
GlcNAcylation was increased when exposed to GlcNAc in vitro or to hyperglycemia in two models of Diabetes Mellitus (DM) type 1 and type 2. Our hypothesis is that hyperglycemia directly alters the cycling of
O
-GlcNAcylation on key myofilament regulatory proteins, and that excessive
O
-GlcNAcylation is responsible for defective myofilament Ca
2+
-activated response, thus contributing to diabetic cardiomyocyte dysfunction. To test this hypothesis, we have identified OGT localization at the Z-disks where other relevant signaling molecules reside, whereas OGA is localized along the M-band. OGT and OGA affinity for myofilament proteins (Actin, Tm, MLC1 and MLC2) is dramatically up-regulated in the diabetic heart, as assessed by co-immunoprecipitation and Western Blot. We also investigated the effect of
O
-GlcNAc removal from normal and diabetic skinned muscles by an engineered hexosaminidase (CPJ). We found that 1 hour exposure to CPJ restored myofilament Ca
2+
sensitivity exclusively in diabetic cardiac muscles (EC
50
4.17±0.48 µM pre-CPJ vs 2.73±0.22 µM post-CPJ,
n
=5
vs
n
=4, p=0.029 ). These results establish a direct link between cardiac protein hyper-
O
-GlcNAcylation and diabetic altered cardiac myofilament Ca
2+
sensitivity. Furthermore, OGT and OGA are abundant, and located at strategic signaling compartments, such as the Z-Disk. Overall these data suggest that a specific increase of OGT/OGA affinity towards key myofilament regulatory proteins, and subsequent hyper-
O
-GlcNAcylation may lead to dysfunctional regulation of myocardial contractility.
Abstract P253: Transcriptional Regulation of the IGFBP-1 Gene by RTEF-1 in Endothelial Cells
Authors: Messmer-Blust Angela F , Wu Jiaping , Jin Yi , Guo Shaodong , Li Jian
Published: March 2022

Background and Objective:
Cardiovascular disease is the most important complication of type 2 diabetes, however, its underlying mechanism(s) are poorly understood. Our lab previously reported that RTEF-1 is a transcription factor that promotes angiogenesis via up-regulating HIF-1α and VEGF. In addition to these studies, we recently determined that RTEF-1 upregulates insulin-like growth factor binding protein-1 (IGFBP-1) expression in endothelial cells in vitro and in vivo. IGFBPs are key regulators of insulin-like growth factor (IGF) type 1 and 2 bioavailability at the cellular level. In addition, IGFBP-1 is implicated in cardiovascular health, in terms of blood pressure and glucose tolerance.

Methods and Results:
RTEF-1 upregulates IGFBP-1 expression in endothelial cells in vitro and in vivo through selectively binding and promoting transcription from the insulin response element (IRE) site on the IGFBP-1 promoter, demonstrated by ChIP assays. In addition to insulin inhibition of IGFBP-1 levels, insulin also dose-dependently inhibits RTEF-1 promoter activity (-2.4 ± 0.3-fold) and expression. In vivo data suggest that Tie2-Cre:RTEF-1 flox/flox transgenic mice, RTEF-1 EC-specific knockout mice, show a significant decrease decrease in IGFBP-1 mRNA levels compared with controls (0.21 ± 0.09 vs. 1.0, respectively), while VE-Cadherin/RTEF-1 overexpressing transgenic mice have increased IGFBP-1 mRNA levels (25.4 ± 0.02-fold). Increased IGFBP-1 levels relate physiologically in our RTEF-1 EC-specific knockout by showing decreased blood glucose tolerance as well as increased insulin resistance as calculated by HOMA-IR compared with littermate controls, (4.1 ± 0.5 vs. 1.1 ± 0.2, respectively). Furthermore, the RTEF-1 EC-specific knockout mice showed a significant increase in systolic blood pressure (10mmHg ± 6mmHg) compared with littermate controls.

Conclusion:
These results demonstrate that RTEF-1-stimulated IGFBP-1 expression may be central to the mechanism by which RTEF-1 attenuates blood glucose levels and potential cardiovascular function. These findings provide the basis for novel insights into transcriptional regulation of IGFBP-1 and contribute to understanding the role of vascular endothelial cells in metabolism.
Abstract P256: Epigenetic Changes by Nuclear Calcium/Calmodulin-Dependent Protein Kinase II in the Heart
Authors: Poizat Coralie , Little Gillian , Volkers Mirko , Toko Haruhiro , Bai Yan
Published: March 2022

Calcium is central in the regulation of many cellular functions and alteration of calcium signaling leads to a variety of cardiac disorders. An important question remaining in cardiac biology is how calcium-dependent mechanisms are regulated and more specifically how calcium activates intracellular signaling pathways and regulates gene activity in the cell nucleus. The nuclear isoform of calcium/calmodulin-dependent protein kinase II (CaMKIIB) plays a protective role in the heart under basal conditions, and alteration of its level or activity is associated with pathological events such as cell death and cardiac hypertrophy. We recently made a novel discovery showing that CaMKIIδB couples calcium signals to the genome by targeting specific histones in chromatin. Our preliminary results show that CaMKIIδB selectively phosphorylates histone H3 in vitro and in primary cardiomyocytes. Activation of CaMK activity after α-adrenergic stimulation of primary cardiac cells increases histone H3 phosphorylation at serine 10 (Ser-10), whereas specific elimination of the kinase has the opposite effect. Enhanced phosphorylation of histone H3 Ser-10 is observed in primary fibroblasts and also in primary ventricular myocytes and culminates at 24 hours of stimulation, suggesting that signaling to histone H3 is not limited to highly dividing cells and is not transient. Importantly, Enhanced histone H3 Ser-10 phosphorylation can be detected in the heart of mice subjected to myocardial infarction. These results reveal a new role for CaMK enzymes in chromatin remodeling the heart and that epigenetic changes may contribute to the development cardiac diseases.
Abstract P268: Nanoparticle-Mediated Delivery of Pitavastatin into Reperfused Myocardium Reduces Myocardial Ischemia/Reperfusion Injury
Authors: Nagaoka Kazuhiro , Matoba Tetsuya , Nakano Kaku , Sunagawa Kenji , Egashira Kensuke
Published: March 2022

Background:
In acute myocardial infarction, early myocardial reperfusion is the most effective therapeutic strategy. The reperfusion, however, induces ischemia-reperfusion (I/R) injury that paradoxically cancels the beneficial effects of reperfusion. Statins are known to activate reperfusion injury salvage kinase (RISK) pathway, but its efficacy is not enough to be used as cardioprotective therapy. Local delivery of statins might optimize cardioprotective effects of statins. Hence we tested the hypothesis that nanoparticle (NP)-mediated delivery of pitavastatin into ischemic myocardium at the time of reperfusion ameliorates myocardial I/R injury.

Methods and Results:
In a rat model of 30-min ischemia followed by reperfusion, significant FITC signals were detected in cardiomyocytes within infarct areas after intravenous (IV) injection of FITC-NP at the time of reperfusion (
Figure
A). No significant FITC signals were noted after IV injection of FITC only. IV injection of pitavastatin-NP that contained 1.0 mg/kg pitavastatin at reperfusion reduced infarct size (
Figure
B). The therapeutic effects of pitavastatin-NP were associated with activation of RISK pathway such as Akt signals in the reperfused myocardium. Wortmannin blunted the therapeutic effects of pitavastatin-NP (Figure B). On the other hand, no therapeutic effects were noted after IV injection of pitavastatin alone (1.0, 10 mg/kg).

Conclusions:
In I/R injury, NPs were delivered selectively into reperfused myocardium possibly via enhanced permeability and retension effects. NP-mediated delivery of pitavastatin at the time of reperfusion reduced myocardial infarct size by activating RISK pathway.
Abstract P270: Resin-Assisted Capture Reveals Unique Targets of Oxidation in Ischemic Preconditioning
Authors: Kohr Mark J , Sun Junhui , Aponte Angel , Wang Guanghui , Gucek Marjan , Steenbergen Charles , Murphy Elizabeth
Published: March 2022

Proper maintenance of cellular thiol redox status is critical in the regulation of many processes that dictate normal myocardial function. Cell death signaling, in particular, is especially susceptible to alterations in thiol redox state. Myocardial ischemic preconditioning (IPC) is a redox sensitive process that is thought to provide protection from ischemia-reperfusion (IR) injury, in part, via reversible oxidative signaling. Protein
S
-nitrosylation (SNO) is a reversible, thiol-based modification that we and others have shown to provide cardioprotection by modulating the activity of target proteins and by shielding cysteine residues from irreversible oxidation. Additional thiol-based modifications are also thought to play an important role in cardioprotection, and these include disulfide bridges and glutathiolation. Therefore, it is of interest to identify these redox sensitive targets that are unique to IPC. Langendorff perfused mouse hearts were subjected to various perfusion protocols (control, IPC, IR, IPC-IR) and homogenized. Specific sites of oxidation were identified using oxidation-resin assisted capture (Ox-RAC) with mass spectrometry. Consistent with a role for oxidative signaling in cardioprotection, Ox-RAC analysis identified 56 proteins that were oxidized with IPC, but not IR or IPC-IR. These proteins are likely to be reversibly oxidized, as evidenced by the fact that they are oxidized with IPC, but are no longer oxidized following reperfusion (IPC-IR). These proteins may represent important targets in cardioprotective signaling and include F-actin-capping protein β, Egl nine homolog 1, and numerous members of the heat shock family (HSP60, HSP70, HSP90β). Heat shock proteins are important in the cellular stress response and have been shown to play an important role in cardioprotection. Pathway analysis also revealed that more than 30% (19 of 56) of the proteins uniquely oxidized following IPC were involved with cell death pathways. These proteins include Aminoacyl tRNA synthase complex-interacting multifunctional protein 1, Charged multivesicular body protein 5, and Lamin-A/C. These results indicate that the cardioprotective effects of IPC may occur, in part, through the reversible oxidation of key protein targets.
Abstract P271: Genetically Induced Moderate Inhibition of the Proteasome in Cardiomyocytes Exacerbates Myocardial Ischemia-Reperfusion Injury in Mice
Authors: Tian Zongwen , Zheng Hanqiao , Wang Xuejun (XJ)
Published: March 2022

Proteasome dysfunction is implicated human ischemic heart disease and observed in experimental myocardial ischemia-reperfusion (I/R) injury. Altered proteasome activities in I/R hearts were reported but it is unclear whether proteasome function in I/R hearts is adequate. Moreover, effects of pharmacological inhibition of the proteasome on I/R injury remain controversial. Hence we sought to determine the adequacy of proteasomal function in I/R hearts and the impact of moderate cardiomyocyte-restricted proteasome inhibition (CR-PSMI) on I/R injury in intact animals. First, myocardial I/R was created by ligation (30min) and subsequent release of the left anterior descending artery in the transgenic (tg) mice overexpressing GFPdgn, a previously validated surrogate proteasome substrate. Compared with the sham controls, myocardial GFPdgn protein levels in the remote area, the board zone, and the
a
rea
a
t
r
isk (AAR) of the ventricle 24hrs after reperfusion were significantly increased, indicative of proteasome functional insufficiency (PFI) in the I/R heart. The most important proteasome peptidase resides in β5 subunits of the 20S proteasome. The clinically used proteasome inhibitor bortezomib targets specially the β5 subunit. To achieve CR-PSMI in intact animals, we engineered and tested a catalytically inactive mouse β5 subunit mutant (T60A-β5) and created multiple tg mouse lines in which T60A-β5 was overexpressed under the control of an attenuated mouse
mhc6
promoter. Baseline analyses of the tg lines confirm the ability of T60A-β5 to replace endogenous β5 and inhibit proteasome chymotrypsin-like activities in the heart in a dose-dependent manner. A stable tg line with ∼40% replacement was used in this study. The same I/R procedure caused a significantly greater infarct size in T60A-β5 tg mice (66.2% of AAR) than in the littermate Ntg mice (53.4% of AAR). Under either the sham surgery or the I/R condition, a decrease in phosphorylated AKT and an increase in PKCδ proteins were evident in the tg hearts, compared with the respective Ntg groups. These results show that I/R causes PFI in the heart and PFI plays a pathogenic role in I/R injury. A potential mechanism by which PFI contributes to I/R injury is to suppress AKT activation and exacerbate PKCδ signaling.
Abstract P272: Physiological and Pathological Functions of Nox2 and Nox4 in Ischemia/Reperfusion Injury
Authors: Matsushima Shouji , Kuroda Junya , Ago Tetsuro , Zhai Peiyong , Sadoshima Junichi
Published: March 2022

NADPH oxidases (Noxes) represent an enzyme system whose primary function is to produce reactive oxygen species (ROS). Both Nox2 and Nox4 play an important role in regulating oxidative stress and growth/death of cardiomyocytes (CMs). However, the role of Nox2 and Nox4 during ischemia/reperfusion (I/R) is poorly understood. In order to elucidate the function of the Nox isoforms, wild type (WT), Nox2 KO (Nox2-/-), and cardiac specific Nox4 KO (cNox4-/-) mice were subjected to 30 minutes ischemia followed by 24 hours reperfusion. Myocardial infarction size/area at risk (MI/AAR) as evaluated by TTC staining was significantly smaller in Nox2-/- and cNox4 -/- than in WT mice (32 ± 3.1% and 28.0 ± 6.1% vs. 40.3 ± 8.3%, p < 0.05). O
2
-
production in Nox2 -/- and cNox4 -/- hearts was significantly lower than in WT hearts, as evaluated with the lucigenin-chemiluminescence assay (433 ± 52, 580 ± 106 vs. 1250 ± 236 RLU, p < 0.05). The I/R experiment was also conducted with mice in which both Nox2 and Nox4 are deleted (double KO mice). O
2
-
production in the double KO heart was significantly lower than in the single KO ones (257 ± 42 RLU, p < 0.05). The MI/AAR in double KO mice was, however, significantly greater than that in WT mice (58.0 ± 6.3% vs. 40.3 ± 8.3%, p < 0.05). These results raised the possibility that marked suppression of ROS by combined downregulation of Nox2 and Nox4 exacerbates I/R injury. To elucidate the underlying mechanism, we examined expression of hypoxia inducible factor-1alpha (HIF-1
α
), using DN-Nox transgenic (Tg) mice, which we have shown also exhibited marked suppression of O
2
-
and significantly greater MI/AAR after I/R. HIF-1
α
is lower in DN-Nox Tg mice than in WT mice in both the ischemic and non-ischemic areas. A genetic cross between DN-Nox Tg and mice lacking prolyl hydroxylase 2, an enzyme mediating hydroxylation and downregulation of HIF-1
α
, partially rescued I/R injury in DN-Nox mice (35.8 ± 5.9% vs 65.2 ± 10.3%, p<0.05). Taken together, these data show that both Nox2 and Nox4 mediate increases in oxidative stress and myocardial injury in response to I/R. However, combined downregulation of Nox2 and Nox4 induces marked downregulation of ROS, which in turn exacerbates I/R injury possibly through downregulation of HIF-1α and consequent impairment of hypoxic adaptation.
Abstract P282: Increased Nuclear Localization of Fyn May Limit the Cardioprotective Effects of Hydrogen Sulfide by Interfering with Nrf2 Signaling
Authors: Moody Bridgette F , Nicholson Chad K , Hood Rebecca L , Calvert John W
Published: March 2022

Background:
Hydrogen sulfide (H2S) therapy reduces myocardial infarct size by up to 56% in non-diabetic mice. Here, we sought to examine if H2S could provide cardioprotection in the setting of diabetes.

Methods and Results:
Diabetic mice (db/db) were subjected to 30 min of left coronary artery occlusion (LCA) followed by reperfusion for 24 hours at which time the extent of myocardial infarction was evaluated. Sodium sulfide (Na2S, 100 μ g/kg) administered at the time of reperfusion decreased infarct size relative the area-at-risk by 18% compared to vehicle treated animals (p<0.001). Previously, Na2S has been shown to increase the nuclear localization of Nrf-2 and upregulates its downstream target, heme oxygenase (HO)-1, in non-diabetic mice. To investigate if this cascade contributed to the observed cardioprotection, Na2S was administered (tail vein) to non-diabetic and diabetic mice and heart tissue was excised 1 hr and 24 hrs later. One hr following the administration of Na2S, the nuclear expression of Nrf2 was increased in both groups. In contrast, Na2S only increased the expression of HO-1 (24 hrs) in the non-diabetic heart. Further studies investigating Fyn, a tyrosine kinase known to inhibit Nrf2 signaling, revealed an upregulation of nuclear Fyn expression in the diabetic heart compared to the non-diabetic heart.

Conclusion:
This data demonstrates the complexity of therapeutic intervention for diabetics following myocardial ischemia, as the robust cardioprotective effects of H2S in the non-diabetic state were found to be diminished in the diabetic state. This data also suggests that interference of Nrf2 signaling by Fyn may be responsible for the loss of protection.
Abstract P286: Acetylation Affords Enhanced Proteolytic Function in Normal and Diseased Myocardium
Authors: Zong Nobel , Fang Caiyun , Wang Ding , Yu Hongxiu , Zhang Jun , Yang Pengyuan , Ping Peipei
Published: March 2022

Proteasome complexes have essential implications on protein homeostasis and myocardial viability under stress. In the heart, the dynamics of proteasome complexes is regulated by post-translational modifications. In particular, acetylation contributes to the adjustment of cardiac functional proteome. However, the regulation of proteasome complexes by acetylation remains largely unknown. We characterized the acetylome (N-terminal and lysine acetylation) of cardiac 20S proteasomes using proteomic technology. With targeted enrichment, eight sites of lysine acetylation and nine sites of N-terminal acetylation were captured, which constituted the first comprehensive acetylome map of cardiac 20S proteasomes. Among them, six sites of lysine acetylation were inducible with pharmacological inhibitors of histone de-acetylases (HDACs). Both
in vitro
and
in vivo
assays demonstrated that HDACs inhibition led to elevated proteasome function. With purified 20S proteasomes, this impact directly related to acetylation modifications. Significantly, this endogenous regulatory mechanism manifests in both clinical and murine model of diseased myocardia. Proteasome biology is modulated by acetylation modifications in the heart. The potency of this regulatory mechanism was demonstrated in both mouse and human species. Moreover, this inherent rheostat effects in both acutely pathological murine model of ischemia-reperfusion and chronically pathological model of end-stage heart failure. With the novel observation made in this investigation, restoration of proteasomal function via pharmacological application after stress turns feasible.
Abstract P291: The Ubiquitin Ligase Siah2 in Cardiac Response to Ischemia
Authors: Scimia Maria C , Kim Hyungsoo , Bowtell David , Mercola Mark , Ronai Ze'ev A
Published: March 2022

Background:
The E3 ubiquitin ligases SIah1 and 2 mediate a cellular response to hypoxia through its control of prolyl-hydroxylases and consequently HIF1α stability. Given the impact of hypoxia on heart function under stress, we have evaluated the effect of genetic deletion of Siah1 and 2 on cardiac function in response to myocardial infarction.

Objective:
To determine the physiological significance of the ubiquitin ligase Siah genes under in vivo ischemia condition.

Materials and methods:
To substantiate the role of Siah2 in ischemia-induced cell death, we tested difference in the response of myocardial tissue to ischemia using

Siah1a
+/−
/Siah2
−/−

mutant mice and their wildtype littermates. Analysis of heart tissue from

Siah1a
+/−
/Siah2
−/−

mice subjected to a model of 24 hours anterior descending coronary artery occlusion revealed their resistance to ischemic injury when compared with their littermates. Cardiac function, measured as EF %, was significantly less impaired in the mutant mice. Heart tissue obtained from

Siah1a
+/−
/Siah2
−/−

mice subjected to myocardial ischemia, exhibited a significantly lower degree of apoptosis (Tunel Staining), and myocardial infarct size (Evans blue, TTC).

Conclusions:
Collectively, these data establish that reduced expression of Siah1/2 exerts a cardioprotective function under ischemic conditions. The underlying molecular mechanisms linking cardioprotection to ischemic challenge will be discussed.
Abstract P294: Cardiomyocyte-Specific RBPJ Regulates Angiogenesis and Stress Response in the Adult Heart
Authors: Diaz Trelles Ramon , Scimia Maria Cecilia , Ruiz Lozano Pilar , Mercola Mark
Published: March 2022

Regulation of adult myocardial angiogenesis is critical for an appropriate cardiac function. We found that lack of RBPJ on isolated mouse adult cardiomyocytes increases angiogenic factors gene expresión and promoter hyperacetylation and hypermethylation of Notch target gene promoters (HEY2) and angiogenic factors. Accordingly, cardiomyocyte specific RBPJ KO adult mice show a denser microvasculature. RBPJ KO mice suggest an angiogenesis repressive role of RBPJ during homeostasis. Stress induced by myocardial infarction (MI) or cardiac overload (TAC) activate an angiogenic response to compensate the increased oxygen demand. We have found that Notch pathway is activated and RBPJ accumulated in the nucleus after MI and TAC. On isolated mouse adult cardiomyocytes, RBPJ is also able to control angiogenesis by mediating a hypoxia-induced response independent of Notch receptor activation. Mice lacking RBPJ in cardiomyocytes present a blunted angiogenic response. Suprisingly RBPJ KO mice had a better cardiac performance alter MI. We propose that RBPJ is a critical effector of the hypoxic response in the heart and the mechanisms by which RBPJ KO could protect from injury will be discussed on the presentation.
Abstract P303: Panaxnotogensengsaponins Promotes Angiogenesis and Enhances the Buildup of Effective Coronary Collateral Circulation in Ischemic Myocardium
Authors: Zhang Teng , Wang Wen-jian
Published: March 2022

Background:
Ischemic heart disease (IHD) is a condition that the blood vessels are narrowed or blocked due to the deposition of cholesterol plaques on the blood vessel walls. This affects the supply of blood to the heart and reduces the supply of oxygen and nutrients to the heart musculature, which has been regarded as the largest threat to the public health worldwide. PNS is a purified natural product extract that has been proved effective and widely used clinically in preventing and treating IHD in China. However, the detailed mechanisms of action of PNS remain to be addressed. The present study aims to explore the effect of PNS on modulating angiogenesis in vitro and in vivo to better understand its mechanism of action in treating IHD.

Methods and Results:
In this study, myocardial ischemia rat model and endothelial cell derived from human capillary blood vessel (ECV304) were utilized. Our data revealed that PNS significantly increased capillary blood vessel density in isoproterenol-induced rat ischemic myocardium model. It also decreased the pathological damage and ameliorated the ultrastructure of myocardial cells in rat ischemic myocardium as assessed by light microscopy, transmission electron microscopy and immunohistochemistry. At cellular level, PNS significantly decreased the number of apoptotic cells in rat ischemic myocardium model. Mechanistic study utilizing microarray and real-time PCR were further performed to examine the gene expression profile. The data revealed that PNS significantly upregulated the mRNA expression of a panel of angiogenic growth factors and their receptors such as VEGF, VEGFR2 and FGFR1. In vitro study revealed that PNS did not exhibit significant effect on the proliferation of ECV304 cells under normoxia culture condition; however, it facilitated the proliferation of ECV304 cells under hypoxia condition, suggesting the selective effect of PNS on angiogenesis under ischemic/hypoxic conditions.

Conclusions:
Our study demonstrated for the first time that PNS exerts its therapeutic effect on IHD through the mechanisms that in part involve regulating the expression of important angiogenic growth factor, which coordinately enhance the build-up of effective coronary collateral circulation in ischemic myocardium.
Abstract P306: Maintenance of Intracellular Zinc Homeostasis Contributes to the Mechanism by Which Postconditioning Protects the Heart from Ischemia/Reperfusion Injury
Authors: Xu Zhelong , Kim Seok Jai , Zhou Juan , Huh Jin , McIntosh Rachel , Zvara David
Published: March 2022

Myocardial ischemia/reperfusion causes zinc depletion and supplementation of zinc can attenuate ischemia/reperfusion injury. The purpose of this study was to determine the role of the intracellular homeostasis in the cardioprotective mechanism of ischemic postconditioning. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2 h of reperfusion. Postconditioning was elicited by six cycles of 10 second reperfusion and 10 seconds ischemia. Zinc concentration was measured with inductively coupled plasma optical spectroscopy (ICPOES). Upon reperfusion cytosolic zinc levels were dramatically decreased but this was prevented by postconditioning, indicating that postconditioning can prevent reperfusion-induced zinc loss. Moreover, postconditioning could also attenuate mitochondrial zinc loss upon reperfusion. In agreement with these observations, the anti-infarct effect of postconditioning was reversed by the zinc chelator N,N,N’,N’-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and exogenous zinc given at reperfusion mimicked the effect of postconditioning by reducing infarct size, suggesting that intracellular zinc account for the cardioprotective effect of postconditioning. Finally, postconditioning enhanced phosphorylation of Akt, ERK, and GSK-3β upon reperfusion, and these effects of postconditioning were inhibited by TPEN, indicating that zinc may mediate the cardioprotective effect of postconditioning by activating the RISK pathway. In conclusion, these data suggest that postconditioning may protect the heart by maintaining intracellular zinc homeostasis and activation of the RISK pathway may account for zinc-mediated cardioprotection of postconditioning.