bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 052: Epicardial-derived Adrenomedullin Drives Cardiac Hyperplasia During Embryogenesis
Authors: Wetzel-Strong Sarah E , Li Manyu , Nishikimi Toshio , Caron Kathleen M
Published: July 2021

The multi-functional peptide adrenomedullin (
Adm
= gene, AM = protein) plays important roles in embryonic development and disease. Previous studies demonstrated that
Adm
knockout mice die at embryonic day 13.5 with small, disorganized hearts and hypoplastic lymphatic vessels, highlighting the importance of this peptide in normal cardiovascular development. Since
Adm
knockout animals are embryonic lethal, our goal was to generate and characterize a novel model of
Adm
over-expression to study the role of
Adm
during development and disease processes. Through gene targeting techniques, we generated a novel mouse model of
Adm
over-expression, abbreviated as

Adm
hi/hi

. When we assessed gene expression of
Adm
from 10 different tissues, we found

Adm
hi/hi

mice express 3- to 15-fold more
Adm
than wildtype littermates. Additionally, peptide levels of AM in lung and kidney, as well as circulating plasma levels of AM were elevated 3-fold over wildtype mice, indicating a functional increase in AM. Our initial analysis revealed that adult

Adm
hi/hi

mice have larger heart weight to body weight ratios than wildtype littermates (4.93±0.23 vs. 5.96±0.29, n = 11-12). We found that compared to wildtype,

Adm
hi/hi

embryos have more proliferating cells during heart development (14.46±1.11 vs. 31.97±2.84, n=4), indicating that hyperplasia drives

Adm
hi/hi

heart enlargement. By crossing the

Adm
hi/hi

line to different tissue-specific Cre lines, we were able to excise the stabilizing bovine growth hormone 3’UTR, thereby returning
Adm
expression levels back to wildtype in cells with active Cre recombinase. Using this approach, we identified the epicardium as a major source of AM during cardiac development. In conclusion, we found that AM derived primarily from the epicardium drives cardiac hyperplasia during embryonic development resulting in persistent, enlarged hearts of adult

Adm
hi/hi

mice. Since our

Adm
hi/hi

mice recapitulate the 3-fold plasma elevation of AM observed during human disease, this mouse line will be a useful tool for studying the role of elevated AM during disease.
Abstract 054: Deficiency Of Nuclear Receptor Interaction Protein Causes Cardiac Hypertrophy
Authors: Chen Show-Li
Published: July 2021

Previously, we demonstrate a novel gene, nuclear receptor interaction protein (NRIP, also named as DCAF6 or IQWD1). We also identify NRIP as a Ca2+- dependent calmodulin binding protein that activates calcineurin phosphatase activity. To investigate insights into in vivo function of NRIP, we generated NRIP-null mice and found that loss of NRIP impairs cardiac function and lead to cardiac hypertrophy progressively. Furthermore, NRIP-/- mice display weaker muscle strength, reduced cardiac function, and cardiac fibrosis compared with WT. To verify the regulation mechanism, we found that α-actinin-2 (ACTN2), which is a biomarker of muscular Z-disc complex, is one of NRIP-interacting proteins from the yeast two-hybrid system. ACTN2 cross-links with actin filament to stabilize sarcomeric structure and muscle contraction, which is an essential constituent of sarcomere. Through the in vitro and in vivo binding assays, we further confirm the interaction and define the interacting domains between NRIP and ACTN2. Plus co-localization of NRIP and ACTN2 is discovered in cardiac tissue by immunofluorescence assays, we firstly define NRIP as a Z-disc protein. Although the Z-disc has been viewed as a passive constituent of the sarcomere traditionally, increasing numbers of mutations in Z-disc proteins leading to disruption and malfunction of the contractile apparatus have been shown to cause cardiomyopathies and/or muscular dystrophies. Hence, we analyzed the sarcomeric structure of NRIP-/- cardiomyocytes and found reduction of I-band width and extension of Z-disc. Besides, we know that NRIP is a Ca2+- dependent calmodulin binding protein. In cardiomyocytes, calmodulin interacts with multiple calcium ion channels or proteins to directly or indirectly regulate the variation of calcium concentration during muscle contraction. Therefore, we isolated and measured the calcium transient of cardiomyocytes. Then, we found that deficiency of NRIP decreases the amplitude of calcium transient. In a conclusion, we speculated that loss of NRIP impairs the structure of sarcomere, the amplitude of calcium transient during muscle contraction and the function of muscle contraction resulting in cardiomyopathy.
Abstract 055: β-Catenin Mediates an Interplay between Genetic Background and Growth-Regulating Effects of Estrogen in the Healthy Heart
Authors: Kararigas Georgios , Nguyen Ba Tiep , Zelarayan Laura C , Hassenpflug Maike , Toischer Karl , Sanchez-Ruderisch Hugo , Hasenfuss Gerd , Bergmann Martin W , Jarry Hubertus , Regitz-Zagrosek Vera
Published: July 2021

In diseased hearts, estrogen (E2) has been shown to exert anti-hypertrophic actions. Little is known about the role of E2 in the healthy heart. Our initial aim was to characterize structurally and molecularly the effects of E2 in the healthy mouse heart. Two-month-old female C57Bl/6J mice were ovariectomized and randomized to an E2-containing or soy-free (control, CON) diet (
n
= 17-18/group). The three-month physiological dose of E2 led to a higher relative heart weight compared with CON (
P
< 0.001). We also confirmed increased cardiomyocyte cross-sectional area by E2 (
P
< 0.01). No activation of the fetal gene program and no fibrosis were observed. Transcriptome analysis revealed induction of growth-related pathways by E2, such as the Wnt signaling pathway (
n
= 5/group; adjusted
P
< 0.05). To further confirm activation of Wnt/β-catenin signaling, we verified increased nuclear β-catenin protein levels by E2 compared with CON (
P
< 0.01) and hypothesized that β-catenin mediates the actions of E2. Cardiac deletion of β-catenin blunted the E2 effects on cardiac growth (
n
= 13/group). Surprisingly, in wild-type littermates with the background C57Bl/6N, E2 decreased the relative heart weight and cardiomyocyte cross-sectional area compared with CON (
n
= 7-11/group;
P
< 0.001). This was underlain by decreased nuclear β-catenin protein levels by E2 compared with CON (
P
< 0.001). Furthermore, E2 increased glycogen synthase kinase 3β (GSK3β) phosphorylation and the endosomal/autophagosomal markers Rab5, Rab7 and LC3-II in C57Bl/6J but not C57Bl/6N mice. Assessing a polymorphism linked to
Snap29
, we confirmed higher Snap29 protein levels in C57Bl/6J than C57Bl/6N mice (
P
< 0.01). This could lead to distinct regulation of endosomes and could be the potential cause of the strain difference. In conclusion, E2 regulates cardiac growth through β-catenin in a strain-specific manner. Collectively, we identified a molecular mechanism that demonstrates a divergent response of mouse sub-strains to E2.
Abstract 057: β-arrestin1-Biased β1-adrenergic Receptor Signaling Regulates MicroRNA Processing
Authors: Kim Il-man , Wang Yongchao , Park Kyoung-mi , Traynham Christopher J , Mao Lan , Koch Walter J , Rockman Howard A
Published: July 2021

MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary transcripts (pri-miRs), they are processed in the nucleus by Drosha into hairpin intermediates (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes following activation by a variety of signals such as those stimulated by β-adrenergic receptors (βARs). Initially discovered to desensitize βAR signaling, β-arrestins are now appreciated to transduce multiple effector pathways independent of G protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the β-arrestin-biased βAR agonist carvedilol activates cellular pathways in the heart. Here, we tested the hypothesis that carvedilol could activate β-arrestin-mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects. In human cells and mouse hearts, carvedilol (Carv) upregulates a subset of mature and pre-miRs but not their pri-miRs in a β1AR-, G protein-coupled receptor kinase (GRK) 5/6- and β-arrestin1-dependent manner (see figure). Mechanistically, β-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1, a component of the Drosha microprocessor complex (see figure). In conclusion, our findings indicate a novel function for β1AR-mediated β-arrestin1 signaling in miR biogenesis, which may be linked to its cell survival mechanism and beneficial adaptive remodeling in the failing heart.
Abstract 068: Age-associated, Vinculin-mediated Remodeling of the Intercalated Disc Results in Localized Myocardial Stiffening but Preserved Mechanical Function
Authors: Kaushik Gaurav , Sessions Ayla O , Spenlehauer Alice , Nishimura Mayuko , Ocorr Karen , Bodmer Rolf , Cammarato Anthony , Engler Adam J
Published: July 2021

Molecular and ultrastructural remodeling of the intercalated disc occurs with advanced age and onset of cardiomyopathy, yet the precise effect of this remodeling on mechanical function remains unclear. We employed recently-developed biophysical techniques to measure passive and active cardiac mechanics to investigate how intercalated disc remodeling affects structure and mechanical function with age. Nanoindentation of intact, living Drosophila heart reveals that juvenile myocytes are mechanically homogeneous; however, aging induces mechanical heterogeneity, indicated by increased transverse passive stiffness proximal to the intercalated disc (ID). Localized stiffening up to a 111% increase correlates with an 18% age-related decline in diastolic dimension (DD) as well as increased fascia adherens expression. On the other hand, aged specimens with stiffened IDs were found to have preserved systolic function, assessed by measuring shortening velocity under acute hemodynamic load, as compared to their juvenile counterparts. Aged specimens with no change in ID stiffness experienced reduced shortening velocities at all loads. Cardiac-specific vinculin overexpression stiffened IDs and increased shortening velocity and fractional shortening at all hemodynamic loads. Cardiac specific, RNAi-mediated knockdown of vinculin softened the IDs of intact hearts and reduced fractional shortening at all loads. These data indicate that increased expression of vinculin at the ID results in a stiffer cortical cytoskeleton which acts as a more stable anchor against which the myocytes can shorten. These findings have potential implications for geriatric patients with diastolic heart failure, in whom increased vinculin expression at the intercalated disc, concomitant with increased diastolic stiffness but preserved ejection fraction, has been observed.
Abstract 069: Mitochondrial Mechanisms Of Reverse Remodeling In Heart Failure
Authors: Ahuja Preeti , MacLellan William R , Wang Yibin
Published: July 2021

Enhancement of myocardial mitochondrial (mt) function resulting in efficient energy production by means of Left ventricular assist device (LVAD) has been suggested in heart failure (HF) which could have important clinical implications and may represent a novel therapeutic target. However, the basis for this improvement remains unknown. To characterize mt biogenesis, mt genomic integrity and mitophagy in reversing pathological remodeling, we investigated LV tissue from post-LVAD human hearts and after reversal of transaortic constriction (TAC) in mice. In Post-LVAD human hearts there was increased expression of mt fusion and biogenesis, mtDNA levels were normalized and deletion mutation rates were significantly reduced with reverse remodeling and these changes were associated with enhancement of mt ETC complex I and II activities and improved cardiac-myocyte morphology. To better understand the mechanisms underlying mt repair/remodeling with LVAD support, we developed a model of aortic banding (AB) and debanding (DB) in mice. C57BL/6 mice were subjected to 2 weeks of AB and subsequent DB for period of 1 to 20 days and cardiac function and hypertrophy were evaluated by echocardiography and real-time PCR, respectively. Compared with control animals, mice that had undergone banding had a robust hypertrophic response with decline in cardiac function. These parameters were reversed following removal of pressure overload by DB. Even 1 day of unloading led to significant increase in the expression of mt fusion and biogenesis genes. Hearts from AB (2 weeks) mice showed a 3.7-fold (P<0.05) increase in frequency of mtDNA deletions. However, mtDNA deletions were significantly reduced in frequency with DB when compared with AB hearts alone. Increase in expression of autophagy related genes could also be observed after hemodynamic unloading in mouse failing hearts. Removal of pressure overload by DB led to 2.58-fold (P<0.05) increase in expression of LC3B when compared to sham and AB mice. Thus, our data strongly suggest that protective effect of enhanced mt biogenesis, fusion/mtDNA repair and removal of damaged mitochondria by mitophagy could play an important role in maintaining mt integrity and function in the adult heart with reverse remodeling.
Abstract 074: GRK5 Increases Cardiac NFAT Transcriptional Activity
Authors: Hullmann Jonathan , Chuprun J K , Gao Erhe , Koch Walter J
Published: July 2021

Introduction:
G protein-coupled receptor (GPCR) kinase-2 and -5 (GRK2 and GRK5) have been shown to be up-regulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been shown that GRK5, unlike GRK2, can reside in the nucleus of cardiomyocytes and exert GPCR-independent effects that promote maladaptive cardiac hypertrophy and heart failure (HF). Previously, our lab has shown that GRK5 increases hypertrophic gene transcription through the phosphorylation of histone deacetylase 5 (HDAC5) and subsequent disinhibition of the transcription factor, myocyte enhancer factor-2 (MEF2). Use of GRK5 knockout (KO) mice has recently proved that GRK5 is indeed a physiological HDAC kinase during hypertrophic stress. Through experiments described below we have now identified the nuclear factor of activated T cells (NFAT) pathway as another potential target of GRK5 during cardiac hypertrophy that may play a role in its facilitation of HF.

Methods and Results:
Cardiac-specific NFAT-luciferase reporter mice were crossed with mice that overexpress wild-type GRK5 in myocytes to determine the role for GRK5 in NFAT signaling. These double-transgenic mice along with controls were stressed using the hypertrophic α
1
-adrenergic agonist phenylephrine (PE) as well as ventricular pressure-overload via transverse aortic constriction (TAC). NFAT activity was determined by both in vivo and ex vivo luciferase assays as well as RT-PCR for the NFAT target gene RCAN. Cardiac specific GRK5 overexpression leads to an increase in NFAT activity in the basal state as well as after both forms of hypertrophic stress. This was reproduced in cultured myocytes using a NFAT-reporter construct. Overexpression of a mutant GRK5 that cannot enter the nucleus appears to not activate NFAT demonstrating a nuclear-dependence. Consistent with this, GRK5 KO mice after TAC showed a decrease in the up-regulation of RCAN transcription as compared to wild-type mice. Studies are ongoing to determine the mechanism of GRK5’s regulation of cardiac NFAT activity.

Conclusions:
This study provides another non-canonical role for GRK5 in activating the hypertrophic NFAT pathway in cardiomyocytes.
Abstract 096: Activation of Delta-Opioid Receptors Regulates Cardiac MicroRNA Expression Under Hypoxia
Authors: Zhi Feng , Yang Yilin , He Xiao-Zhou , Sandhu Harleen K , Kang Xuezhi , Chao Dongman , Wang Rong , Kim Dong H , Xia Ying
Published: July 2021

Delta-opioid receptors (DOR) are involved in cardioprotection under hypoxia/ ischemia. However, the underlying mechanism is not well understood yet. We have recently shown that DOR activation significantly modify the expression of hypoxia-sensitive microRNAs (miRNAs) in the brain and kidney. In extension, we further investigated if DOR activation can regulate cardiac miRNAs in hypoxia. We compared global changes in miRNA gene expression in the rat heart following exposure to hypoxia for 10 days with those in normoxia using a low density miRNA microarray to delineate hypoxia-induced changes in the miRNA expression. The up-regulated miRNAs from the microarray analysis were selected for further investigating their response to hypoxia and DOR activation with a DOR agonist, UFP-512 after 1, 5, and 10 days. We found that 1) miR-376a increased after 1-day hypoxia and progressively increased with hypoxic duration; 2) miR-7b, miR-107, miR-134, miR-135a, miR-193a-3p, miR-196a and miR-324-3p progressively increased after 1-day hypoxia and reached a peak after 5-days; 3) miR-7a, miR-141, miR-196c, miR-200a, miR-200b, miR-203a and miR-324 increased only after 1-day hypoxia and significantly decreased after 5-10 days of hypoxia; 4) miR-128a did not significantly change after 1-10 days of hypoxia. After DOR activation with UFP-512, miR-128a, miR-135a, miR-193a-3p, miR-196a, miR-200a, miR-324-3p and miR-338 continued to increase after 1-day hypoxia, and then decreased after 5-10 days of hypoxia; 5) miR-7b and miR-134, in the condition of DOR activation, increased after 1-day hypoxia and further increased after 5-day hypoxia, while significantly decreased after 10-day exposure. In contrast, some miRNAs like miR-350 decreased in response to hypoxia, while DOR activation almost completely reversed such hypoxic reduction. These results suggest that cardiac miRNAs differentially respond to hypoxic stress with a variable degree directly related to the duration of hypoxic exposure, and DOR activation can regulate such hypoxic responses.

Supported by
HD034852
,
AT004422
, NSFC31071046, CS20102010,
ZD201007
, CY 20119004,CY20120003, STCSM10DZ1975800,NBRP09CB522901 & 12CB518502.
Abstract 097: Recombinant Human Erythropoietin Prevents Reactive Oxygen Species - Induced Apoptosis Via Akt and p38 MAPK Pathway During Hypoxia/ Reperfusion Injury
Authors: Allaudeen Asiya Parvin , Devendran Ajay , Baker John E , Dhanasekaran Anuradha
Published: July 2021

Erythropoietin (EPO) is a cytokine produced primarily in the kidney that is essential for red blood cell production. Apart from playing a role in hematopoiesis, EPO also has a protective role in heart myocytes, ovarian, glial cells brain and retinal diseases. In this study we observed that recombinant human EPO (rhEPO) reduces Hypoxia/ Reperfusion (H/R) injury by virtue of its effect on EPO receptor prosurvival signaling pathway, which ultimately leads to reduced expression of apoptotic proteins and increased survival of cardiomyocytes. H9C2 cells were exposed to H/R with or without pretreatment using 10, 15 and 20 U/ml of rhEPO. We determined viability using MTT, nuclear fragmentation by Hoechst staining, apoptotic nuclei by Acridine orange and Ethidium bromide, Reactive Oxygen Species (ROS) by Dicholorofluoresin Diacetate and activity of late apoptotic protease, Caspase-3 by colorimetric Caspase-3 assay. The expression of mitochondrial superoxide dismutase (MnSOD) by RT-PCR and Western blot, phospho-Akt and p38 MAPK by Confocal microscopy were analyzed. Cell viability is increased in cells pretreated with rhEPO compared to cell exposed to H/R. Cells subjected to H/R showed early apoptotic and late apoptotic cells but showed normal nuclei with intact cell membrane in cells pretreated with rhEPO. Intracellular production of ROS and Caspase-3 activity was decreased in cells pretreated with rhEPO compared to cells exposed to H/R. The expression of MnSOD RNA and protein was up-regulated in response to rhEPO, but not in H/R. The phosphorylative activation of Akt, p38MAPK progressively diminished during H/R but increased in rhEPO pretreated cells. We show that rhEPO prevents apoptosis in cardiomyocytes, subjected to H/R injury via phosphorylation of Akt and p38MAPK. These results it is hoped would help us distinguish the cell signaling pathways involved in cardioprotection and thus would open new avenues in cardiovascular therapy.
Abstract 098: Blocking Cardiac Gq-Receptor Coupling Prevents Adverse Remodeling and Preserves Myocardial Function after Ischemic Injury in Mice and Promotes Repair via Cardiac Regeneration
Authors: Gao Erhe , Zuo Lin , Shang Xiying , Boucher Matthieu , Lei Yonghong , Huang Meggie Z. , Chuprun Kurt J , Koch Walter J
Published: July 2021

Background:
Studies have shown that the heterotrimeric G protein, Gq, is a critical molecule in the development of myocardial hypertrophy and heart failure (HF). In the present study, we examined whether Gq signaling is also a critical pathological trigger for adverse remodeling and HF after ischemic injury. Using cardiac-specific transgenic mice with expression of a specific Gq inhibitory peptide (GqI) that blocks Gq-GPCR coupling allowed us to investigate whether Gq inhibition can alter cardiac structure and functional changes after myocardial infarction (MI).

Methods and Results:
Cardiac-specific GqI transgenic (TG) and non-transgenic littermate control (NLC) mice were subjected to MI. Serial echocardiography performed before ischemia as well as 2, 4, 8 weeks after MI injury show that TG mice develop less progressive LV enlargement and dysfunction. The % LV EF and FS were significantly improved in the TG compared to NLC mice at 8 weeks post-MI. Consistently, cardiac contractility (+dP/dt) and relaxation (-dP/dt) were significantly improved in TG mice also when stimulated with isoproterenol. The LVEDP, the heart weight to body weight (BW) and lung to BW ratios were significantly less in TG mice, indicating that HF was limited with GqI expression. Moreover, TG mice have smaller infarcts, less LV dimensions and lower fibrosis in both the border zone and remote area with clearly more viable tissue in the scar region. Constantly, there are more micro-vessel and more BrdU(+) cardiomyocytes in the scar area and border zone at the 2 week post-MI in TG group. Western blot shows a consistent decrease in phosphorylated PKC substrates and higher levels of activated Akt in TG mice. Over expression of the GqI in cultured H9c2 myoblasts did lead to higher level of Ki67 positive cells (38.06±2.46%) than a control group (21.78±2.64%) and FACS analysis showed that Gq inhibition increased the % of H9c2 cells in S phase and G2/M phase compared to control cells.

Conclusion:
These data indicate cardiac specific Gq inhibition protects the heart against adverse LV remodeling and HF progression after MI. Mechanisms attributed to GqI expression appear to include improved myocyte survival, less fibrosis, and promotion of angiogenesis and cardiac repair through regeneration.
Abstract 103: Mechanisms Underlying Mitochondrial Translocation Of GSK-3β, A Crucial Inducer Of Mitochondrial Permeability Transition: GSK-3β Activity, Interaction With VDAC2 And A Mitochondrial Targeting Sequence.
Authors: Tanno Masaya , Kuno Atsushi , Ishikawa Satoko , Ogasawara Makoto , Tobisawa Toshiyuki , Murase Hiromichi , Sato Tatsuya , Kouzu Hidemichi , Miki Takayuki , Miura Tetsuji
Published: July 2021

Background:
Although glycogen synthase kinase-3β (GSK-3β) is mainly localized in the cytosol in cardiomyocytes, mitochondrial GSK-3β has been shown to trigger permeability transition and cell death. In this study, we examined molecular mechanisms of the mitochondrial translocation of GSK-3β.

Methods and Results:
H9c2 cells were transfected with EGFP-tagged GSK-3β (WT) or kinase-deficient GSK-3β (K85R) and stained with MitoTracker Red. Observation by time-lapse microscopy (Eclipse Ti-E, Nikon) revealed that WT, but not K85R, translocated from the cytosol to mitochondria and induced cell death after exposure to H2O2. Two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates obtained from H9c2 cell lysates indicated that exposure to H2O2 down-regulated 4 spots and up-regulated 9 spots. LC/MS analysis revealed that one of the up-regulated spots contained voltage-dependent anion channel (VDAC) 2. Transfection of H9c2 cells with VDAC2-siRNA attenuated mitochondrial translocation of WT in response to H2O2 challenge (% of GSK-3β-positive mitochondria to total mitochondria =36±3% vs. 76±3%, p<0.05). Furthermore, observation with a super-resolution microscope (N-SIM, Nikon) revealed that knockdown of VDAC2 reduced mitochondrial swelling and fragmentation caused by repetitive laser exposure compared with control cells (78±3% vs. 53±12%, p<0.05). Based on similarities to known mitochondrial targeting sequences (MTSs), we hypothesized that GSK-3β contains an MTS consisting of N-terminal 15 amino acid residues dotted with positively charged amino acid residues, R4, R6 and K15. To test this hypothesis, one of these amino acid residues was replaced with alanine. R6A and K15A, but not R4A, showed significant attenuation in mitochondrial translocation after exposure to H2O2 compared with WT (51±5%, 45±5%, 75±7% vs. 76±3%, respectively).

Conclusion:
Kinase activity of GSK-3β and interaction with VDAC2 significantly promote translocation of GSK-3β to mitochondria, contributing to cell death. An N-terminal domain of GSK-3β, possibly as an MTS, mediates its mitochondrial translocation in response to oxidative stress.
Abstract 105: The nitroso-redox-induced SR Ca
2
+ leak is temperature-sensitive: Role of Nitric Oxide Synthase 1
Authors: Dulce Raul A , Mayo Vera , Rangel Erika B , Balkan Wayne , Hare Joshua M
Published: July 2021

Sarcomeric reticulum (SR) Ca
2+
leak contributes to an impaired Ca
2+
cycling and contractile dysfunction of the heart. Leak is thought to be regulated by post-translational modifications of RyR2 associated with nitroso-redox (NO/redox) imbalance. Ca
2+
cycling is affected by both NO signaling and temperature.

We hypothesized that NO/redox balance modulates SR Ca
2+
leak in a temperature–dependent manner.

We used tetracaine to investigate SR Ca
2+
leak in cardiomyocytes (CMs) using a model of NO/redox imbalance (NOS1
−/−
mice) or WT mice. Reactive oxygen species (ROS) were measured by H
2
DCF. The expression of NOS isoforms was assessed by real time PCR. Experiments were carried out at 23°C - 37°C.

The SR Ca
2+
leak increased as the temperature decreased from 34°C to 23°C in WT CMs whereas, in NOS1
−/−
CMs, the leak suddenly increased when the temperature surpassed 30°C (Fig. A). SR Ca
2+
content in NOS1
−/−
CMs was reduced at 37°C compared to 23°C (42.7 mol/L vs. 115.6 mol/L, respectively;
p
=0.001), whereas it was unaffected in WT CMs. Differences in Ca
2+
leak between WT and NOS1
−/−
were magnified according the SR Ca
2+
load increased. Contrarily to WT, there was a trend to rise in ROS production at temperatures over 30°C in NOS1
−/−
CMs (Fig. B). There was a linear association between ROS and leak in both strains. The NOS isoforms were unaffected by temperature in WT.

Our observations show that Ca
2+
leak increases when the temperature drops below 34°C; however, in the absence of NOS1 activity, the leak rises at temperatures greater than 30°C, and is associated with more ROS production. These results suggest that Ca
2+
leak from the SR is regulated by a temperature-dependent balance between NO and ROS.
Abstract 110: Adiponectin Inhibits TNF-a-Induced Vascular Inflammatory Response via Caveolin-Mediated Ceramidase Recruitment and Activation
Authors: Wang Yajing , Lau Wayne , Gao Erhe , Koch Walter , Ma Xin
Published: July 2021

Anti-inflammatory and vascular protective actions of adiponectin (APN) are well-recognized. However, many fundamental questions remain unanswered. The current study attempted to identify the APN receptor subtype responsible for APN’s vascular protective action, and investigate the role of ceramidase activation in APN anti-inflammatory signaling. Wild type (WT) or gene manipulated HUVEC were treated with TNFα in the presence and absence of APN. The effect of APN on TNFα-induced inflammatory and oxidative/nitrative stress was determined. In WT HUVEC, APN significantly reduced TNFα-induced ICAM-1 expression and attenuated TNFα-induced superoxide and peroxynitrite formation (P<0.01). These anti-inflammatory actions were virtually abolished by AdipoR1-, but not AdipoR2-, knockdown (KD). Treatment with APN significantly increased neutral ceramidase (nCDase) activity (3.7-fold, P<0.01). AdipoR1-KD markedly (P0.05), reduced APN-induced nCDase activation. More importantly, siRNA mediated nCDase-KD markedly blocked the effect of APN upon TNFα-induced ICAM-1 expression (P0.05), and modestly inhibited APN anti-inflammatory effect (P87% of APN-induced nCDase activation was lost. Whereas APN treatment failed to inhibit TNFα-induced ICAM-1 expression, treatment with S1P or SEW (S1P receptor agonist) remained effective in Cav1-KD cells in reducing TNFα-induced ICAM-1 expression (P<0.01). AdipoR1 and Cav1 co-localized and co-precipitated in HUVECs. APN treatment did not affect this interaction. Moreover, re-expression of WT Cav1 in Cav1-KD cells restored nCDase activation in response to APN (P<0.01 vs. vehicle), whereas re-expression of a mutated Cav1 blocking AdipoR1/Cav1 interaction failed to restore APN-mediated nCDase activation. Finally, there is weak basal Cav1/nCDase interaction, which significantly increased following APN treatment. These results demonstrate for the first time that APN inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1- dependent fashion.
Abstract 111: Blood Lipoproteins May Be Implicated in Blood Pressure Regulation
Authors: Lendner Janna , Krohn Stephan , Jumar Agnes , Grün Daniel , Zakrzewicz Andreas , Ermilov Eugeny , Siegel Günter
Published: July 2021

Background:
An increase in triglycerides, which comprise the main component of VLDL, belongs to definition criteria of the metabolic syndrome. We investigated the influence of VLDL (30 mg/dL) on flow-dilatation without and with α- (10
-7
mol/L) and β-blockers (10
-7
mol/L) to find out whether the VLDL effect is partly due to a possible α- and β-receptor interaction.

Methods:
Isometric tension, membrane potential and cAMP-cGMP were measured in 16 coronaries from heart transplantations.

Results:
Flow-dependent relaxation of 0.432 g in Krebs solution (T
3
- T
100
) was reduced under VLDL by 15.3% to 0.366 g, and under VLDL + α/β-blockers by 20.1% to 0.345 g, thus practically no change. However, calculation of the difference in tension for the individual flow rates between Krebs and VLDL on the one hand, and Krebs and VLDL + α/β-blockers on the other, results in significant differences (Table). Setting the difference between Krebs and VLDL equal to 100%, leads to a 12.2% to 45.4% change in difference under α/β-blockers. Experiments under blockers yielded that α-receptor blockade alone is sufficient to explain the reduction of the VLDL-effect. β-effects were reflected by variation in [cAMP], α-effects in [cGMP].

Conclusion:
About one third of the constricting VLDL-effect is due to a direct VLDL-inhibition of the β- receptor and/or stimulation of the α- receptor. We prove for the first time that blood lipoproteins exert their influence on arterial tone also via an interaction with the autonomic nervous system. We conclude that blood lipids may be implicated in blood pressure regulation. This unveiling could help in navigating the treatment options of both hypertension and hypercholesterolemia.
Abstract 112: A Pathological Role of Endothelial p53 in the Regulation of Endothelial Function and Glucose Metabolism
Authors: YOKOYAMA Masataka , KOBAYASHI Yoshio , MINAMINO Tohru
Published: July 2021

Cellular senescence is a state of irreversible growth arrest induced by various stresses such as oncogenic stimuli. This response is controlled by negative regulators of the cell cycle like the p53 tumor suppressor protein. Accumulating evidence has suggested a role of p53 activation in various age-associated conditions including atherosclerosis, heart failure and diabetes. Here we show that endothelial p53 activation plays a pathological role in the regulation of endothelial function and glucose metabolism under diabetic conditions. Endothelial expression of p53 was markedly up-regulated in a streptozotocin-induced diabetes model. Endothelial function such as acetylcholine-dependent vasodilatation was markedly impaired in this model. Although hyperglycemia was not altered, impairment of endothelial function was significantly improved in mice with endothelial cell-specific p53 deficiency. In same way, p53 was markedly activated in ischemic vessels, and endothelial p53 deficiency enhanced ischemia-induced angiogenesis. Mechanistically, endothelial p53 up-regulated the expression of PTEN that negatively regulated the Akt-eNOS pathway, and therefore disruption of p53 improved endothelial dysfunction. We also found that endothelial p53 was markedly activated, and the Akt-eNOS pathway was attenuated in a diet-induced obesity model. Disruption of endothelial p53 activation improved dietary inactivation of eNOS that up-regulated the expression of PGC-1α in skeletal muscle, thereby increasing mitochondrial biogenesis and oxygen consumption. Inhibition of endothelial p53 also improved dietary impairment of glucose transport into skeletal muscle by up-regulating endothelial expression of glucose transporter 1. Consequently, mice with endothelial cell-specific p53 deficiency fed a high-calorie diet showed improvement of insulin sensitivity and less fat accumulation compared with control littermates. These results indicate that endothelial p53 negatively regulates endothelium-dependent vasodilatation, ischemia-induced angiogenesis, and mitochondrial biogenesis by inhibiting the Akt-eNOS pathway and suggest that inhibition of endothelial p53 could be a novel therapeutic target in diabetic patients.
Abstract 115: Nuclear CaMKII is a Histone H3 Kinase that Remodels Chromatin During Cardiac Hypertrophy
Authors: Mahmoud Salma , Kunhi Muhammad , Little Gillian H , Bai Yan , An Woojin , Kedes Larry , Poizat Coralie
Published: July 2021

Background and Purpose:
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine/threonine kinase implicated in pathological events such as cardiac hypertrophy. In this study we investigated the role of a specific nuclear isoform of CaMKII in chromatin remodeling and in transcriptional regulation in cardiac muscle.

Methods:
Comprehensive experimental approaches performed in primary cardiomyocyte cultures were used including chromatin immunoprecipitation assays (ChIP), q-PCR, chromatin remodeling assays, in vitro phosphorylation/transcription assays, production of recombinant adenovirus, siRNA technology, fluorescence microscopy and mass spectrometry.

Results:
We found that CaMKIIδB targets specific components of chromatin during cardiac hypertrophy and binds to nucleosomes through its association domain in a cooperative model. CaMKIIδB also increased chromatin relaxation, and this action was dependent on its kinase activity. The observation that CaMKIIδB interacts with chromatin suggested to us that histones maybe novel substrates of the kinase in cardiac muscle. To test this hypothesis, we performed in vitro kinase assays and found that histone H3 is a bona fide CaMKIIδB substrate and Ser-10 appears to be a predominant phosphorylation site. Increased histone H3 Ser-10 phosphorylation was observed following hypertrophic stimulation and was not associated with cellular proliferation, whereas depletion of CaMKIIδB significantly reduced histone H3 Ser-10 phosphorylation in primary cardiomyocytes. Interestingly, we found that H3 S10 phosphorylation and recruitment of CaMKIIδB occur at promoters of fetal cardiac genes. To establish the functional link between H3 phosphorylation by CaMKIIδB, chromatin remodeling and transcription activation, we developed an in vitro transcription system and using it we found that CaMKIIδB increased chromatin accessibility and mediated transcription of the Mef2 transcription factor.

Conclusion:
Taken together, these findings highlight a new role of CaMKIIδB as relevant histone H3 kinase and link for the first time epigenetic changes by CaMKII to cardiac hypertrophy.
Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia
Authors: Zhao Helong , Anand Appakkudal , Ganju Ramesh
Published: July 2021

Abstract

Introduction:
Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation.

Hypothesis:
We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses.

Methods and Results:
Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart.

Conclusions:
Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.
Abstract 126: Functional Properties of Cardiac Stem Cell Niches in the Young and Old Heart
Authors: Kim Junghyun , Zheng Hanqiao , Cappetta Donato , Sanada Fumihiro , Arranto Christian , Gurusamy Narasimman , Goichberg Polina , Hosoda Toru , Rota Marcello , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

Cardiac stem cells (CSCs) are clustered in interstitial structures, in which cardiomyocytes and fibroblasts act as supporting cells. However, the effects of the niche microenvironment on the functional properties of CSCs are largely unknown and whether young and senescent myocytes affect differently CSC fate remains to be determined. To address this issue, cardiac niches were examined in young and old Fischer 344 rats. Clusters composed of two or more lineage negative CSCs were included in the analysis. Unexpectedly, from 4 to 28 months of age, the number of CSCs per niche increased nearly 2-fold. This expansion of the CSC pool took place despite the increase in the fraction of dying CSCs with age. Apoptosis of CSCs was restricted to cells expressing the senescence-associated protein p16
INK4a
. However, the fraction of CSCs positive for p16
INK4a
increased significantly, documenting that the process of clearance of senescent CSCs was inefficient in the old myocardium. The fraction of p16
INK4a
-positive CSCs undergoing apoptosis decreased 3.5-fold with age, resulting in accumulation of senescent cells within the niche. These observations indicate that the pool of functionally competent CSCs able to contribute to myocyte turnover decreased with age. In an attempt to compensate for the higher myocyte loss occurring in aged hearts, p16
INK4a
-negative CSCs proliferated 4-fold more in old than in young rats, experiencing progressive telomeric shortening. To define whether the fate of CSCs was dictated by the properties of the neighboring myocytes in the senescent heart, myocytes and CSCs were co-cultured for 3 days. With respect to CSCs plated alone, old myocytes enhanced 2.2-fold BrdU incorporation in co-cultured CSCs. This effect was less apparent with young myocytes. No significant differences in CSC apoptosis were seen. Comparable results were obtained when CSCs and myocytes were plated in Transwell systems. Importantly, old myocytes released more IGF-1 than young myocytes; blockade of IGF-1 receptor in CSCs attenuated significantly the positive impact of myocytes on CSC replication. Collectively, our findings indicate that the rate of accumulation of old CSCs is greater than the rate of their death leading to the formation of senescent niches and organ aging.
Abstract 129: Human Mesenchymal Stem Cells Augment Contractile Function of Human Engineered Cardiac Tissues
Authors: Cashman Timothy , Turnbull Irene C , Karakikes Ioannis , Da Silva Jose , Hare Joshua M , Hajjar Roger J , Costa Kevin D
Published: July 2021

Mesenchymal stem cells (MSC) have demonstrated efficacy for improving cardiomyocyte (CM) function
in vitro, in vivo
and in clinical trials, but the mechanism of this enhancement remains elusive. The objective of this study was to test the hypothesis that human engineered cardiac tissues (hECT) offer a viable model system to investigate the effects of human MSC on CM contractile function.

Human CM (hCM) were produced from embryonic stem cells (hESC, H7 line) using a small-molecule based differentiation approach. Blebbistatin and BMP4 were added to hESC suspended in StemPro34 differentiation media for 24 h, followed by BMP4 and Activin A to day 4.5, followed by addition of IWR-1 Wnt inhibitor for at least 4 days. To create hECT, approximately 1 million hCM were mixed with 2.0 mg/ml bovine type I collagen and 0.9 mg/ml Matrigel, and pipetted into a mold fabricated from polydimethylsiloxane with integrated cantilever end-posts. To model hMSC cell therapy, two types of hECT were created: hCM-only control hECT, and hMSC-CM hybrid hECT containing hCM mixed with 5-10% of human bone marrow-derived MSC. Over several days in culture, the hECT self-assembled and started beating; end-post deflection was tracked in real time to compute twitch force using beam theory.

Human CMs were produced with high efficiency (>70% cTnT+) with a predominantly ventricular phenotype (MLC2v+). Resulting hECTs exhibited spontaneous beating (1.3±0.4 Hz), cellular alignment, registered sarcomeres, and expression of cardiac specific genes cTnT, α-MHC, β-MHC and SERCA2a. After 11±2 days in culture, developed stress (force/area) was over 10-fold higher in hMSC-CM hybrid tissues (0.27±0.048 mN/mm
2
) compared to hCM-only controls (0.02±0.006 mN/mm
2
; p=0.04, n=5 per group). This reflected significantly greater twitch force (0.11±0.004 mN vs 0.033±0.016 mN, p=0.016) and smaller cross-sectional area (0.19±0.12 mm
2
vs 0.49±0.10 mm
2
; p=0.003) in hMSC-CM hybrid vs hCM-only hECT.

In conclusion, human ECT offer a novel system to study MSC-CM interactions. The findings suggest hMSC supplementation improves contractility compared to CM-only hECT. Investigating the mechanisms of hMSC-mediated enhancement of hECT function may yield insights into MSC-based therapies for cardiac regeneration.
Abstract 136: Distinct Temporal Requirements for Scl to Repress Cardiomyogenesis in Endothelial-derived Cells in Hemogenic Tissues and the Heart
Authors: Van Handel Ben , Org Tonis , Montel-Hagen Amelie , Nakano Haruko , Nakano Atsushi , Evans Sylvia , Mikkola Hanna
Published: July 2021

Identification of precursors with the capacity to generate cardiomyocytes is critical for advancing cardiac regenerative medicine. By analyzing knockout embryos for the bHLH factor Scl, we demonstrated that endothelial cells in hematopoietic tissues and the heart possess latent cardiomyogenic capacity. Furthermore, analysis of tamoxifen-inducible Rosa26-Cre
ERT2
Scl
fl/fl
embryos suggested that the time window during which Scl is required for cardiac repression extends later in the heart versus the yolk sac. However, the cell types in which Scl acts remained elusive. We then deleted Scl in a cell-type specific manner in early mesoderm using Mesp1-Cre and in endothelial cells using Tie2-Cre. Lineage tracing in Mesp1-Cre Rosa26-YFP embryos demonstrated that at E9.5, a large majority of hematopoietic and endothelial cells in the yolk sac and heart were labeled. Moreover, deletion of Scl in Mesp1-Cre Scl
fl/fl
embryos phenocopied the germline knockout, essentially abrogating hematopoiesis and promoting the emergence of CD31
+
PDGFRα
+
cardiomyogenic precursors and ectopic expression of the cardiomyocyte genes
Myl7
and
Tnnt2
in yolk sac vasculature. In contrast, deletion of Scl after endothelium had been specified in Tie2-Cre Scl
fl/fl
embryos did not grossly affect yolk sac hematopoiesis, nor did it induce ectopic cardiomyogenesis in hemogenic tissues. However, endothelial-derived cells in the hearts of Tie2-Cre Scl
fl/fl
embryos evidenced profound expansion of CD31
+
PDGFRα
+
cardiogenic precursors at E11.5 and E13.5, as well as displayed dramatic upregulation of
Myl7
and
Tnnt2
, showing that the requirement for Scl to repress the cardiomyogenic program extends longer in endothelial derivatives in the heart than in the yolk sac. These data demonstrate that endocardial-derived cells in the heart retain latent cardiomyogenic potential until mid-gestation and nominate Scl as a critical regulator of endocardial fate.