bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P077: Ischemic Preconditioning Improves Postischemic Cardiac Function by Preserving PTEN Activity
Authors: Zheng Xiaoxu , Zu Lingyun , Cai Zheqing P
Published: March 2022

Background
Ischemic preconditioning (IPC) increases post-ischemic functional recovery. Although reduced infarct size is associated with the better outcome, its underlying mechanism is not fully understood. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) promotes cell death and increases myocardial contractility. We recently reported that IPC attenuates loss of PTEN activity in post-ischemic hearts. In the present study, we have investigated the hypothesis that IPC improves post-ischemic cardiac function by preserving PTEN activity.

Methods and Results
Isolated mouse hearts were exposed to no ischemia as control (CON) or IPC, consisting of 10-min ischemia and 5-min reperfusion (I-10/R-5), followed by I-30/R-120. bpV(phen) (10 µM), a PTEN inhibitor, or vehicle was added into the perfusion line in IPC group following reperfusion. Isolated hearts from muscle-specific PTEN knockout mice (

Pten
loxp/loxp
;ckm
+/−

) and control mice (

Pten
loxp/loxp
;ckm
−/−

) were exposed to I-30/R-30. Left ventricular (LV) pressure was monitored by a pressure catheter. Myocardial infarct size was measured by triphenyltetrazolium chloride staining. After I-30/R-120, IPC increased LV developed pressure (LVDP) and maximal +dp/dt (+dp/dtm) compared with CON. bpV blocked the effects of IPC (LVDP: 42±4 vs. 63±4 mmHg, p<0.01; +dp/dtm: 1209±101 vs. 1880±155 mmHg/sec, p<0.01). However, it did not increase infarct size compared with vehicle. Muscle-specific PTEN knockout modestly decreased LVDP and +dp/dtm in isolated perfused hearts under basal conditions, but it markedly impaired their recovery after I-30/R-30 compared with control (LVDP: 21±4 vs. 45±5 mmHg, p<0.01; +dp/dtm: 784±146 vs. 1795±154 mmHg/sec, p<0.01). Myocardial infarct size was decreased in PTEN knockout hearts after I/R.

Conclusions:
PTEN inhibition blocks the improvement of cardiac functional recovery induced by IPC. Loss of PTEN exacerbates post-ischemic dysfunction in isolated hearts. Therefore, our studies suggest that IPC attenuates post-ischemic cardiac dysfunction by preserving PTEN activity.
Abstract P091: Transcriptional Profiling of Pluripotent Stem Cell--Derived Neuropilin-1+CD34- Vascular Precursors Reveals Significant Overlap with Ventral Mesoderm and Identifies Signaling Pathways in Differentiation from Vascular Cells
Authors: Lang Jennifer K , Sim Fraser J , Cimato Thomas
Published: March 2022

Background:
Pluripotent stem cells are a model to understand the mechanisms of vascular differentiation in humans. We previously showed that the onset of Neuropilin-1 (NRP1) expression identifies vascular precursors in mouse and human embryonic stem cells. Our goal was to determine the degree of overlap between NRP1+CD34- cells and embryonic primitive streak, and identify novel growth factor signaling pathways controlling their differentiation.

Methods:
Human pluripotent stem cells were differentiated, and NRP1+CD34- cells were isolated. Following RNA extraction, whole genome transcriptional profiling was performed. Microarray data were compared with undifferentiated stem cells and human umbilical vein endothelial cells (HUVECs) using differential gene expression analysis with a false discovery rate of 5%, and greater than two-fold change between groups. We compared significantly enriched transcripts in NRP1+CD34- cells against genes expressed in the primitive streak in Xenopus embryos using hypergeometric testing. Functional annotation-based clustering was used to identify putative cell signaling pathways.

Results:
A total of 785 transcripts were significantly enriched in NRP1+CD34- vascular precursors vs. hESCs, and 605 transcripts were unique to NRP1+CD34- vascular precursors. NRP1+CD34- cells shared a significant number of transcripts (46 of 357 human homologs) with ventral mesoderm isolated from Xenopus embryos, (p ≤ 1 x 10-14). We then identified CXCL14, Neuregulin 2, Leptin and Apelin receptors as significantly upregulated in NRP1+CD34- vascular precursors, potentially controlling human ventral mesoderm and new vessel formation.

Conclusions:
NRP1+CD34- vascular precursors have significant transcript identity with ventral mesoderm from embryonic primitive streak, and a unique transcriptional profile compared with HUVECs. These findings suggest NRP1+CD34- human vascular precursors significantly resemble ventral mesoderm, and provide a model of human vascular development. The identification of a comprehensive database of cell-surface receptors expressed by the human ventral mesoderm analogs permits future pharmacological approaches to induce vascular differentiation from human pluripotent stem cells.
Abstract P094: Anesthetic Preconditioning in Human Cardiomyocytes Derived from Type II Diabetic Patient-Induced Pluripotent Stem Cells in a Varying Glucose Environment
Authors: Canfield Scott , Sepac Ana , Sedlic Filip , Muravyeva Maria , Bai Xiaowen , Bosnjak Zeljko
Published: March 2022

Volatile anesthetic-induced preconditioning (APC) has been shown to pharmacologically precondition the myocardium, providing protection from an ischemia-reperfusion injury. However, APC is attenuated or even eliminated in diabetic individuals and in the presence of acute hyperglycemia with the underlying mechanism(s) being unknown. In this study, we used ventricular cardiomyocytes (CMs) differentiated from non-diabetic and type II diabetic-induced pluripotent stem cells (N-iPSCs and DM-iPSCs, respectively) to investigate the influence of a high glucose environment and genetic background on APC. Differentiated CMs were identified using cardiac-specific immunostaining and expression of green fluorescent protein under the transcriptional control of cardiac promoter myosin light chain-2v, a genetic construct delivered by lentiviral vector. N-iPSC- and DM-iPSC-derived CMs were exposed to varying glucose environments (11 mM and 25 mM). Confocal microscopy was utilized to measure mitochondrial membrane potential and mitochondrial permeability transition pore opening (mPTP) in CMs. The volatile anesthetic isoflurane depolarized mitochondria in N-iPSC-derived CMs via opening of the mitochondrial adenosine triphosphate-sensitive potassium channel; however, isoflurane depolarized mitochondria to a significantly lower level in diabetic-derived cardiomyocytes and in the presence of 25 mM glucose. APC delayed mPTP opening in both N-iPSC- and DM-iPSC-derived CMs in 11 mM glucose environment only. We have established an
in vitro
model based on directed differentiation of ventricular CMs from N-iPSCs and DM-iPSCs that allows us the unique opportunity to conduct comparative studies to address the inability of diabetic individuals to be preconditioned with anesthetics. Our preliminary results indicate for the first time that both a high glucose environment and a diabetic background have detrimental effects on the efficiency of APC to protect CMs from an ischemia-reperfusion injury.
Abstract P108: Direct Evidence of Postnatal Cardiomyocyte Generation in Murine Models of Aging and Cardiac Injury
Authors: Ali Shah R , Ardehali Reza , Hippenmeyer Simon , Luo Liqun , Weissman Irving
Published: March 2022

The dogma of the adult mammalian heart as a post-mitotic organ has recently come under question. Radiolabeled isotope studies have demonstrated that the human heart exhibits a low rate of renewal of cardiomyocytes throughout one’s lifespan. Furthermore, a recent study has elegantly shown that a neonatal mouse can regenerate its ventricle if the chamber is resected within the first week of birth. However, a number of questions remain unanswered about the nature of the cell type that gives rise to cardiomyocytes postnatally. The field has yet to clonally address whether cardiomyocytes divide symmetrically upon birth, or if a resident progenitor differentiates into cardiomyocytes. Rather than rely on proxies for cell division (e.g. BrdU incorporation studies), we use genetic mouse models in which cell division results in asymmetric, indelible labeling of the daughter cells (“Mosaic analysis of double markers” (MADM)) and a stochastic multi-color Cre reporter in the Rosa26 locus akin to the “Brainbow” mouse to investigate postnatal cell division clonally in order to identify which cell type(s) generate cardiomyocytes. Our studies have demonstrated limited symmetric division of cardiomyocytes during normal aging up to six weeks, with a significantly high rate of cardiomyocyte division during the first postnatal week. While we provide evidence for limited cell division in the peri-infarct region of a myocardial infarction model in the 24 hours after the infarct, the extent of cell division and proliferation over a longer time frame after the infarct remains to be explored.
Abstract P121: Cardiac Deletion of Aryl Hydrocarbon Nuclear Translocator (Hypoxia-Inducible Factor 1-β) in the Adult Heart Results in Cardiomyopathy
Authors: Wu Rongxue
Published: March 2022

Rongxue Wu, Kusum Chawla, Yoshihiko Ichikawa, Mohsen Granefar Hossein Ardehali

Background:
Aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins and serves as a binding partner for a number of other family members. ARNT is also a required dimerization partner of HIF1a. Although HIF1a is known to be required for normal cardiac development, the role of ARNT in basal cardiac function in the adult heart is not known. We hypothesized that ARNT is required for normal cardiac physiology, and its deletion in adult heart results in cardiomyopathy.

Methods and Results:
The deletion of ARNT gene in the heart of 3 month old mice was achieved by crossing ARNT
flox/flox
mice with αMHC-MCM (tamoxifen-inducible heart specific Cre) transgenic mice followed by administration of tamoxifen chow. MHC-MCM/ARNT
+/+
(WT) littermates were used as a control. The ARNT knock out (KO) mice exhibited enlarged left ventricle with a significant reduction in ejection fraction (KO vs. WT: 35.8 ± 3.6% vs. 61.2 ± 2.8%, n=12, p<0.01) and fractional shortening (KO vs. WT: 18.3 ± 1.5 % vs. 30.1 ± 1.1%, p<0.01), assessed by Echocardiography. Closed-chest catheterization also demonstrated reduced +dP/dt (KO vs. WT: 4355 ± 538 vs. 9426 ± 180 mmHg, p<0.01) and increased left ventricular end diastolic pressure (KO vs. WT: 3.12 ± 1.1 vs. 8.47± 1.2 mmHg, P<0.05) in KO mice but not in WT mice. The worsened cardiac function in the KO mouse heart was associated with increase in ANF and BNP expression, interstitial fibrosis, and apoptosis as determined by TUNEL staining. Furthermore, electron microscopy reveals a variety of degenerative changes and some lipid droplet in the KO hearts, and histological studies demonstrated intramyocardial lipid accumulation in the perivascular area in the KO mouse hearts. Analysis of gene expression in the KO heart revealed up-regulation of peroxisome proliferator-activated receptor alpha.

Conclusion:
ARNT is essential for the maintenance of structural and functional homeostasis in the adult heart, and its inactivation leads to cardiac contractile dysfunction. Our findings implicate a novel critical transcriptional requirement for ARNT in the maintenance of adult cardiac function.
Abstract P126: Calpain Protects the Heart from Hemodynamic Stress
Authors: Taneike Manabu , Hikoso Shungo , Yamaguchi Osamu , Nishida Kazuhiko , Mochizuki Naoki , Komuro Issei , Otsu Kinya
Published: March 2022

Although calpains are well-known Ca
2+
-dependent intracellular cysteine proteases, the pathophysiological function of calpains in the heart remains to be elucidated. Previous reports have suggested that calpains play detrimental roles to induce apoptotic or necrotic cell death in pathological Ca
2+
-overloaded conditions such as ischemia-reperfusion injury or myocardial infarction. Recent loss of function studies have suggested that calpains play important physiological roles in development, cell migration, organization of actin cytoskeleton, and plasma membrane repair. In the present study, we generated and analyzed cardiac-specific calpain 4-deficient mice (CKO) to answer unresolved questions as to in vivo function of calpains in the heart. Ubiquitous calpain is consisted of a common regulatory subunit (calpain 4) and a large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain). In agreement with previous reports, cardiac-specific deletion of calpain 4 markedly resulted in a simultaneous decrease in protein levels of calpain 1 and 2, indicating that calpain activity was almost absent in CKO hearts. CKO showed no cardiac phenotypes under basal conditions. Then, we subjected CKO and control mice (CTL) to pressure overload by means of transverse aortic constriction (TAC). One week after TAC, CKO showed left ventricle dilatation (LVDd, CKO 3.64 ± 0.2 mm versus CTL 2.66 ± 0.05 mm), and contractile dysfunction (FS, CKO 33.2 ± 3.9% versus CTL 47 ± 0.7%). CKO hearts took up Evans blue, a membrane-impermeant dye, within cardiomyocytes after TAC, whereas CTL hearts or sham-operated CKO hearts did not. This indicates plasma membrane was disrupted in CKO hearts in response to pressure overload. We performed membrane repair assays on isolated cardiomyocytes from CKO hearts using a two-photon laser-scanning microscope. CKO cardiomyocytes continued to take up FM1-43FX dye for at least 480 sec after disruption of a plasma membrane by laser irradiation, although CTL cardiomyocytes resealed within 250 sec. These data indicate that plasma membrane of cardiomyocytes disrupted by pressure overload failed to be resealed in CKO hearts. Thus, we conclude that calpains protect the heart from hemodynamic stresses by promoting membrane repair.
Abstract P128: Type II Deiodinase Upregulation in Patients with End-Stage Dilated Cardiomyopathy
Authors: Henderson Kyle K , McElligott Andrew , Chu Miensheng , Patel Nilam , Paul Jennifer T
Published: March 2022

Background:
Dilated cardiomyopathy (DCM) is the most common form of heart disease, with a 50% survival rate 5yrs after diagnosis. Low thyroid hormone levels are an independent risk factor for heart failure progression. Recent studies in animal models with heart failure have shown re-expression of a fetal enzyme, type III deiodinase (D3) that functions to reduce thyroid hormone concentrations. Alternatively, an increase in β-adrenergic stimulation during heart failure may increase expression of type II deiodinase (D2) that would function to increase thyroid hormone concentration. We hypothesized that human hearts in end-stage dilated heart failure would have increased D3/D2 expression and lower myocardial and serum thyroid hormone concentrations.

Methods and Results:
Serum and left ventricular (LV) tissue from patients with dilated heart failure undergoing heart transplantation, and organ donors not meeting the criteria for heart donation were obtained with full consent and Institutional Review Board approval. Deiodinase expression was measures with real time rtPCR and thyroid hormone concentrations measured with radioimmunoassay. Interestingly, D3 expression and protein levels were not elevated in DCM patients vs. “non-failing” heart samples. On the other hand, D2 expression and protein levels were increased in DCM hearts and tended to have greater myocardial thyroid hormone concentrations (P =0.10). In spite of the increase in D2 expression in DCM patients, serum thyroid hormone concentrations were significantly lower than serum obtained from healthy volunteers, (P<0.05). Changes in thyroid hormone receptors and thyroid hormone dependent downstream signaling changes for Akt, mTOR, and ERK are currently being investigated.

Conclusions:
Contrary to our hypothesis, patients with end-stage DCM have a reduced D3/D2 expression ratio and tend to have elevated myocardial thyroid hormone concentrations. Compensatory increases in D2 expression may enhance myocardial T3 production in DCM patients; however changes in thyroid hormone receptors and downstream signaling may negate this effect. Importantly, serum T3 concentrations were significantly reduced which likely contributed to patients progression toward end stage heart failure.
Abstract P131: An Akt-Phosphomimetic Sequence of the Cavb2 C-Terminal Region Protects L-Type Calcium Channels from Protein Degradation
Authors: Rusconi Francesca , Cazade Magali , Caprari Silvia , Polticelli Fabio , Chemin Jean , Condorelli Gianluigi , Catalucci Daniele
Published: March 2022

Alteration in the density or function of L-Type Calcium Channels (LTCCs) has been related to cardiovascular diseases such as heart failure and diabetic cardiomyopathy. It could therefore be envisioned that increasing LTCC density may improve cardiac function in heart failure. Recently, we determined that the Ser-Thr kinase Akt plays a key role in regulating cardiac inotropism through the modulation of LTCC density and function. Specifically, we found that the LTCC pore-forming channel subunit Cava1.2 contains highly evolutionary conserved PEST sequences (signals for rapid proteolytic degradation) that are responsible for direct Cava1.2 protein degradation. Phosphorylation of the C-terminal coiled coil of the Cavb2 chaperone subunit enhances LTCC protein stability by preventing PEST-mediated Cava1.2 degradation.

The aim of this study was to further dissect this Akt-dependent fine-tuning mechanism regulating LTCC density, searching for potential Akt-phosphomimetic (APM) molecules that could enhance LTCC density.

Using yeast two-hybrid screening, we found that APM Cavb2 sequences interact with the globular domain of Cavb2 itself in a solvent-exposed region that we named Tail Interacting Domain (TID). Biochemical and functional assays as well as site-specific mutagenesis in TID identified the minimal aminoacid sequence responsible for the TID-tail interaction.

In addition, through an approach comprising western blot analyses, fluorescent-based calcium assays, calcium current (ICaL) measurements, molecular modeling and peptide arrays, we identified the minimal APMs that efficiently protects Cava1.2 from protein degradation.

Based on our in vitro results, we suggest that the identified APM sequence/peptide could be used as a “therapeutic approach”; for increasing or reestablishing impaired cardiac contractility in mouse models of cardiomyopathy in which the LTCC density is altered, thus enhancing inotropism.
Abstract P143: Activation of GPR30 Attenuates Diastolic Dysfunction and LV Remodeling in Oophorectomized mRen2.Lewis Rats
Authors: Wang Hao , Jessup Jewell A , Lindsey Sarah , Lin Marina S , Chagas Clarissa , Groban Leanne
Published: March 2022

The incidence of diastolic dysfunction increases in women after menopause but the mechanisms involved are not completely understood. GPR30 is a novel estrogen receptor expressed in various tissues including the heart. Studies have shown that activation of GPR30 by its agonist G1, improves contractile function and reduces infarct size in isolated rat and mouse hearts subjected to ischemia/reperfusion injury and attenuates diastolic dysfunction in a salt-sensitive rat model of hypertension. We hypothesized that the activation of GPR30 by G1 protects against the development of diastolic dysfunction and cardiac hypertrophy in oophorectomized mRen2.Lewis rats, an established model that emulates the cardiac phenotype of the postmenopausal woman. Surgical bilateral oophorectomy (OVX) was performed in female mRen2.Lewis rats at 4 weeks of age, and G1 (50 or 100 µg/kg/day) was given subcutaneously via minipump starting at 13 weeks of age for 2 weeks. Both doses of G1 significantly improved lusitropic function and structure, independent of changes in blood pressure. Compared to vehicle-treated OVX rats, G1 reduced the tissue Doppler-derived index of left ventricular filling pressure (E/e’), left ventricle mass, wall thickness, and the biomarkers of hypertrophy, ANF and BNP mRNA levels. Using cultured H9c2 cardiomyocytes, in vitro studies further showed that 1) G1 inhibited angiotensin (ANG) II-induced hypertrophy, evidenced by reductions in cell size, and ANF and BNP gene expression; 2) the antagonist of GPR30, G15, inhibited estrogen’s protective effects on ANG II-induced hypertrophy; and 3) G1 induced phosphorylation of Erk and Akt. These data demonstrate the protective role of GPR30 in a model of diastolic dysfunction and cardiac hypertrophy, and provide insight into the underlying mechanisms.
Abstract P146: Fas-ligand Induces Cardiomyopathy via the Activation of the Erk1/2 Pathway
Authors: Huby Anne-Cecile , Purevjav Enkhsaikhan , Towbin Jeffrey A
Published: March 2022

Fas receptor and its ligand, Fas-L, are the most well characterized co-stimulatory molecules playing an essential role in the induction of programmed cell death or apoptosis. Increase of circulating Fas-L in serum and in cardiomyocytes are described in different models of cardiac diseases such as myocarditis, pressure overload, AND myocardial infarction/ischemia. In addition, associated activation of inflammatory pathwayshas been observed. However, no studies have yet revealed mechanisms underlying the pathogenecity of FasL in the heart.

Transgenic mice (C57/Bl6) with cardiac-specific over-expression of Fas-L were investigated. We characterized cardiac function by echocardiography. Cellular and molecular studies were performed on heart tissue using genomic, histologic, immunological, and protein analysis techniques.

Fas-L transgenic mice had a significant rate of mortality (p<0.0001) compared to WT due to heart failure. Histological studies of Fas-L hearts revealed predominant perivascular fibrosis associated with inflammation. Expression studies of secondary messengers of the activated Fas/FasL system demonstrated up-regulation of an active CASP-3 confirming induction of apoptosis in myocardium. Down-regulation of FLIP, NIK and NF-κB was evident. Interestingly, RIP was up-regulated leading to the activation of ERK1/2 (4.27±0.158 vs 10.086±1.591 in WT and FasL mice hearts, respectively, p=0.022). Analysis of inflammatory mediators revealed increase in secretion of interleukines (IL-17, IL-6, IL-1β and TNF-α) and diffuse infiltration of CD3 positive cells. Studies of fibrotic pathways showed an increased expression of periostin and osteopontin, but not activation of TGFβ1, showing that periostin and osteopontin can be activated independently of TGFβ1 via the ERK1/2 pathway,

Our findings suggest that Fas-L induces cardiac failure, via an activation of the ERK1/2 pathway, which is an important player involved in the hypertrophic response in the heart. Targeting the ERK1/2 pathway in cardiac diseases associated with Fas-L expression may therefore be able to prevent progression to cardiomyopathy.
Abstract P147: Antiangiogenic Effect of Doxorubicin on Human Cardiac Microvascular Endothelial Cells Does Not Result from Reduced Vascular Endothelial Growth Factor Production
Authors: Konorev Eugene A , Sun Zuyue
Published: March 2022

Clinical use of the potent anticancer agent doxorubicin (DOX) is limited by a dose-dependent cardiomyopathy. Although cardiac muscle has been thought to be a primary target of the cardiotoxic effect of doxorubicin, our studies indicate that adult cardiomyocytes may be resistant to the direct effects of this agent. These studies have prompted us to identify alternative targets for doxorubicin in adult heart that may contribute to the development of cardiomyopathy. Additionally, recent literature report (
Lee et al. PNAS 2009;106:2353–2358
) indicated that DOX inhibited tumor angiogenesis due to the reduced production of vascular endothelial growth factor (VEGF) by tumor cells. We set out therefore to test the hypothesis that DOX inhibits angiogenesis by human cardiac microvascular endothelial cells (HCMVEC) via inhibition of VEGF production. We detected reduced proliferation/viability of HCMVEC cultures treated with DOX. DOX increased caspase-3-like activity and development of apoptosis in HCMVEC, and inhibited the formation of vascular tubule formation in a co-culture of HCMVEC with human cardiac fibroblasts (HCF) with an IC
50
of 8 nM. HCF cultures released 7-fold greater amounts of VEGF into the culture media as compared with HCMVEC under the normoxic conditions while 1% hypoxia and 100 μM CoCl
2
further increased the production of VEGF by HCF. DOX moderately inhibited hypoxia- and CoCl
2
-induced VEGF release by these cells at concentrations above 500 nM whereas inhibition of vascular tubule formation by DOX in a co-culture system occurred at much lower concentrations and was not associated with the reduced production of VEGF. We conclude that DOX is a very potent antiangiogenic agent in the human cardiac microvascular endothelial cell system. However, this effect of DOX is not caused by the reduced production of VEGF. Inhibition of vascular network formation in the heart is likely to contribute to the development of doxorubicin cardiomyopathy in treated patients.
Abstract P148: Urotensin II Impairs Cardiac Functions and Proliferation of Cardiac Side Population Cells in Mice During Chronic Pressure Overload.
Authors: Zou Yunzeng , Gong Hui , Ma Hong , Zhang Guoping , Chen Zhidan , Jiang Guoliang , Yang Chunjie , Yan Yuan , Yuan Jie , Li Lei , Wu Jian , Ge Junbo
Published: March 2022

Urotensin II (UII), a potent vasoactive “somatostatin-like” peptide, has been known to play a role in cardiovascular diseases including cardiac hypertrophy and heart failure. But its effect on cardiac side population cells (CSPs), one of somatic stem cells in the heart potentially participating in cardiac protection after injury, is unclear. The present study was therefore conducted to examine influences of UII on the differentiation, proliferation and function of CSPs. CSPs were isolated from neonatal rat hearts by fluorescence-activated cell sorting (FACS) and cultured in the absence or at the presence of UII. The expressions of alpha-cardiac myosin heavy chain, alpha-smooth muscle actin and Von Willebrand factor at mRNAs and protein levels were analyzed by reverse transcriptional PCR (RT-PCR) and immunofluoresence to evaluate the differentiation of CSPs into cardiomyocytes, smooth muscle cells and endothelial cells, respectively. The proliferation of CSPs was assessed by Luminescent Cell Viability Assay. The influence of UII on the proliferation of CSPs
in vivo
was also evaluated by FACS. Our results revealed that UII did inhibit the proliferation of CSPs through up-regulation of phosphorylated c-Jun N-terminal protein kinase (JNK), although it didn’t affect the differentiation of cultured CSPs into cardiaomyocytes, smooth muscle cells and endothelial cells.
In vivo
experiments also showed that injection of UII reduced the number of CSPs and impaired cardiac functions compared with vehicle injection in mice subjected to a chronic pressure overload, and an UII antagonist urantide induced a preserved cardiac function with an increased number of CSPs. These data indicate that UII reduces the number of CSPs by inhibiting the proliferation of CSPs possibly through increase of JNK phosphorylation, and blockage of UII may be a useful strategy for cardiac protection in the hypertensive.
Abstract P163: Rescue of Preexisting Severe Pulmonary Hypertension and Right Ventricular Failure by Intralipid
Authors: Umar Soban , Matori Humann , Foroughi Reza , Partownavid Parisa , Eghbali Mansoureh
Published: March 2022

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling leading to right ventricular (RV) hypertrophy and failure (RVF). Intralipid (ILP) is the first safe lipid emulsion for human use. Several constituents of ILP, like γ-linolenic acid and soy- phytoestrogens are protective for lungs and heart. Previously we showed that ILP prevents the development of PH. Here we test a more clinically relevant hypothesis if ILP can rescue pre-existing severe PH and RV dysfunction. To induce PH, male rats received single monocrotaline (MCT, 60mg/kg) injection. At day 21, when severe PH had been well-established (PH group, n=7), rats were given ILP (1mL of 20% ILP/day, i.p., n=7) from day 21-30. Other MCT rats were left untreated to develop RVF (RVF group, n=9) by day 30. Saline treated rats were control (CTRL, n=5). Serial echocardiography was done to monitor cardiopulmonary hemodynamics. Cardiac catheterisation was performed just before sacrifice to record RV pressure (RVP). CD31 staining for RV capillary density and smooth muscle actin staining for pulmonary arteriolar medial hypertrophy were performed. Rats developed severe PH and RV dysfunction 21 days after MCT in PH group [RVP=67±1 vs. 31±1mmHg in CTRL, RV-ejection fraction (RVEF)=39% vs. 65±1%; RV/(LV+IVS)=0.64±0.05 vs. 0.24±0.02; medial hypertrophy=2.8 fold vs. CTRL; all p<0.05]. Interestingly, we found that even 10-day ILP therapy not only prevented the progression of PH to RVF but also rescued pre-existing PH and RV dysfunction [RVP=44±1 mmHg vs. 70±2 mmHg in RVF; RVEF=53±1 vs. 30±1%; RV/(LV+IVS)=0.39±0.02 vs. 0.66±0.08; medial hypertrophy=3 fold less vs. RVF; all p<0.05]. ILP therapy resulted in 100% survival compared to only 25% survival in RVF. Next we investigated if ILP rescue was associated with stimulation of RV neoangiogenesis. We found that RV capillary density was decreased in PH (0.7±0.07 vs. 1±0.07 capillaries per myocyte in CTRL; p<0.05) and worsened in RVF (0.52±0.07 capillaries per myocyte; p<0.05 vs. CTRL). ILP therapy resulted in stimulation of neoangiogenesis (capillary density=0.98±0.01 capillaries per myocyte; p<0.05 vs. PH and RVF). ILP therapy rescues severe pre-existing PH and retards the development of RV failure possibly via stimulation of RV neoangiogenesis.
Abstract P167: CXCR4 Antagonism Is Equally Effective as Sirolimus in Reducing Neointima Formation After Arterial Injury Without Impairing Reendothelialization
Authors: Hamesch Karim , Subramanian Pallavi , Li Xiaofeng , Thiemann Anna , Heyll Kathrin , Dembowsky Klaus , Chevalier Eric , Weber Christian , Schober Andreas
Published: March 2022

The chemokine receptor CXCR4 is crucial in neointima formation which is a problem after stent implantation. Drug-eluting stents reduce the restenosis risk but delayed re-endothelialization promotes late stent thrombosis. We compared CXCR4 antagonism with sirolimus treatment in preventing neointima formation after arterial injury in mice. Apolipoprotein E
-/-
mice after carotid wire injury were given a CXCR4 antagonist (POL5551 (P), a Protein Epitope Mimetic (PEM)) or sirolimus (n=6-8/group). The drugs were administered continuously (P: 2 and 20 mg/kg/d; sirolimus: 1.25 mg/kg/d; osmotic pumps) or intermittently (P: 20 mg/kg; IP, once daily) for 28 days. The neointimal area, smooth muscle cell (SMC) content, macrophage content, and endothelial coverage were quantified by planimetry from injured arteries stained with Elastica van Giesson, or after immunostaining for α-smooth muscle actin, Mac-2, and von Willebrand Factor, respectively. Peripheral Sca-1
+
/Lin
-
smooth muscle progenitor cells (SPCs) were measured by flow cytometry. Treatment with sirolimus as well as with a continuous (C) or intermittent (I), high (H) or low (L) dose (D) of POL5551 (P) reduced the neointimal area compared to corresponding vehicle (Sirolimus: 69%, CHD-P: 70%, CLD-P: 63%, IHD-P: 53%). The relative neointimal content of macrophages increased by sirolimus application (70%). In contrast, the macrophage content decreased by CHD-P and IHD-P application (57% and 37%, respectively). However, CLD-P application did not alter the macrophage content. Sirolimus, CHD-P, and IHD-P highly diminished the neointimal SMC content (78%, 85%, and 67%, respectively), while CLD-P treatment did not. SPC increase in the circulation 1 day post injury was prevented by sirolimus, CHD-P, and IHD-P, but not by CLD-P. The endothelial coverage was not reduced by P, but significantly by sirolimus (14%). In conclusion, CXCR4 antagonism by POL5551 is equally effective as sirolimus in reducing neointima formation after arterial injury in mice. It might be more beneficial than sirolimus because it does not impair re-endothelialization and leads to a more stable lesional phenotype. These results indicate that POL5551 might be a promising alternative for restenosis prevention after stent implantation.
Abstract P200: Transcriptional Regulation of
tcap
Conveys an Inhibitory Mechanotransductive Response to Modulate Cardiomyocyte Size
Authors: Yang Jingchun , Ding Yonghe , Sun Xiaojing , Xu Xiaolei
Published: March 2022

To sense and to respond to mechanical stimuli is a fundamental process of cardiomyocytes during both heart development and diseases. However, how mechanical stimuli control cardiomyocyte size remains elusive. Here, we report a novel inhibitory mechanotransductive response suggested by loss-of-functions studies of α-Actinin2 (Actn2), a predominant sarcomeric Z-disc protein. During zebrafish cardiogenesis, depletion of Actn2 results in a defective Z-disc, while other sarcomeric substructures remain normal. As a consequence, ventricle chamber size is severely reduced. Interestingly, this chamber size reduction can be rescued by the cessation of heart contraction, suggesting the involvement of an inhibitory mechanotransductive reponse. At the molecular level, both
tcapa and tcapb,
two zebrafish
tcap
homologues that are transcriptionally responsive to stretch, are transcriptionally activated in
actn2
knockdown embryos while activation of these gene results in smaller cardiomyocytes. Depletion of either gene rescues the reduced cardiomyocyte size in
actn2
knockdown embryos. Consistent to embryonic studies, tcaps are transcriptionally activated during pathogenesis of anemia-induced cardiomyopathy, while over expression of
tcapa
via transgenics exerts a cardioprotective effect. Together, we conclude that transcriptional regulation of
tcaps
conveys a novel inhibitory mechanotransductive response that modulates cardiomyocyte size during both zebrafish cardiogenesis and adult cardiac remodeling.
Abstract P203: Nuclear PKA Compartmentation Manages Hypertrophic Responses to β-Adrenergic Signaling
Authors: Yang Jason H , Saucerman Jeffrey J
Published: March 2022

Protein kinase A (PKA) directly mediates several cardiac behaviors in response to β-adrenergic stimulation. However, it remains unclear how PKA makes context-dependent decisions to select between contractile and hypertrophic β-adrenergic signaling responses. We have previously shown PKA activation by isoproterenol is spatially heterogeneous in neonatal rat cardiac myocytes, indicating subcellular β-adrenergic signaling compartmentation. We hypothesize hypertrophic β-adrenergic signaling responses are specifically regulated by nuclear compartmentation of PKA activity. We expressed spatially targeted FRET reporters for PKA activity and found PKA responses to isoproterenol to be slower and less sensitive in the nucleus than in the cytosol. To investigate mechanisms underlying these differences, we constructed a biochemically mechanistic computational model of cytosolic and nuclear PKA activity in rat cardiac myocytes. Using this model, we found that nuclear PKA dynamics are limited by diffusional transport barriers, whereas nuclear PKA sensitivity to isoproterenol is limited by protein kinase inhibitor (PKI) inhibition. Moreover, our model predicts these differences in compartmented PKA activity may help PKA select between different substrates, sensitive to the temporal characteristics of β-adrenergic stimulation. In the model, short, transient isoproterenol treatment fully phosphorylates phospholamban while CREB is only partially phosphorylated. In contrast, long, sustained isoproterenol treatment fully phosphorylates both phospholamban and CREB. To test the model prediction that compartmented PKA can regulate different cardiac behaviors, we over-expressed PKA targeted to the cytosol and nucleus. Myocytes over-expressing PKA in the nucleus were 24.8% larger than control (n = 908 cells, p = 0.0002), while myocytes over-expressing PKA in the cytosol were the same size as control cells (n = 987 cells, p = 0.9971). Together, these results highlight an important role for nuclear PKA compartmentation in selecting hypertrophic responses to β-adrenergic stimulation.
Abstract P205: The Role of Nuclear Nox4 in Mediating Oxidation of HDAC4 in Response to Phenylephrine Stimulation
Authors: Matsushima Shouji , Kuroda Junya , Ago Tetsuro , Sadoshima Junichi
Published: March 2022

Reduction and oxidation (Redox) stress is involved in the pathogenesis of cardiac hypertrophy and heart failure. We have shown previously that oxidation of conserved cysteine residues in histone deacetylase 4 (HDAC4) induces nuclear-to-cytoplasmic translocation of HDAC4, thereby mediating phenylephrine (PE)-induced cardiac hypertrophy. NAD(P)H oxidase 4 (Nox4) is a major source of oxidative stress in cardiomyocytes (CMs) in response to pressure overload. We hypothesized that Nox4 plays an essential role in mediating cardiac hypertrophy by regulating the redox state of HDAC4. Nox4 exists on intracellular membranes, including the mitochondria and nucleus in CMs. A subpressor dose of phenylephrine (PE, 20 mg/kg per day) or saline alone (control) was continuously infused into wild type (WT) and cardiac specific Nox4 knockout (Nox4-/-) mice subcutaneously via an osmotic minipumps. After 14 days, mean aortic pressure was similar between WT and Nox4-/- mice. Left ventricular (LV) wall thickness (1.04±0.03 vs. 1.27±0.06 mm, p<0.05) and LV weight/ tibial length (LVW/TL 5.7±0.14 vs. 6.4±0.05 mg/mm, p<0.05) were significantly lower in Nox4-/- than in WT mice. CM cross sectional area (MCA: 223±13 vs. 258±12 μm
2
, p<0.05) was significantly smaller in Nox4-/- than in WT mice. Superoxide production from the nuclear membrane, as evaluated by lucigenin chemiluminescent assays, was also significantly lower in Nox4-/- than in WT mice (4116±314 vs. 7057±1710 RLU, p<0.05). On the other hand, PE infusion induced significantly greater cardiac hypertrophy in transgenic mice with cardiac specific overexpression of Nox4 than in control mice. In cultured neonatal rat CMs, PE (100μM) treatment induced upregulation of Nox4 (2.8 fold vs. control, p<0.05) within 5 min, which was accompanied by increases in superoxide production (6 fold, p<0.01) and rapid (< 5min) nuclear export of HDAC4. Knockdown of Nox4 with shRNA-Nox4, but not Nox2 knockdown, attenuated superoxide production in the nucleus and prevented PE-induced nuclear export of HDAC4. These results suggest that Nox4 plays an essential role in mediating PE-induced cardiac hypertrophy. Nox4 mediates PE-induced superoxide production in the nucleus and nuclear export of HDAC4, possibly through cysteine oxidation.
Abstract P208: Depletion of Dapper-1 Acts Antihypertrophic on Cardiomyocytes and Inhibits Wnt3a- and Wnt5a-Mediated Activation of the Wnt Signaling Pathway
Authors: Hagenmueller Marco , Katus Hugo A , Hardt Stefan E
Published: March 2022

Background:
Cardiac development is regulated by the evolutionary conserved Wnt signaling pathway. While Wnt activity is silenced in the adult heart under normal conditions this pathway is activated during myocardial remodeling following pathological injury such as myocardial infarction. Among other members of this pathway Dapper-1 was suggested to antagonize Wnt activation but its role in Wnt signaling is poorly understood. In a murine myocardial infarction model as well as in a cardiomyocyte hypoxia model we found a robust over-expression of Dapper-1. To elucidate its function in cardiomyocytes we performed Dapper-1 specific siRNA mediated knockdown experiments.

Results:
Depletion of Dapper-1 leads to reduction of cardiomyocyte surface area (-10% ± 4%, p<0.001 vs. control), attenuation of global protein content (-20% ± 3%, p<0.02 vs. control) and diminishes global protein synthesis (-27% ± 10%, p<0.02 vs. control). Augmentation of protein synthesis by β-adrenic Isoproterenol stimulation is widely inhibited. Wnt3a induced stimulation of protein synthesis and enlargement of cardiomyocyte surface area is blocked in Dapper-1 knockdown cardiomyocytes. Furthermore, we observed reduced total- and active-β-catenin protein levels. Moreover, Dapper-1 knockdown inhibits β-catenin from translocation to the nucleus following Wnt3a stimulation. TCF/LEF reporter activity was reduced in Dapper-1-diminished -and elevated in Dapper-1-overexpressed cardiomyocytes after Wnt3a treatment. Robust Ser9 -and weak Tyr216 phosphorylation of GSK3β suggested a GSK3β independent inhibition of canonical Wnt signaling. In addition, activation of non-canonical Wnt/JNK pathway by Wnt5a conditioned medium was completely inhibited in Dapper-1 knockdown cells.

Summary:
Dapper1 is an essential modulator for global protein synthesis. Furthermore, Dapper-1 is required to activate canonical and noncanonical Wnt signaling pathways in cardiomyocytes.
Abstract P216: PRAS40 Is a Critical Regulator of Cardiomyocyte Growth
Authors: Volkers Mirko , Konstandin Mathias , Gude Natalie , Din Shabana , Sussman Mark
Published: March 2022

Background:
Proline-rich Akt substrate of 40 kDa (PRAS40) is a component of the mammalian target of rapamycin complex (mTORC) 1. Whereas, PRAS40 was originally characterized as substrate for PKB/Akt, subsequent studies identified PRAS40 both as inhibitor and substrate of mTORC1. These studies identify PRAS40 as a negative regulator of mTORC1 activity and cell growth. Phosphorylation of PRAS40 by AKT, which results in dissociation of PRAS40 from mTORC1, relieves the inhibitory constraint on mTORC1. The physiological role and pathophysiological function of PRAS40 in the heart is unknown.

Methods and Results
PRAS40 is expressed in cardiomyocytes and highly phosphorylated in embryonic and neonatal hearts. PRAS40 phosphorylation and expression is decreased in adult cardiomyocytes. Pathological challenge in mice induced phosphorylation of PRAS40 in the border zone after myocardial infarction and globally after transaortic constriction. Subcellular, phosphorylated PRAS40 is localized in the cytoplasma after pathological challenge, whereas stimulation with Insulin or IGF1 induced nuclear localization of PRAS40. Adenoviral mediated overexpression of PRAS40 completely blocks pathological hypertrophy after stimulation with phenylephrine as well as physiological growth after stimulation with IGF1 or Insulin. Molecular, mTORC1 activity was significantly reduced after PRAS40 overexpression. Conversely, silencing of PRAS40 resulted in significant increased mTORC1 activity. Gain and loss of function studies of PRAS40 in vivo will be performed to test the therapeutically potential of PRAS40.

Conclusion
PRAS40 is a novel regulator of cardiomyocyte growth. Manipulation the activity or expression of PRAS40 could represent a novel therapeutic target to treat cardiac diseases.
Abstract P218: PKCε Regulates PI3K/mTOR Complex-2-Dependent AKT Activation Independent of c-Raf/MEK/ERK Pathway
Authors: Moschella Phillip , McKillop John , Harston Rebecca , Kuppuswamy Dhandapani
Published: March 2022

Mammalian target of rapamycin (mTOR) has been shown to influence hypertrophic growth of the myocardium through two unique complexes, mTOR complexed with Raptor (mTORC1) and mTOR complexed to Rictor (mTORC2) with downstream activation of S6K1 and AKT respectively. Previously, we characterized the role of novel PKC isoforms (epsilon and delta) on mTORC1-dependent activation of S6K1 downstream of both PI3K-dependent and independent pathways in adult cardiomyocytes. PKC epsilon (PKCε) has been implicated in AKT activation during ischemia-reperfusion injury and has been co-immunoprecipitated in transgenic PKCε overexpressing murine myocardium. In the present study, we explored the role of PKCε on PI3K-dependent AKT S473 phosphorylation. As such, insulin stimulated pAKT S473 was significantly reduced by pretreatment with wortmannin, a selective PI3K inhibitor. However, insulin stimulated activation of AKT was not reduced by pretreatment with either Gö6976 blockade of the classical PKC isoforms (PKC alpha/beta) or U0126 blockade of MEK. Thus, insulin stimulated activation of AKT is mediated primarily through a PI3K-dependent pathway independent of the classical PKC isoforms. To begin to evaluate the role of the novel PKCs, we utilized adenoviral expression of dominant negative PKCε (dnPKCε). After 48 hours of expression of dnPKCε, insulin stimulated pAKT S473 was significantly reduced as compared to B-gal infection controls. Next we identified for the first time in isolated cardiomyocytes a direct link between PKCε and AKT through immunoprecipitation of a unique signaling complex comprised of PKCε bound to mTORC2 and pAKT (S473) only during insulin stimulation. These data establish for the first time in adult isolated cardiomyocytes: i) the necessity of PKCε during insulin stimulated PI3K-dependent AKT activation and ii) a signaling complex containing mTOR, Rictor, PKCε, and pAKT that forms during insulin stimulation. Given the importance of AKT in physiologic growth and survival, these studies indicate that PKCε plays a pivotal role during AKT activation and for the first time displays a direct link between PKCε, pAKT and mTORC2 in isolated cardiomyocytes.
Abstract P227: Metformin Inhibits Glucose-Induced mTOR Activation and ER Stress in Rodent Heart
Authors: Sen Shiraj , Guthrie Patrick , Taegtmeyer Heinrich
Published: March 2022

Background:
When subjected to increased workload, the heart remodels both metabolically and structurally by increasing glucose metabolism and activating the mTOR signaling pathway. Specifically, glucose activates mTOR in a TSC2-dependent manner. We now examined whether metformin regulation of AMPK, a major upstream regulator of TSC2, modulates mTOR activation and endoplasmic reticulum (ER) stress when hearts are subjected to increased workload.

Methods:
Working hearts from 12 week old male Sprague Dawley rats were perfused ex vivo (5 mM glucose) at physiologic pre- and afterload. At 30 minutes, the workload was doubled. Hearts were freeze-clamped at 60 minutes. Hearts received either the AMPK activator metformin (10 mM) or the ER stress relieving agent sodium 4-phenylbutyrate (10 mM) directly in the perfusate. Rates of glucose uptake and oxidation were measured with radiolabeled substrate (3H, 14C). Cardiac power, the mTOR signaling pathway, and markers of ER stress were assessed.

Results:
With increased workload rates of glucose uptake and oxidation doubled, with rates of glucose uptake exceeding those of glucose oxidation. Neither metformin nor sodium 4-phenylbutyrate significantly changed those rates. In control hearts, there was a downregulation of AMPK, an activation of mTOR, an induction of ER stress, and a decline in contractile function. Perfusion with media containing metformin activated AMPK, prevented glucose-mediated mTOR activation, alleviated ER stress and improved cardiac function (25% increase in cardiac power). In a separate group of animals nearly identical findings were obtained when metformin (300 mg/kg/day) was administered in vivo for one week. The functional improvement was also observed when ER stress was directly relieved with sodium 4-phenylbutyrate, as well.

Conclusion:
Our results implicate a critical role for glucose metabolism and AMPK in load-induced mTOR activation. Metformin improves contractile function in rat hearts subjected to increased workload by relieving ER stress. The relief of ER stress by metformin suggests previously unrecognized beneficial effects of AMPK activation in the hemodynamically stressed heart.