bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 334: Complementary Atherosclerosis Prophylaxis in Patients After Coronary Bypass Surgery
Authors: Siegel Günter , Winkler Karl , Malmsten Martin , Schmidt Annette , Zimmermann Elke , Ermilov Eugeny , Just Sören
Published: July 2021

Background and purpose:

Atherosclerotic coronary arteries can be well-treated with innovative medicaments, through PCI or by aortocoronary bypasses. Some of the stents and bypasses close again in spite of blood-thinning. We examined in a clinical pilot study of 8 patients who had undergone a CABG operation whether atherosclerosis prophylaxis can be practised through an additional phytochemical approach with ginkgo (EGb 761, 2 × 120 mg/d). Furthermore, our trial aimed to inquire whether the bypasses remain open.

Methods:

As a biomarker for plaque development, atherosclerotic nanoplaque formation and size were monitored by laser ellipsometry (patent EP 0 946 876). Lipoproteins, cytosolic SOD, oxLDL, and [cAMP] and [cGMP] were determined by established methods.

Results:

After 2-month medication, the decrease in nanoplaque formation amounted to 11.9 ± 2.5% (p < 0.01) and in nanoplaque size to 24.4 ± 8.1% (p < 0.02), after 5 years 25.0 ± 3.7% (p < 0.01) and 45.8 ± 9.5% (p < 0.01), respectively. A multiple correlation between nanoplaque reduction and the risk factors oxLDL/LDL, Lp(a) and cGMP yielded an estimate of their relative contribution to nanoplaque diminution (34.7%, 43.3%, 22.0%). In the 5 year follow-up examination using high-resolution CT, all arterial and 86.7% of the venous bypasses were open, whereby the increase in blood nucleotides seemed to have played a prominent role.

Conclusion:

Ginkgo may be used as complementary drug with potentially preventive character after coronary-artery bypass graft.
Abstract 337: Alternative Angiogenic Pathway Driven by Stimulus-Dependent Phosphorylation of Profilin-1
Authors: Fan Yi , Fox Paul L
Published: July 2021

Angiogenesis, the outgrowth of new blood vessels from pre-existing ones, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances directional endothelial cell (EC) migration to initiate angiogenesis, which can be driven by a multistep signaling cascade that directs actin polymerization. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr129 is critical for EC migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and its immediate downstream kinase Src directly induce Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Interestingly, Pfn-1 phosphorylation is robustly and preferentially elevated in blood vessels during tissue repair after myocardial infarction (MI) in humans. Conditional endothelial knock-in of phosphorylation-deficient Y129F Pfn-1 mutant in mice reveals that Pfn-1 phosphorylation is required for VEGF-A-induced EC sprouting migration and critical for angiogenesis in response to wounding and ischemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signaling events to initiate EC migration and angiogenesis, and may serve as a selective therapeutic target for angiogenesis-related disorders including ischemic heart disease.
Abstract 347: Targeted FAK Activation in Cardiomyocytes Protects the Heart from Doxorubicin-Induced Cardiomyopathy
Authors: Cheng Zhaokang , DiMichele Laura A , Hakim Zeenat S , Rojas Mauricio , Mack Christopher P , Taylor Joan M
Published: July 2021

OBJECTIVE:
We recently reported that cardiac-restricted activation of focal adhesion kinase (FAK) attenuated myocardial injury following ischemia/reperfusion using transgenic mice that express a FAK variant (termed SuperFAK) in cardiomyocytes. Here we interrogated whether targeted elevation of myocardial FAK activity could protect against cardiomyopathy induced by the highly effective chemotherapy drug Doxorubicin (DOX).

METHODS AND RESULTS:
Eight- to twelve-week-old male mice were given a single injection of DOX (20mg/kg, i.p.). At day 14, SuperFAK mice exhibited better survival (62.5%, n=8) than littermate control mice (37.5%, n=8). Serial echocardiography revealed that DOX administration markedly decreased cardiac function and ventricular wall thickness in control mice, whereas both parameters were better preserved in SuperFAK mice at day 5 (fractional shortening: 52.7±1.5% in SuperFAK
vs
. 38.9±3.7% in control,
p
<0.01; posterior wall end-systolic thickness: 1.83±0.12mm in SuperFAK
vs
. 1.43±0.12mm in control,
p
<0.05). Importantly, SuperFAK hearts exhibited a dramatic increase in FAK activity (as assessed by phospho-FAK Y397 immunoblotting) and a reduction in myocyte apoptosis (as assessed by TUNEL staining) in comparison with control hearts. DOX also induced apoptosis in cultured neonatal rat cardiomyocytes and adenoviral-mediated expression of SuperFAK ameliorated DOX-induced toxicity as assessed by MTT and TUNEL assays. Over-expression of SuperFAK also enhanced expression of the pro-survival NF-κB transcriptional targets Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis, whereas pharmacologically blockade of the NF-κB pathway completely abolished the up-regulation of these anti-apoptotic molecules by SuperFAK.

CONCLUSIONS:
Ventricular dysfunction and myocyte apoptosis induced by DOX was attenuated by enhancing cardiac FAK activity, which may represent a novel strategy to reduce anthracycline mediated cardiotoxicity in cancer patients undergoing chemotherapy.
Abstract 355: Systems Epigenomics of Chromatin Structure in the Heart
Authors: Franklin Sarah , Chen Haodong , Karbassi Elaheh , Monte Emma , Vondriska Thomas M
Published: July 2021

Except during metaphase, endogenous chromatin structure is unknown. DNA - invariant between cells - and the cell type-specific modifiers of the genome establish chromatin structural features, both local (e.g. at the scale of individual nucleosomes) and global (e.g. chromosomal territories). A fundamental question is how these cell type-specific modifiers, including DNA modification, non-coding RNAs, and proteins, establish the chromatin environment conducive to gene expression for the correct cell type: in cardiac muscle, how is the genome structurally poised to confer cardiac (and not, say, renal) transcriptomes and proteomes, and what physical reprogramming events occur during disease?

To address these questions, we are conducting a systems analysis of the epigenetic features of the healthy and diseased heart. In adult mice, we have used quantitative mass spectrometry to dissect distinct fractions of the nucleus and reveal the itineraries of chromatin structural proteins, enzymatic nucleosome remodelers, histone molecules and histone post-translational modifications. These studies have revealed rules for global reprogramming of gene expression, which involve altered abundance of non-histone chromatin structural proteins, a shift from hetero- towards euchromatic post-translational marks and a decreased linker to core histone ratio in heart failure. Furthermore, interrogation of genome-wide binding patterns of known cardiac transcription factors within the genes that encode proteins operative in cardiac genomic structure reveals hierarchical relationships between these protein, transcript and gene networks. Complementary investigations in isolated myocytes are characterizing the global rearrangement of chromatin following hypertrophic agonist treatment using conventional and super-resolution microscopy to directly visualize the chromatin backbone. Lastly, a combination of multiple genomic scale sequencing studies have revealed regions under control of specific chromatin structural proteins. Together, these studies aim to address the basic question of how global chromatin structure is maintained in cardiac myocytes and how diseases like heart failure result from deranged chromatin structure on a genomic scale.
Abstract 357: Cardiac Fibroblasts as a Support Cell for Endothelial Cell Sprout Formation in Engineered Cardiac Tissue
Authors: Twardowski Rachel L , Black Lauren D
Published: July 2021

Cardiac tissue engineering offers a promising approach to regenerating post-MI myocardial tissue; however, a primary impediment lies in the inability to adequately vascularize a tissue scaffold. Endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are able to support the formation of mature tubules/vessels. Cardiac fibroblasts (CFs) provide important electrical and mechanical components to the myocardium and are present in the native cardiac cell population, but to date have not been sufficiently studied for their role in angiogenesis in the heart. As a model of myocardial tissue, we co-cultured different concentrations of various cell types in fibrin hemispheres in appropriate combinations of their specific media, to determine the optimal sprout formation through DNA analysis, flow cytometry and immunohistology (IH). For flow cytometry, endothelial cells and mesenchymal stem cells were marked with CD31 and CD90, respectively. The highest total cell count was found in the fetal CF co-cultured populations (Fig 1A p<0.05), but ECs proliferated best with the adult and neonatal CFs (Fig 1B p<0.001). Analysis of IH images (Fig 1C, D) demonstrated that ECs formed the longest (Fig 1E p<0.05) and most numerous sprouts (p<0.002) with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs (Fig 1F p<0.001). In summary, CFs provide a better support system for EC proliferation and sprout formation than MSCs, while MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.
Abstract 359: Higher ROS Generation and Lower Threshold of mPTP Opening May Underlie Increased Vulnerability of Late Pregnant Heart to Ischemia-Reperfusion Injury
Authors: Li Jingyuan , Iorga Andrea , Youn Ji-Youn , Cai Hua , Regitz-Zagrosek Vera , Eghbali Mansoureh
Published: July 2021

Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In-vivo female rat hearts (non-pregnant (NP) or LP) or Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion. The infarct size was ∼4 fold larger in LP compared to NP both in the in-vivo rat model and ex-vivo mouse model. The hemodynamic parameters were similar between NP and LP before ischemia. However, the postischemic functional recovery was extremely poor in LP mice comparing to NP mice. RPP was reduced from 12818±1213mmHg*beats/min in NP to 1617± 287mmHg*beats/min in LP mice at the end of reperfusion. Interestingly, all of the hemodynamic parameters almost fully recovered in hearts seven days post-partum (PP7)( RPP= 9604±1215 mmHg*beats/min). To explore the mitochondrial function involvement in the higher vulnerability of LP hearts to I/R injury, mitochondrial respiration and ROS production were measured. Respiratory control index(RCI) were significantly decreased in LP subjected to I/R compared to NP and PP7 (RCI=1.9±0.1 in LP, 4.0±0.5 in NP and 3.9±0.5 in PP7, P<0.05 LP vs. NP and PP7). The superoxide production was also significantly higher in isolated cardiac mitochondria from LP hearts subjected to I/R injury (10.7±1.7mM/min/mg protein in NP; 21.3±3.1mM/min/mg protein in LP and 9.3±3.3mM/min/mg protein in PP7; p<0.05 LP vs. NP and PP7). The threshold for opening of mitochondrial permeability transition pore (mPTP) in response to Ca2+ overload was much lower in LP hearts (calcium retention capacity(CRC)=167±10 nmol/mg-mitochondrial protein) compared with NP (233±18 nmol/mg-mitochondrial protein) and PP7 (260±12 nmol/mg-mitochondrial protein, P<0.01). In conclusion, the higher susceptibility of LP hearts to I/R injury is associated with a lower threshold for triggering the mitochondrial permeability transition pore (mPTP) opening in response to Ca2+ overload which may at least be in part due to higher ROS generation and lower mitochondrial respiration.
Abstract 362: Proteomic Analysis of Low Oxygen Tension-Induced Secretome of Adipose Tissue-Derived Stem Cells
Authors: Bosnjak Zeljko , Wakim Bassam , Yan Yasheng , Canfield Scott , Kikuchi Chika , Bai Xiaowen
Published: July 2021

Growing evidence from animal studies shows that adipose tissue-derived stem cells (ASCs) improve cardiac function of infarcted hearts. It is commonly accepted that therapeutic potential of ASCs may depend more on their paracrine effects than differentiation potential. The underlying mechanisms remain unclear. However, most data regarding paracrine factors were obtained from ASCs cultured in normoxic condition (20%). The present study investigated how in vivo physiological oxygen (4%) tension influenced the secretome of ASCs.

ASCs were isolated from three 8-week-old BALB/c mice. ASCs were confirmed by the expression of stem cell markers (CD44 and CD90) and their capacity to differentiate into adipocytes and osteocytes. ASCs at passage 5 were cultured in normoxic (20%) and lower oxygen (4%) incubators and conditioned for 24 h (3 cultures/group). The conditioned media (CM) from ASCs were subjected to trypsin digestion followed by analysis using automated nano-flow liquid chromatography tandem mass spectrometry. The collected LC/MS/MS data were searched against the rodent subset of the Uniprot database and the total proteomes were identified. The data were from 6 technical replicates.

A total of 28 proteins were identified and 7 proteins were unique to normoxic CM. Of the 21 common proteins detected in both normoxic and lower oxygen CM, 9 were extracellular matrix proteins. The abundance of 6 of these proteins (e.g., collagen I and laminin) differed noticeably between normoxic and lower oxygen CM. In addition, a greater amount of cytokine CXCL5 and matrix metalloproteinase (MMP)-2 was detected in lower oxygen CM than in normoxic CM while tissue inhibitor of metalloproteinase (TIMP)-1 was only detected in normoxic CM.

These results indicate that lower oxygen tension differentially regulates the secretome of ASCs. Extrapolating the results of this study to the in vivo setting, it would appear that injected ASCs may exert their anti-fibrotic and trophic effect by 1) directly regulating the balance of MMP/TIMP production and preventing collagen accumulation in ischemic hearts to decrease fibrosis, and 2) secreting trophic factors including CXCL5. These data suggest that proteomic analysis of CM is useful for elucidation of the paracrine effect of ASCs in vivo.
Abstract 364: Valproic Acid Induced Reprogramming of Adult Somatic Cells for Cardiac Differentiation
Authors: Pasha Zeeshan , Ashraf Muhammad
Published: July 2021

Aims:

Owing to the ethical concerns for use of embryonic stem cells (ESC), adult somatic cells are attractive stem cell sources for reprogramming to pluripotency. We report here non-viral approach for reprogramming of skeletal myoblasts (SM) using a small molecule.

Methods and Results:

SM purified from young male Oct3/4-GFP+ transgenic mouse were treated for 5 days with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. Three weeks later, GFP+ colonies of SM derived iPSC (Sk-iPS) resembling with mouse embryonic stem cells were observed and propagated in vitro. SiPS were positive for alkaline phosphatase, had normal karyotype, expressed SSEA1, and induced teratomas in nude mice containing tissue comprising all three germ layers. RT PCR analysis showed that Sk-iPS cells expressed Oct4, Sox2, KLF4, c-Myc, Nanog and ESC specific pluripotency genes. HDAC1 activity was significantly reduced in Sk-iPs generated with valproic acid treatment as compared to ES cells. Sk-iPS derived embryoid bodies (EBs) yielded spontaneously contracting cardiomyocytes with morphological, molecular, and ultrastructural features of developing cardiomyocytes. These cells were also positive for early and late cardiac markers such as myosin heavy chain, Gata4, Mef-2c and Nkx2.5, Connexin-43 (P<0.01vs native SM). Micro RNA (miR) profiling showed abolition of let-7 family in Sk-iPS whereas ESC specific family of miR-290-295 was upregulated which indicated that Sk-iPS possessed miR profile similar to ESC. However muscle specific miRNAs (miR -133, -206) were identified in Sk-iPS cells as compared to ES cells indicating that the Sk-iPS retained the epigenetic memory of myogenic origin.

Conclusions:

We conclude that SM with endogenous expression of Sox2, KLF4, and cMyc are suitable candidates to generate iPS cells without viral vectors using a single small molecule.
Abstract 66: The Role of the Viral Nef Protein as a Mediator of HIV-1--Induced Endothelial Dysfunction
Authors: Wang Ting
Published: July 2021

With the prevalence of antiviral therapy in the developed world, many HIV-1-infected people die of diseases other than AIDS. One of the emerging major causes is cardiovascular disease, leading to the prediction that the majority of HIV-1 patients are expected to develop cardiovascular complications. Endothelial dysfunction is thought to be a key event in the development of cardiovascular diseases, particularly atherosclerosis. Assays testing the effect of HIV-1 on endothelial activation shows that direct contact with HIV-1 infected T cells enhance endothelial cell activation to a greater extent than HIV-1 alone, suggesting an intracellular HIV-1 protein is responsible for endothelial activation. The HIV-1 viral protein Nef, which is responsible for T cell activation and maintenance of high viral loads
in vivo
, has been shown to mediate its own transfer to bystander cells. We demonstrate here for the first time that Nef induces nanotube-like conduits connecting T cells and endothelial cells. We also show that Nef is transferred from T cells to endothelial cells via these nanotubes, and is necessary and sufficient for endothelial cell activation. Moreover, we show that SIV-infected macaques exhibit endothelial Nef expression in coronary arteries. Nef expression in endothelial cells causes endothelial apoptosis, ROS and MCP-1 production. Interestingly, a Nef SH3 binding site mutant abolishes Nef-induced apoptosis and ROS formation and reduces MCP-1 production in endothelial cells, suggesting that the Nef SH3 binding site is critical for Nef effects on endothelial cells. Nef induces apoptosis of endothelial cells through an NADPH oxidase- and ROS-dependent mechanism, while Nef-induced MCP-1 production is NF-kB dependent. Taken together, these data suggest that Nef can mediate its transfer from T cells to endothelial cells through nanotubes to enhance endothelial dysfunction.Thus, Nef is a promising new therapeutic target for reducing the risk for cardiovascular disease in the HIV-1 positive population.
Abstract 67: A Rare Variant in
Myh11,
R247C, Affects Smooth Muscle Cell Phenotype and Acts as a Modifier for Aortic Disease
Authors: Kwartler Callie , Kuang Shao-Qing , Prakash Siddharth , Byanova Katerina , Pham John , Huang Jian , Kamm Kristine , Stull James , Sweeney H Lee , Milewicz Dianna
Published: July 2021

Thoracic aortic aneurysms and dissections (TAAD) affect more than 30,000 Americans annually. One-fifth of TAAD patients have a first-degree relative with the disease, suggesting a genetic component to its pathogenesis. Missense mutations in a number of genes lead to TAAD, including
MYH11
,which encodes the smooth muscle isoform of myosin heavy chain. However, potential genetic contributors in the four-fifths of patients without affected relatives have not been explored. Variants of unknown significance in
MYH11
are prevalent in the population, and are enriched in patients with TAAD and other vascular diseases. The goal of this study was to evaluate one of these variants as a potential contributor to TAAD.

A knock-in mouse model of the
Myh11
R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility.
Myh11
R247C/R247C
mice also have increased neointimal proliferation following carotid ligation and increased proliferation of vascular smooth muscle cells (SMCs)
in vitro. Myh11
R247C/R247C
SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, adhesions are smaller in
Myh11
R247C/R247C
SMCs, and there is increased Rac1 and focal adhesion kinase (FAK) activation with decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of
Myh11
R247C/R247C
SMCs, and FAK inhibition reduces proliferation.

Since this rare variant occurs in the general population, we sought to determine if it acts as a modifier gene for aortic disease. The
Myh11
R247C
allele was bred into
Acta2
-/-
mice, which develop aortic aneurysms, and into
Acta2
+/-
mice, which do not. In both cases, the presence of the R247C allele increases aortic diameter. These preliminary data suggest that the
MYH11
R247C rare variant may be a genetic contributor to TAAD in patients without an inherited mutation, or may affect the clinical presentation of TAAD in patients with Mendelian disease.
Abstract 70: mTOR Is Essential for Normal Heart Development and Compensatory Cardiac Growth in Fetal Mice
Authors: Hennig Maria , Magarin Manuela , Yin Kom Voy , Black Mary Jane , Thierfelder Ludwig , Drenckhahn Jörg-Detlef
Published: July 2021

Remarkable growth plasticity enables the prenatal mammalian heart to counteract various unfavorable intrauterine conditions and build a functional and normally sized organ at birth. We have recently shown that the murine embryonic and fetal heart has a substantial regenerative capacity in response to tissue mosaicism for mitochondrial dysfunction caused by heart specific inactivation of the X-linked holocytochrome c synthase (Hccs) gene. In heterozygous Hccs knockout (Hccs+/-) embryos, hyperproliferation of healthy cardiomyocytes compensates for the functional loss of 50% cardiac cells, ensuring formation of a functional heart at birth. However, we hypothesized that embryonic heart regeneration alters peri- and postnatal cardiac growth mechanisms. Indeed, neonatal Hccs+/- hearts are hypoplastic containing a reduced number of cardiomyocytes, whereas in adult Hccs+/- hearts compensatory cellular hypertrophy normalizes morphology and size. We aimed at identifying postnatal adaptive growth mechanisms utilized by the hypoplastic Hccs+/- heart to restore organ size and allow normal heart function throughout lifetime. Microarray RNA expression analyses revealed numerous genes involved in amino acid metabolism, protein homeostasis and translational control being differentially expressed in neonatal Hccs+/- hearts. Subsequently, western blot analyses evidenced significantly increased mammalian target of rapamycin (mTOR) activity, a major regulator of translation and cell growth, in Hccs+/- hearts compared to controls. To clarify its role for compensatory growth of the Hccs+/- myocardium, we inhibited mTOR in fetal and neonatal mice by rapamycin treatment of pregnant dams. Rapamycin treated Hccs+/- neonates (n=17) show significantly reduced heart weight to body weight ratios compared to controls (n=18) or vehicle treated animals (n=12 and n=13). Furthermore, Hccs+/- hearts after prenatal mTOR inhibition are morphologically characterized by developmental delay. In conclusion, our data revealed mTOR being essential for normal as well as compensatory growth of the fetal heart. Thus, metabolic adaptations converging on mTOR might be required to prevent postnatal heart disease after impaired intrauterine development.
Abstract 72: Ca
2+
and Calmodulin-Dependent Protein Kinase II Mediate a Temporal Switch Between Cardiotoxic and Cardioprotective Signaling by β2-Adrenergic Receptors
Authors: Rajagopalan Viswanathan , Fajardo Giovanni , Zhao Mingming , Urashima Takashi , Bernstein Daniel
Published: July 2021

Background:
ß-adrenergic receptors (AR) play a critical role in cardiac function but also mediate adverse remodeling associated with chronic sympathetic stimulation in heart failure. Some studies have suggested cardiotoxic signaling through ß1-AR and cardioprotective through ß2-AR. However, ß2-AR can signal through both Gs & Gi, partially dictated by the time course of activation. We sought to determine whether this temporal effect influenced the differential roles of ß1- & ß2-AR in the pathogenesis of cardiomyopathy.

Methods:
We compared two models of oxidative stress-induced cardiomyopathy: acute doxorubicin (AD) 15 mg/kg in a single dose vs. chronic doxorubicin (CD) 2 mg/kg/wk x 8 wks in WT, ß1-AR-/- & ß2-AR-/- mice. Endpoints were survival and cardiac size and function. Activation of AR signaling pathways was assessed by Western blot.

Results:
With AD, 100% of ß2-/- die within 20 min, an effect not seen in ß1-/- or WT. With CD, in contrast, ß2-/- were protected: WT survival t½ 42 d, 100% mortality 70 d, n=33; ß2-/- t½ 56 d, 100% mortality 109 d, n=44; ß1-/- t½ 54 d, 100% mortality 99 d, n=31 (log rank p<0.0001). Fractional shortening declined and LV diastolic dimension increased by 2 wks in WT (13.9±2.5, 10.3±2.2%) whereas both were preserved in ß2-/- (5.0±1.7, p<0.01; 4.8±1.5%) and ß1-/- (2±1.6, p<0.001; 0.8±1.8, p<0.01) at least until 4 wks. Total Ca2+ and calmodulin-dependent protein kinase II (CaMKII) was increased at baseline in both ß1-/- & ß2-/- vs. WT. However, in ß2-/-, CaMKII was decreased with AD (0.74±0.1; p<0.01), and increased with CD (1.3±0.05; p<0.01). Similarly, CaMKII phosphorylation was decreased with AD (1.27±0.16) vs. baseline (1.95±0.19) and increased with CD (2.35±0.18, p<0.001). With AD, p-p38 increased 20-fold in ß2-/-, but was normal in CD. In contrast, p-JNK was increased in CD in both ß2-/- (0.34±0.09) and ß1-/- vs. WT (0.1±0.04; p<0.001).

Conclusion:
The differential role of ß2-AR in mediating cardioprotection/cardiotoxicity are more complex than initially thought. ß2-AR signaling exhibits a temporal switch depending on whether a stress is acute (cardioprotective) vs. chronic (cardiotoxic). This switch is mediated by differential activation of CaMKII and JNK (mediating cardioprotection) and p38 (mediating cardiotoxicity).
Abstract 76: Ablation of Parkin Attenuates Acute Cardiotoxicity of Doxorubicin Associated with Mitophagy
Authors: Huang Chengqun , Andres Allen , Ramil Jennifer , Gottlieb Roberta
Published: July 2021

Parkin is an E3 ubiquitin ligase expressed in heart, skeletal muscle and brain. However, the function of Parkin in the heart is largely unknown. We previously showed in cardiomyocytes and the ischemic heart that Parkin translocates to mitochondria to selectively remove damaged mitochondria through autophagy. It has been shown that Doxorubicin (Dox) induces cardiotoxicity characterized by impaired mitochondrial respiration and energetic failure (PLoS One. 2010; 5:e12733). Here, we investigated the role of Parkin in acute Doxorubicin-induced cardiac dysfunction in mice.

Acute cardiotoxicity was induced by IP injection of a single dose of 15 mg/kg Doxorubicin hydrochloride in 10-12 wk-old C57BL/6J (WT) mice and Parkin knock out (PKO) mice matched by age, sex and body weight. Analysis of histology, hemodynamics, and mitochondrial content was performed 7d after Dox.

Dox induced significant loss of body weight but less in PKO mice (20% in WT vs 12% in PKO). Dox toxicity was more severe in male mice. Signs of discomfort (decreased mobility and hunched posture) were noted 4d after Dox in 90% of WT male mice vs. 20% of PKO males. Mortality at 7d was 30% in WT mice vs. 10% in PKO mice. Dox caused reduction in heart size to a greater extent (10-20%) in WT than in PKO mice. Significant deterioration of hemodynamics was seen in WT mice 7d after Dox; this was attenuated in PKO mice. Because Parkin mediates mitophagy, we examined autophagy and mitochondrial content 7d in Dox treated hearts. Dox resulted in extensive autophagy and decreased mitochondrial protein content in WT mice; this was attenuated in PKO mice.

These results suggest that high-dose Dox triggers extensive Parkin-dependent mitophagy which may contribute to acute cardiac dysfunction. We suggest that therapy directed at the PINK1-Parkin mitophagy pathway may be beneficial in the setting of doxorubicin overdose.
Abstract 84: Atrial Fibrillation Causes a Progressive, Time-Dependent Reduction in Myocardial Neuronal NOS in Humans and Goats: Implications for AF-Induced Electrical Remodeling
Authors: Reilly Svetlana , Liu Xing , Jayaram Raja , Verheule Sunder , Schotten Uli , Casadei Barbara
Published: July 2021

Rationale:
Nitric oxide (NO) plays a key role in the regulation of cardiac and endothelial function and thrombogenesis. Atrial fibrillation (AF) has been associated with reduced NO availability but the mechanisms and implications of this finding remain to be fully investigated.

Methods and Results:
We evaluated NO synthase (NOS) activity and localization in right atrial (RA) tissue from 30 patients with permanent AF (vs. 65 controls in sinus rhythm, SR), and in the RA and left atrial (LA) tissue of 48 goats after 2 weeks (2W) and 6 months (6M) of pacing-induced AF. NOS activity was uncoupled in RA tissue from patients and goats in 6M-AF, and was caused by a reduction in BH4 tissue concentration and by an increase in arginase activity (HPLC). Although BH4 and arginine supplementation re-coupled NOS, it did not abolish the difference in NOS activity between AF and SR. Immunoblotting and immunolocalization revealed a progressive reduction in bi-atrial neuronal NOS (nNOS) protein with the duration of AF (by 65% at 2W, 86% at 6M in goats and by 62% in patients with AF) and a reduction in eNOS in long-standing AF. nNOS was reduced in atrial myocytes but not in neuronal tissue. The mRNA expression of NOS (qRT-PCR) was unaltered; however, the reduction in nNOS protein in AF was associated with an increase in nNOS ubiquitination which was partially reversed by inhibition of proteosomal activity with MG132; inhibition of the autophagy-lysosomal pathway with bafilomycin A1 did not restore nNOS protein. To investigate the electrophysiological consequences of a reduced nNOS in LA and RA myocytes, we compared electrical properties of the isolated atrial myocytes from nNOS-/- mice (n=18) and their wild type (WT) littermates after nNOS inhibition with SMTC. Both nNOS gene deletion and inhibition impaired myocytes' relaxation in both RA and LA, and result in a slower rate of decay of [Ca2+]i transient in the LA myocytes only.

Conclusions:
A reduction in bi-atrial nNOS activity and protein level is an early event in the natural history of AF that results in a chamber-specific effect on electrical properties of the myocytes.
Abstract 87: The Impact of Coxsackievirus Infection on Cardiac Stem Cells and Postnatal Heart Development
Authors: Sin Jon , Tabor Godwin Jenna , Huang Chengqun , Gottlieb Roberta A , Feuer Ralph
Published: July 2021

Coxsackievirus B (CVB) is an enterovirus that causes a self-limited febrile illness in infants, but can also cause myocarditis. Long-term consequences of mild CVB infection are unknown, although an association between heart failure and seropositive status has been previously described. We have shown that early postnatal exposure to doxorubicin depleted c-Kit+ cardiac stem cells (CSCs). Cardiac dysfunction in adult mice only developed after exercise challenge, or experimentally-induced myocardial infarction. We now report that CSCs from neonatal mouse hearts were susceptible to cytopathic CVB infection. To learn if mild CVB infection might deplete the CSC population in the heart, we inoculated newborn BALB/c mice with a recombinant CVB expressing eGFP. We observed CVB-infected CSCs 2 days post-inoculation and documented a reduction in the number of c-Kit+ cells in the hearts of animals at 4 and 11 weeks of age despite the absence of detectable virus. Moreover, CVB infection induced differentiation in the surviving CSCs. These results indicate that CVB can infect CSCs in the neonatal heart and trigger their destruction. Premature differentiation of surviving cells may also explain the reduced CSC population in adult mice. Despite normal histologic features in adult hearts, prolonged exercise challenge elicited cardiac hypertrophy and fibrosis in mice infected with CVB as pups. We suggest that the loss of CSCs after mild neonatal CVB infection may predispose to late-onset heart failure.
Abstract 9: The Virtually Mature BNP1-32 Is a Precursor for the Biologically More Active BNP1-30
Authors: Schwiebs Anja , Wang Yong , Zhu Xudong , Siems Wolf E , Walther Thomas
Published: July 2021

The mature B-type natriuretic peptide (BNP1-32), mainly released from ventricular myocardium, has antifibrotic effects and exerts vasorelaxant and natriuretic activity leading to a reduction of blood volume and pressure. Thus, BNP is used as a drug for the treatment of decompensated heart failure.

Here we show the discovery of a new murine BNP metabolite, BNP1-30, exerting higher biological activity compared to the mature BNP1-32, in vitro and in vivo.

By mass spectrometric analysis of murine lung tissues incubated with BNP1-32 and of elution fractions of BNP1-32-perfused livers, we discovered a distinct BNP1-32 cleavage product, identified as the C-terminally truncated BNP1-30. We identified endothelin-converting enzyme (ECE) as the peptidase generating BNP1-30 from BNP1-32 by the hydrolysis of the two C-terminal leucine.

This new metabolite is significantly more efficient compared to BNP1-32 in stimulating the natriuretic peptide receptor (NPR) A. Interestingly it is also stimulating the C-type natriruretic peptide specific receptor NPRB. In parallel, dose-response curves in NPRA & -B co-transfected cell lines and stimulation of primary smooth muscle cells, endothelial cells and mesangial cells showed better efficacy for BNP1-30. In vivo, BNP1-30 reduced the mean arterial blood pressure of normotensive mice after acute infusion significantly more than BNP1-32. Additionally, BNP1-30 was also significantly more potent compared to the rodent derived pro-BNP cleavage product BNP1-45. Even more important, in a 3-day infusion experiment using spontaneously hypertensive rats (SHR), BNP1-30 was able to reduce systolic blood pressure by 30 mmHg and decreased cardiac BNP mRNA, a marker for cardiac failure, while BNP1-32 was without significant effect.

We show for the first time the occurrence of a BNP metabolite being a more efficient antihypertensive peptide than the virtually mature BNP1-32 that has been described as ‘the’ circulating biologically active form of BNP. Our results suggest that BNP1-32 is only the precursor for the more biologically active BNP1-30 leading to a fundamental revision of the natriuretic peptide system and also opens the avenue for the identification of more effective targets in the treatment of cardiovascular diseases.
Abstract 002: Blinded Head-to-Head Comparison of Intracoronary Allogeneic Cardiosphere-Derived Cells (icCDCs) Versus Allogeneic Mesenchymal Stem Cells (icMSCs) in Swine with Hibernating Myocardium
Authors: Weil Brian R , Suzuki Gen , Leiker Merced M , Fallavollita James A , Canty John M
Published: July 2021

Background:
Autologous icCDCs and icMSCs each improve myocardial function by stimulating endogenous myocyte proliferation. Since few myocytes arise directly from the injected cells, we evaluated the feasibility and comparative efficacy of allogeneic icCDCs vs. icMSCs.

Methods:
Studies were conducted in a blinded fashion. Immunosuppressed swine (cyclosporine 200 mg/day, N=26) with hibernating myocardium from a chronic LAD stenosis were randomized to receive vehicle, ~35 x 10
6
icMSCs, or ~35 x 10
6
icCDCs. Cells were infused into the 3 major coronary arteries at ~10
6
cells/min under continuous flow conditions. LAD function and pathologic indices of myocyte regeneration and proliferation were assessed 1 month later.

Results:
Baseline LAD wall thickening (%WT) was reduced in all animals (LAD 38 ± 2% vs. 83 ± 5% in remote, p<0.01) but not different between treatment groups. One month later, LAD %WT remained depressed in vehicle-treated animals but increased to a similar extent following icCDCs and icMSCs (Table). Intracoronary cell therapy also produced large reciprocal increases in nuclear density and reductions in myocyte size with no differences between cell types. Indices of myocyte proliferation (Ki67 and phospho-Histone H3) remained elevated following icCDC treatment but had returned towards control in icMSC-treated animals.

Conclusion:
Our data demonstrate the feasibility of intracoronary allogeneic icCDC and icMSC infusion to treat viable dysfunctional myocardium without employing the “stop-flow” technique. Both cell types produce an equivalent increase in myocyte number and LAD function after 4-weeks but proliferative indices remain elevated after icCDCs.
Abstract 004: Microrna-22 Regulates Smooth Muscle Cell Differentiation From Stem Cells By Targeting Methyl Cpg Binding Protein 2
Authors: Zhao Hanqing , Huang Yuan , Yu Xiaotian , Wen Guanmei , Wang Wen , Ye Shu , Xiao Qingzhong
Published: July 2021

Objectives:
To investigate the role of microRNA-22 (miR-22) in smooth muscle cell (SMC) differentiation from stem cells and the molecular mechanism involved.

Methods and Results:
Mouse embryonic stem (ES) cells were seeded on collagen IV-coated flasks and cultured in the absence of Leukemia Inhibitory Factor in differentiation medium for 4 to 8 days in order to induce SMCs differentiation. We found that miR-22 was significantly up-regulated during SMC differentiation. Enforced expression of miR-22 by its mimic in differentiating ES cells significantly up-regulated expression of a panel of SMC-specific genes, while knock-down of miR-22 by its antagomiR decreased these gene expression at both RNA and protein levels. Furthermore, we found over-expression and knockdown of miR-22 up-regulated and down-regulated SMC transcription factor SRF and its co-activator myocardin in a similar manner, respectively, and miR-22 overexpression in stem cells promoted SMC differentiation
in vivo
. Transcription repressor methyl CpG binding protein 2 (MECP2) was predicted as one of the top targets of miR-22 by using several computational miRNA target prediction tools. Interestingly, the gene expression levels of MECP2 were significantly decreased during SMC differentiation in a time dependent manner. Moreover, MECP2 was dramatically decreased in miR-22 overexpressing cells, but significantly increased when miR-22 was knockdown in the differentiating stem cells. Importantly, luciferase assay showed miR-22 substantially inhibited wild type MECP2-3’-UTR-luciferase activity in differentiating ES cells, but not mutant MECP2-3’-UTR-luciferease reporter. In addition, modulation of MECP2 expression levels affect multiple SMC-specific marker gene expression in differentiated ES cells by conducting gain-of-function experiments using MECP2 expression plasmid. Finally, we tested the expression of miR-22 in mouse femoral artery denudation injury model and found that miR-22 was significantly down-regulated in the wire injury-induced neointimal formation as early as day 3 post-injury.

Conculsion:
Using genomic and functional studies, we have discovered miR-22 controls SMC differentiation from stem cells via modulating MECP2 expression and function.
Abstract 009: Cardiac Human Mesenchymal Stromal Cells from Patients with Ischemic Heart Disease are Proinflammatory and Impair Recovery after Myocardial Infarction in Rat
Authors: Naftali-Shani Nili , Itzhaki-Alfia Ayelet , Landa-Rouben Natalie , Kain David , Holbova Radka , Molotski Natali , Asher Elad , Grupper Avishay , Millet Eran , Tessone Ariel , Winkler Eyal , Kastrup Jens , Feinberg Micha S. , Kassif Yigal , Sternik Leonid , Lavee Jacob , Raanani Ehud , Leor Jonathan
Published: July 2021

Introduction:
Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest for cell-based therapies. However, the best source of hMSCs for infarct repair is still unknown.

Hypothesis:
We aimed to test the hypothesis that, because of their proximity to the heart, hMSCs from the heart and epicardial fat would be better cells for infarct repair.

Methods and Results:
We isolated and grew hMSCs from patients with ischemic heart disease (IHD) from four different sources: 1) epicardial fat 2) pericardial fat 3) subcutaneous fat and 4) the right atrium. hMSCs from the right atrium and epicardial fat secreted the highest amounts of trophic and inflammatory cytokines, while hMSCs from pericardial and subcutaneous fat secreted the lowest (p<0.05, n=5-6 per source). Subsequently, the greatest angiogenic effect was induced with conditioned medium from atrium and epicardial fat hMSCs, by tube formation assay. To determine the effect of hMSCs
in vivo
, we allocated rats to hMSC transplantation 7 days after myocardial infarction (MI) (n=8-10 per group). Atrial hMSCs induced the highest number of vessels in the infarct, together with the highest inflammation score and macrophage accumulation 27 days after their injection. However, left ventricular (LV) dysfunction was worst after therapy with atrium and epicardial fat hMSCs, and minimal after subcutaneous fat hMSC therapy. Notably, there was a correlation between levels of tumor necrosis factor-α and hepatocyte growth factor,
in vitro
, and post-transplantation LV systolic dilatation. These surprising findings were supported by gene expression analysis in hMSCs from different sources of a patient with IHD. We found that the relative expression of inflammation and fibrosis-related genes was considerably higher in hMSCs from the right atrium and epicardial fat than in subcutaneous fat hMSCs.

Conclusions:
The origin of hMSCs affects their reparative and immunomodulatory properties. Because of their pro-inflammatory properties, hMSCs from the right atrium and epicardial fat of IHD patients could impair heart function after MI. Our findings might be relevant to autologous MSC therapy and pathogenesis and progression of IHD.
Abstract 019: Diabetic Endothelial Progenitor Cells in Combination with an RGDS Presenting Peptide Amphiphile Enhance Recovery from Critical Limb Ischemia in Diabetic Mice
Authors: Vaughan Erin E , Sleep Eduard , Misener Sol , Klinger Aiko Ito , Ramirez Veronica , Abramova Tatiana V , Mackie Alexander R , Verma Suresh Kumar , Krishnamurthy Prasanna , Stupp Samuel I , Kishore Raj
Published: July 2021

Approximately two million people suffer from critical limb ischemia (CLI) with the prevalence of the disease expected to rise. Thus, there is a crucial need to develop new therapies to enhance angiogenesis and minimize the impact of the blocked vessel(s). Cell therapy using endothelial progenitor cells (EPCs) has shown some promise, however, the cells may not remain at the site of injury long enough to significantly impact the course of the disease. Further, the use of autologous cells may be problematic as the underlying disease, such as diabetes, which resulted in CLI also appears to negatively impact the function of EPCs. Thus, we propose to use a biomaterial in combination with the EPCs to enhance both the retention of cells at the site of injury and also enhance the function of the cells. A self-assembling peptide amphiphile (PA) was developed with an attached functional group consisting of the cell-attachment sequence of peptides identified in fibronectin: RGDS.

We hypothesize that EPCs combined with RGDS PA will improve the angiogenic response in CLI. Indeed, in vitro we found that EPCs from diabetic mice (db/db) exhibited increased survival on an RGDS PA in comparison to a scrambled sequence (DGRS) PA as measured by calcein-AM and ethidium homodimer-1 staining. To test if this enhanced survival would improve critical limb ischemia in diabetic mice, uni-lateral ischemia was induced by ligation of the femoral artery. Three days post surgery ischemia was confirmed by laser Doppler and the following treatments were injected into the ischemic limb of the diabetic mice: (1) PBS (2) scrambled PA (3) RGDS (4) scrambled PA +100,000 diabetic EPCs (5) RGDS PA + 100,000 diabetic EPCs. At four weeks post-injection, blood flow (as measured by laser Doppler) was increased in the group receiving RGDS PA when compared to the other groups (n>6). Further, necrosis was decreased in the RGDS PA group and muscle regeneration, as measured by the number of central nuclei, was increased in the RGDS PA group. Taken together, these results suggest that RGDS PA in combination with db/db EPCs enhances recovery from CLI in diabetic mice.
Abstract 030: Cardiac-specific Expression Of Constitutively Active Sumo-2 In Mice Leads To Cardiomyopathy
Authors: Kim Eun Y , Wei Yu , Ling Qian , Chang Jiang , Xi Yutao , Wang Jun
Published: July 2021

Sumoylation is a versatile posttranslational modification that regulates a wide spectrum of cellular activities and is involved in a number of human diseases. Cardiomyopathy is the leading cause of heart failure. Whether and how sumoylation is implicated in cardiomyopathy has not been well investigated. To address this issue, we generated a number of independent transgenic mouse lines that express constitutively active SUMO-2 in hearts (SUMO-2-Tg). Four of five SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to hypertrophic cardiomyopathy which developed gradually as mice aged. An increase in apoptosis was observed in the SUMO-2-Tg hearts prior to the occurrence of any other discernible heart phenotypes. Adenovirus-mediated expression of SUMO-2 in cultured cardiomyocytes significantly increased Annexin V staining, indicating that SUMO-2 directly promoted the apoptotic process. We further revealed that SUMO-2 was directly involved in regulation of apoptosis at least partially via targeting calpain 2 (Capn2) and its natural inhibitor calpastatin (CAST). SUMO attachment to CAST mainly promoted its turnover, thus inhibiting CAST activity. On the other hand, SUMO-2 linkage to Capn2 increased its capacity to promote apoptosis, as evidenced by the observation that adenovirus-aided expression of SUMO-fused Capn2 (SUMO-Capn2) in cultured cardiomyocytes significantly promoted Annexin V staining compared with free Capn2. We also observed that SUMO-2/3 conjugation was significantly increased in the human failing hearts compared with the control hearts. Correspondingly, elevated levels of SUMO-2/3-conjugated Capn2 and CAST were detected in the human failing hearts vs. control hearts. Thus, elevated SUMO-2 conjugation led to increased apoptosis and cardiomyopathy at least partially via targeting calpain-calpastatin proteolytic system. The present study uncovers a direct regulation of the apoptotic pathway by the SUMO-2/3 conjugation pathway.