bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 169: Angiotensin-II Induced Left Ventricular Remodeling Differs Between C57bl/6 Substrains
Authors: Cardin Sophie , Scott-Boyer Marie-Pier , Picard Sylvie , Reudelhuber Tim L , Deschepper Christian F
Published: July 2021

Background:
Although C57Bl/6 mice are widely used in cardiovascular research, little is known about possible substrain differences. We compared the left ventricular (LV) remodeling induced by angiotensin II (angII) in C57Bl/6J and C57Bl/6N mice, these 2 substrains corresponding to two main branches having diverged since 1951.

Methods and Results:
Male C57Bl/6J and C57Bl/6N mice were treated with angII (350ng/kg/min) or vehicle via mini-osmotic pumps for either 48h (for microarray profiling of gene expression and macrophage cytofluorometry counting) or 15 days (for both gene expression and histology). AngII (15d) induced fibrosis in LV from C57BL/6N (as shown by histology and col1a expression), but not in C57BL/6J. After 48h of treatment, about 100 genes responded in a strain-specific fashion, most responses being specific for C57Bl/6N mice. Among genes showing greater than 2-fold induction by angII (48h) in C57Bl/6N mice, there was enrichment for markers of macrophages activation and M2 polarization (including osteopontin, arginase1 and galectin3). These strain-specific differences were confirmed (both in LV tissues and macrophages isolated from LVs) by Q-RT-PCR, and occurred despite that fact that AngII increased the abundance of Cd11b+ macrophages to the same extent in both strains. Moreover, AngII (48h) increased expression of several markers of fibroblast activation (including Timp1, Lox and tenascin) in C57Bl/6N (both in LV tissue and fibroblasts isolated from LVs), but not in C57Bl/6J. Although one of the best known genetic differences in both substrains is the inactivation of the Nnt gene in C57Bl6/J mice, experiments performed in F2 mice do not indicate that the fibrotic response co-segregates with the Nnt mutation.

Conclusions:
Although angII-treatment induces macrophage recruitment in the LVs of both C57Bl/6J and C57Bl/6N mice, activation of cardiac macrophages and their M2 pro-reparative polarization (with subsequent activation of fibroblasts and synthesis of collagen) occurs only in the genetic C57Bl/6N background. The absence of macrophage activation and cardiac fibrosis in C57BL/6J may possibly explain some differences in experimental results obtained by various investigators using different C57BL/6 substrains.
Abstract 172: Notch Signaling Induces Endocardial Hematopoiesis And Endocardial Cushion Formation Downstream Of Nkx2-5
Authors: Nakashima Yasuhiro , Nakano Haruko , Liu Xiaoqian , Harmon Andrew W , Nakano Atsushi
Published: July 2021

A subset of cardiac progenitors display multipotency to give rise to major cardiovascular lineages during cardiogenesis. In addition, we have recently identified Nkx2-5+ endocardial cells in the outflow and atria that contribute to definitive hematopoietic cells in vivo, which represents phylogenetically conserved pathway of cardio-vasculo-hematopoietic differentiation. Here, we have examined the molecular mechanism underlying the endocardial hematopoiesis. Immunostaining and colony assay suggest that Nkx2-5 mutant mice showed significant decrease in CD31+/41+ hemogenic endocardial cells, while lineage tracing on Nkx2-5-null background revealed that Nkx2-5-derived endocardial cells are present. These data indicate that Nkx2-5 is required not for the formation of the Nkx2-5-derived endocardium but for the hemogenic activity of the endocardium. Notch signaling is also active in cushion endocardium and its mutant embryos show a similar defect with Nkx2-5 mutants as previously reported. Inspired by these similarities between the function of Nkx2-5 and Notch signaling during cardiogenesis, we next investigated the impact of Notch activation on the endocardial hematopoiesis. In vivo forced activation of Notch in the developing heart using Nkx2-5-Cre mice led to massive increase in the endocardial hematopoiesis. As Nkx2-5 binding sites are found in the regulatory region of Notch1, RBPjk, and Jag1 genes, we tested whether Notch regulates endocardial hematopoiesis downstream of Nkx2-5. Surprisingly, Notch activation restored the defective endocardial hematopoiesis as well as endocardial cushion formation in Nkx2-5 knockout mice, suggesting that Notch signaling mediates the Nkx2-5-dependent endocardial hematopoiesis and cushion development. Drosophila dorsal vessel and lymph gland are closely related and regulated by tinman and notch. Therefore, Nkx2-5-Notch regulatory pathway represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway.
Abstract 173: Clonal Analysis Reveals Limited Proliferative Capacity of Mature Cardiomyocytes during Embryonic Development.
Authors: Sereti Konstantina-Ioanna , Kamran Paniz , Ali Shah R , Lee Josh Z , Subat Ali , Contreras-Trujillo Humberto , Zhao Peng , Ardehali Reza
Published: July 2021

There is growing evidence that supports the ability of the heart to generate new cardiomyocytes during development and in response to injury albeit at a very low rate. It still remains unclear whether the newly generated cells originate from cardiac progenitor/stem cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Understanding the cellular mechanisms regulating new cardiomyocyte formation is imperative towards the development of novel clinical strategies. We assessed the hypothesis that the cardiomyocytes generated during cardiac development are derived from cardiac progenitor cells and to a lesser extent form proliferating cardiomyocytes. We performed clonal analysis of cardiomyocyte formation by utilizing a stochastic multicolor reporter system (Rainbow) that allows random labeling of cells with three fluorescent proteins upon Cre-mediated recombination. Rainbow mice were crossed to αMHC-CreER mice for selective marking of cardiomyocytes or to Actin-CreER mice where all types of cells can be labeled. We induced rare recombination events at embryonic day E12.5 and clonal expansion of labeled cells was retrospectively analyzed at different time-points from E15.5 to postnatal day P30. To determine at which developmental stage cardiac cells lose their ability to proliferate, recombination was induced at different time-points from early embryonic to postnatal life and analysis was performed at P30. For each time-point, data was collected from ten mice. In αMHC-CreER;Rainbow mice, we observed mainly single cell labeling, a few doublets and some rare four-cell clones. At the same time-points, Actin-CreER;Rainbow hearts exhibited a large number of clones ranging from doublets to clones of more than twenty cells. Interestingly, clone formation and expansion was reduced with age. In conclusion, our results indicate that αMHC-positive cardiomyocyte proliferation is limited during development. On the other hand, non-αMHC expressing cells exhibited clonal expansion suggesting the possible contribution of cardiac progenitors even during late cardiovascular development.
Abstract 183: Small Molecule Gβ? Inhibition Reduces Pathologic Activation of Cardiac Fibroblasts
Authors: Travers Joshua G , Kamal Fadia A , Blaxall Burns C
Published: July 2021

Heart failure (HF) is a devastating disease characterized by cardiac hypertrophy, fibrosis and inflammation. Excess signaling through Gβγ subunits leads to chronic β-adrenergic receptor (β-AR) downregulation, mediated predominantly by GRK2 in complex with PI3Kγ. Our recent work has demonstrated the therapeutic potential of the small molecule Gβγ-GRK2 inhibitor Gallein in limiting HF progression. Chronic activation of cardiac fibroblasts (CF), critical yet underappreciated myocardial cells, is a key contributor to pathologic cardiac remodeling. We hypothesized that Gβγ-GRK2 inhibition may limit pathologic CF activation.

CFs were stimulated with Isoproterenol (Iso, β-AR agonist), AngII, or vehicle (V), +/- Gβγ inhibition for 24hr. Gallein treatment attenuated the induction of αSMA expression, a marker of pathologic CF activation, and two inflammatory cytokines, IL-1β and IL-6 in response to these pathologic stimuli (Iso, AngII), as assessed by real time PCR. This data suggest that Gallein treatment may reduce pathologic CF activation. Iso stimulation also enhances the phosphorylation of Akt, a kinase downstream of PI3Kγ known to be involved in cellular proliferation. Gβγ inhibition mitigated this induction, decreasing Akt phosphorylation >60% in response to Iso. This phenomenon was also observed in failing human CFs, in which Gallein decreased Akt phosphorylation >70%.

We have recently demonstrated that the protease-activated receptor 1 (PAR1), a GPCR we have implicated in cardiac hypertrophy, is transactivated via chronic β-AR stimulation by induction of MMP-13, a protease we have found to be elevated in HF. Recent data from our lab and others have demonstrated that PAR1 is the most abundantly expressed GPCR in CFs, and that its stimulation in CFs may be pathologic. Interestingly, Gβγ inhibition treatment reduced PAR1 cleavage and activation in response to chronic Iso.

In summary, small molecule Gβγ inhibition appears to reduce pathologic CF activation. The reduction in β-AR-mediated PAR1 cleavage reveals an alternative role for Gβγ inhibition in preventing CF activation and proliferation. These data suggest a potential therapeutic role for small molecule Gβγ inhibition in limiting pathologic CF activation and cardiac hypertrophy.
Abstract 188: Sarcomere Protein Cardiac Myosin Binding Protein C Regulates Cardiomyocyte Proliferation
Authors: Jiang Jianming , Wakimoto Hiroko , Onoue Kenji , Fomovsky Gregory M , Lee Richard T , Seidman Jonathan G , Seidman Christine E
Published: July 2021

Introduction:
Wildtype neonatal murine cardiomyocytes become bi-nucleated, hypertrophy and terminally differentiate, and concurrently develop well-organized striation patterns of sarcomeres of myofibrils. Neonatal mice lacking cardiac myosin binding protein C (Mybpc
t/t
) have subtle abnormalities in myofibrillar structure and ventricular dilation, but remain healthy life-long.

Hypothesis:
Deficits in MybpC impacts formation of bi-nucleated and cell proliferation of cardiomyocytes.

Methods:
We studied the cardiac development in Mybpc
t/t
mice and hearts depleted of cMyBP-C by adeno-associated virus (AAV)-mediated RNAi.

Results:
Mybpc
t/t
hearts presented significant increase in mono-nucleated cardiomyocytes (18.28 ± 1.68%) and decrease in bi-nucleated cardiomyocytes (74.87 ± 87%), compared to wildtype hearts (mono-nucleated: 5.85% ± 1.14%, p<0.001; bi-nucleated: 89.83 ± 2.04%, p<0.001, n=4 in each genotype). Similar distribution of mono-nucleated (25.25 ± 3.15%) and bi-nucleated cardiomyocytes (73.55 ± 3.8%) was also observed in cardiomyocytes depleted of cMyBP-C by AAV-mediated RNAi (n=4). Significant increase in EdU incorporation was observed in Mybpc
t/t
cardiomyocytes (47.02 ± 2.5%) compared to wildtype cardiomyocytes (29.35 ± 3.06%, p<0.005). Phosphorylated histone H3, a marker of mitosis, was approximately two-fold more positive in Mybpc
t/t
than wildtype cardiomyocytes at postnatal day 3, day 7 and day10 (p<0.01). In addition, we observed a 15-fold increase of Aurora B expression within the cytoplasmic bridge between cardiomyocytes undergoing cytokinesis in Mybpc
t/t
hearts compared to wildtype hearts at postnatal day 7 (p<0.001). The predicted consequence of protracted cardiomyocyte proliferation in neonatal Mybpc
t/t
mice, a significantly increased number of cardiomyocytes, was demonstrated. In comparison to wildtype, the number of cardiomyocytes in Mybpc
t/t
hearts was significantly increased by 40% (n=3 in each group, p<0.01).

Conclusions:
Loss of cMyBP-C causes neonatal cardiomyocytes to undergo extra round of cytokinesis and continued progression of the cell cycles. Changes in cell number, rather than cell size, may be responsible for morphologic abnormalities of some cardiomyopathies.
Abstract 194: Phosphodiesterase 3 Controls Basal cGMP Levels And Regulates cGMP/cAMP Cross-talk In Adult Mouse Ventricular Cardiomyocytes
Authors: Götz Konrad , Nikolaev Viacheslav
Published: July 2021

PURPOSE:
cGMP is an important second messenger which is involved in the regulation of cardiac contractility and pathological hypertrophy. In cardiomyocytes, signaling by cGMP is organized in microdomains and is considered cardioprotective. Especially in adult cardiac myocytes, measurements of cGMP have been challenging and little is known about the spatio-temporal dynamics of cGMP. Here we developed a transgenic mouse model to visualize cGMP dynamis in adult cardiac myocytes.

Methods:
We generated transgenic mice with cardiomyocyte-specific expression of a highly sensitive fluorescence resonance energy transfer (FRET)-based cGMP biosensor red cGES-DE5 and performed FRET measurements in freshly isolated adult mouse ventricular myocytes. To analyze cGMP/cAMP crosstalk, FRET experiments were performed in cardiomyocytes isolated from mice transgenically expressing the cAMP sensor Epac1-camps.

Results:
Basal cytosolic cGMP levels were very low (~10 nM), but could be markedly increased by stimulation with natriuretic peptides (CNP>>ANP). In contrast, direct stimulation of the soluble guanylyl cyclase (sGC) with NO-donors such as SNAP showed no effect. However, constitutive activity of this cyclase contributes to basal cGMP production, since a clear decrease of basal cGMP levels was observed after stimulation with the sGC inhibitor ODQ. This basal cGMP production is regulated by phosphodiesterase (PDE) activity. Unexpectedly, PDE3 is most important in controlling basal cGMP levels, whereas PDE2 and PDE5 are much less active. We could also show that cGMP pools produced by GC-B after CNP stimulation are mainly regulated by PDE3, so that the receptor and this PDE form one functional unit important for the regulation of cGMP/cAMP cross-talk.

Conclusion:
In summary, we performed the first FRET-based measurements of cGMP in adult cardiomyocytes and we could highlight the key role of PDE3 in the regulation of basal cGMP levels and cGMP/cAMP cross-talk.
Abstract 201: BMP-pSmad1/5/8 Pathway Activation in a Novel Mouse Model of Calcific Aortic Valve Disease
Authors: Gomez Maria V , Cheek Jonathan D , Wirrig Elaine E , Yutzey Katherine E
Published: July 2021

Calcific Aortic Valve Disease (CAVD) affects >2% of the population over the age of 65, for whom the current standard of care is valve replacement surgery. To date, there are no pharmacologic-based therapies that prevent the progression or inhibit the development of CAVD, thus highlighting the necessity for new therapeutic approaches. Despite its clinical significance, the pathogenic mechanisms that drive the development of CAVD, and that could serve as potential therapeutic targets, remain unknown. We have recently identified Klotho-deficient mice, an established model of premature aging, as a novel model of CAVD that exhibit aortic valve calcification on the fibrosa side of the hinge region, closely mimicking human CAVD pathology. Unlike previous models, calcification occurs independent of inflammation and valve thickening in these mice, supporting a distinct mechanism of age-related calcification common in elderly patients. In bone, BMP-mediated osteogenic gene induction is essential for the process of calcification. Notably, pSmad1/5/8 activation, indicative of active BMP signaling, is observed prior to the onset of calcification and later is localized with calcific nodule formation in klotho-deficient mice with CAVD. Our hypothesis is that activation of BMP-pSmad1/5/8 signaling pathway promotes osteogenic gene induction and aortic valve mineralization in CAVD. Osteogenic factors, Runx2 and Osteopontin, are significantly increased in the aortic valves of klotho-deficient mice, suggesting an osteogenic-like mechanism of disease. Likewise, pSmad1/5/8 activation precedes osteogenic gene induction in these mice. Moreover, increased BMP2/4 ligand expression is detected prior to the onset of disease, as well as during calcific nodule formation, thus supporting an active role for the BMP-pSmad1/5/8 signaling cascade during aortic valve calcification. Our ongoing work includes BMP pathway inhibition studies to determine if this is an effective therapeutic strategy for the treatment of CAVD in the klotho-deficient mice. Altogether our data support a role for the BMP-pSmad1/5/8 signaling cascade as a critical mechanism in the initial onset and progression of aortic valve calcification in a novel mouse model of CAVD.
Abstract 215: Intralipid Protects The Heart In Late Pregnancy Against Ischemia Reperfusion Injury Via Stat3 Signaling Pathway
Authors: Li Jingyuan , Eghbali Mansoureh
Published: July 2021

We have recently reported that the hearts of late pregnant (LP) mice are more prone to ischemia/reperfusion (I/R) injury compared to non pregnant mice. We also found that intralipid, a fat emulsion commonly used for parenteral nutrition, protects the male rodent heart against I/R injury if administrated during reperfusion. The activation of the survivor activating factor enhancement (SAFE) pathway, which includes the activation of the transcription factor signal transducer and activator of transcription-3 (STAT3), has recently been highlighted in cardioprotection. Here we explored the therapeutic potential of intralipid in the protection of LP mouse hearts against I/R injury as well as the involvement of STAT3 signaling pathway. Isolated LP mouse hearts were subjected to 20 min ischemia followed by reperfusion with i) Krebs Henseleit buffer (CTRL group), ii) 1% intralipid (ITLD group) or iii) ITLD+STAT3 inhibitor Stattic (20 µM, Stattic group). The duration of reperfusion was 40 min for heart functional measurements and infarct size assessment. Postischemic administration of intralipid significantly improved the cardiac function of LP mice after ischemia. The rate pressure product (RPP) at the end of reperfusion was improved from 1617±287mmHg*beats/min (n=6) in CTRL to 11556±784 mmHg*beats/min (n=5) in the ITLD group. The infarct size was also significantly reduced by postischemic administration of intralipid to 17±2% (n=5) from 59±5%(n=6) in CTRL (p<0.01). A specific inhibitor of STAT3, Stattic, fully abolished, intralipid-induced cardioprotection, as the RPP at the end of reperfusion in the Stattic group (1345±559, n=3) was similar to CTRL group, and significantly lower compared with ITLD group. Furthermore, the infarct size in the Stattic group was markedly higher compared with ITLD group (48±99% (n=3) vs. 17±2%(n=5) in ITLD group, p<0.01). In conclusion, intralipid protects the late pregnant heart against I/R injury via the STAT3 signaling pathway.
Abstract 231: Decreased Parasympathetic Activity in the Ovulatory Phase of the Menstrual Cycle
Authors: Tweedell Andrew , Tenan Matthew , Hackney Anthony C , Brothers Matthew , Griffin Lisa
Published: July 2021

The Heart rate variability (HRV) power spectrum is a non-invasive index of parasympathetic and sympathetic branches of the autonomic nervous system (ANS). Sex hormone oscillations may affect autonomic nervous system function; however, no systemic difference in HRV across the menstrual cycle has been shown. The purpose of this study is to test the hypothesis that there are changes in HRV spectral power components across the menstrual cycle.

Method:
Eleven women (24.4±3.6 years) volunteered for this experiment. Menstrual cycle phase was determined via basal body temperature mapping. Participants were tested once in each cycle phase. A 3 lead ECG was sampled at 1000 Hz. A piezoelectric force transducer, placed around the chest, recorded breathing rate. After 20 minutes of quiet seated rest, 5 minutes of data was collected in the upright seated position. Power spectral density analysis was performed on the R-R interval variations by fast Fourier transformation. The spectrum was divided into low (0.04-0.15, Hz) and high (0.15-0.40 Hz) frequency components. In order to attain data normality, the components were transformed with a natural logarithm. A repeated measures ANCOVA was used with a piece-wise covariate function for breathing rates higher and lower than 10 breaths per minute.

Results:
A significant decrease in total power spectrum was observed at the ovulatory phase (P=0.04) compared to the early follicular, late follicular and mid luteal phases. Furthermore, high-frequency oscillations were lower (P=0.04) in the ovulatory phase compared to late follicular phase. There were no changes observed (P>0.05) in the low frequency power spectrum across the menstrual cycle.

Conclusions:
The decrease in the high-frequency components of the HRV power spectrum during the ovulatory phase indicates a decrease in parasympathetic regulation of the heart. The ovulatory phase is characterized by an elevated level of progesterone, follicle stimulating and luteinizing hormones, and a moderate level of estradiol. Thus, the changes in these hormones during ovulation appear to affect ANS function.
Abstract 236: HDAC5 is Required for Recruitment of an HDAC1/Sin3a Repressor Complex to the Ncx1 Promoter During Cardiac Hypertrophy
Authors: Harris Lillianne G , Wang Sabina , Mani Santhosh K , Kasiganesan Harinath , Menick Donald K
Published: July 2021

Background:
Pressure overload can lead to aberrant gene expression and subsequent adverse hypertrophic changes in cardiac tissue. Histone deacetylases (HDACs) play an important role in alteration of gene expression during cardiac hypertrophy. We observed that class I/IIb HDAC inhibitors, TSA and Vorinostat prevent upregulation of the sodium calcium exchanger gene,
Ncx1
, in response to cardiac hypertrophy. Chromatin immunoprecipitation (ChIP) demonstrates the class I HDAC, HDAC1, class IIa HDAC, HDAC5 and co-repressor, Sin3a are recruited to the
Ncx1
promoter via Nkx2.5. Class IIa HDACs have very poor catalytic activity and can interact with many specific transcription factors. Thus,
we hypothesized
, that the class IIa, HDAC5 acts as a scaffold to recruit the class I HDAC/Sin3a complex to the
Ncx1
promoter.

Methods and Results:
To test our hypothesis adult cardiomyocytes were treated with either vehicle (control) or class specific HDAC inhibitors: class I (Entinostat), or class IIa (dPAHA). We found that class I HDAC inhibitor, Entinostat, abrogates adrenergic stimulated upregulation of
Ncx1
expression in adult cardiomyocytes to almost control levels (p<0.05 Student’s t; n=4), while class IIa HDAC inhibitor, dPAHA has no effect on adrenergic stimulated upregulation of
Ncx1
expression (p>0.05 Student’s t; n=4). Also, Entinostat inhibits
Ncx1
upregulation in a transaortic constriction (TAC) model of hypertrophy. We assessed the role class IIa HDAC5 in
Ncx1
regulation, by subjecting HDAC5-Knockout mice (HDAC5-KO) to TAC. We saw no induction of
Ncx1
expression post-TAC (n=5). Furthermore, Co-IP and ChIP analyses show that interactions between the Sin3a/HDAC1 complex and Nkx2.5 are lost in the absence of HDAC5 expression and fail to be recruited to the
Ncx1
promoter.
To conclude
, our data suggests the catalytic activity of HDAC5 is not essential but rather HDAC5 is a scaffold that recruits the class I HDAC1–Sin3a complex to the
Ncx1
promoter. Our novel findings provide insight into possible roles of class IIa HDACs in regulation of gene expression and efficacy of HDAC inhibitors to treat cardiac pathologies.
Abstract 240: Temporally-Stiffening Hydrogel Enhances Cardiomyocyte Maturation via Activation of Mechanosensitive Pathways
Authors: Young Jennifer L , Kretchmer Kyle , Engler Adam J
Published: July 2021

As cells migrate and differentiate throughout development, they secrete and assemble extracellular matrix, giving rise to time-dependent, tissue-specific stiffness, i.e. myocardium stiffens ~10-fold during maturation. When mimicked in vitro with a thiolated hyaluronic acid/poly(ethylene glycol) diacrylate hydrogel, myocardial matrix stiffening enhanced cardiac specific gene expression and myofibril organization in isolated embryonic cardiomyocytes vs. static hydrogels. Mechanical cues alone, even when time-dependent, were insufficient to induce cardiogenesis in mouse embryonic stem cell (mESCs), but a combination of precisely timed developmentally-relevant chemical cues (e.g. BMP4, Activin A, DKK-1) and dynamic matrix mechanical cues at the cardiac mesoderm induction stage enhanced maturation of mESC-derived cardiomyocytes vs. static hydrogels. On dynamic hydrogels, measurement of sarcomere length shows mESC-derived cardiomyocytes exhibited greater than 30% more mature myofibrils compared to cells plated on static, stiffer matrices. While active mechanotransduction aided maturation, the specific proteins responsible for responding to time-dependent stiffness remain unknown. In order to assess matrix-mediated mechanotransduction, we examined the expression and phosphorylation state of 890 protein kinases, as well as organization of mechanosensitive proteins, e.g. focal adhesion kinase and vinculin, of embryonic cardiomyocytes plated on matrices with either dynamic or static cardiac tissue-specific stiffness. Microarray analysis of protein kinases showed differential expression as a function of mechanics, confirmed by ratiometric western blotting. Several cardiogenic pathways exhibited time-dependent up-regulation on dynamic vs. static matrices, including PI3K/Akt and p38 MAPK, while GSK3β, a known inhibitor of cardiomyocyte maturation was down regulated. These data indicate that mechanically driven maturation is at least partially achieved via active mechanosensing at focal adhesions. Identifying mechanosensitive pathways that are active in cardiomyogenesis can lead to a better understanding of how stem cell differentiation and development are mediated by extracellular matrix properties.
Abstract 247: Scaffold-stiffness Dependent Notch1 Activation Regulates Cardiogenic Gene Expression In Cardiac Progenitor Cells
Authors: Boopathy Archana V , Che Pao L , Narui Yoshie , Salaita Khalid , Davis Michael E
Published: July 2021

Rationale:
Cardiac progenitor cells (CPCs) are multipotent, self-renewing cells that can regenerate the myocardium and improve cardiac function in animal models of myocardial infarction (MI). However, limited survival of stem/progenitor cells inhibits cardiac regeneration. Force dependent Notch activation promotes cardiac development and cardiac gene expression in many adult stem cells. As dysregulation of Notch signaling leads to embryonic lethal cardiovascular defects, activating this critical pathway during cell transplantation could improve efficacy of stem cell therapy.

Objective:
Investigate i) whether self-assembling peptide scaffolds can be used to activate Notch1 signaling in CPCs to promote cardiogenic differentiation and ii) the effect of scaffold stiffness on Notch1 activation and differentiation.

Methods:
Rat CPCs (c-kit
+
) were cultured for 48h in 3D self-assembling scaffolds of varying stiffness (1% low, 2% high): empty scaffolds (RADA), scaffolds modified with peptide mimicking Notch1 ligand, Jagged1 (RJAG), or scaffolds modified with a scrambled peptide (RSCR) and cardiogenic gene expression measured by qRT-PCR. CHO cells expressing Notch1 responsive YFP were also cultured in the above scaffolds for 48h and YFP expression was determined. Results are mean ± SEM with p<0.05 considered significant by one or two-way ANOVA with appropriate post test.

Results:
In the Notch1 reporter cells, Notch1 activation increased significantly in presence of RJAG (p<0.01) and on increasing scaffold stiffness (p<0.01,n=6) indicating scaffold stiffness-dependent Notch1 activation. Culture of CPCs in RJAG containing 1% scaffolds (low stiffness) significantly increased early endothelial and smooth muscle but not cardiac gene expression while in 2% scaffolds (high stiffness) significantly increased only cardiac and not endothelial or smooth muscle gene expression (p<0.05, n≥4).

Conclusions:
Taken together, these data show that i) Notch1 activation in 3D is dependent on ligand density and scaffold stiffness and ii) stiffness dependent Notch1 activation differentially regulates cardiogenic gene expression in CPCs. Therefore, delivery of CPCs in JAG containing scaffolds could be used to improve cardiac function following MI.
Abstract 252: Endocardial Cushion-derived Cardiovascular Progenitor Cells
Authors: Truong Raymond
Published: July 2021

Introduction:
Regenerative therapies utilizing cardiac progenitor cells (CPCs) derived from human embryonic stem cells (hESC) have the potential to regenerate damaged myocardium post-myocardial infarctions. Previous studies have identified multipotent progenitors localized in the endocardial cushions of the developing mouse heart. Recent data in mice have also shown that the loss of a single transcription factor, Scl, enabled robust cardiomyogenic potential in these progenitor cells. Further analyses have delineated that these progenitors can be identified by co-expression of CD31 and PDGFRα. Recapitulation of this phenotypic progenitor cell from hESCs may have potential for clinical regenerative therapies.

Hypothesis:
We hypothesize that CD31
+
/PDGFRα
+
cells sorted from wild type mouse and human fetal heart tissue possess nascent cardiomyogenic potential. In addition, we believe that close mimicry of the
in vivo
developmental process of cushion formation would generate a hESC-derived CPC expressing CD31 and PDGFRα.

Methods:
CD31
+
/PDGFRα
+
cells sorted from wild type mouse heart and human fetal heart are plated in cardiac differentiation medium. Through a stage-specific differentiation protocol, hESCs are first differentiated into mesodermal progenitor cells, which then are sorted for the endothelial marker CD31. Under cardiac culture conditions, these CD31
+
cells express the CPC marker PDGFRα. The CD31
+
/PDGFRα
+
cells are then isolated and characterized through quantitative PCR, immunostaining, and functional assays.

Results:
Wild type mouse heart CD31
+
/PDGFRα
+
cells, when isolated at E14.5 and plated in cardiac differentiation medium, develop beating colonies. In addition, CD31
+
/PDGFRα
+
cells are present in human fetal hearts. Moreover, hESC-derived CD31
+
/PDGFRα
+
cells, when cultured in cardiac medium, show increased expression of cardiomyogenic markers.

Conclusions:
We conclude that endogenous hESC-derived CD31
+
/PDGFRα
+
cells have cardiogenic potential through
in vitro
differentiation, exhibited by their expression of cardiomyocyte characteristics and ability to beat. These cells may represent putative endocardial cushion-derived CPCs.
Abstract 266: Truncated Glycogen Synthase Kinase 3β Increases Mitochondrial Fragmentation, Reactive Oxygen Species generation, and Cell Injury
Authors: Hurst Stephen , O-Uchi Jin , Jhun BongSook , Force Thomas , Sheu Shey-Shing
Published: July 2021

Introduction:
Oxidative stress during ischemia reperfusion (IR) injury remains a major obstacle during percutaneous coronary intervention after acute myocardial infarction. It often leads to an increased susceptibility for mitochondria permeability transition pore (mPTP) opening leading to cell death and worsened contractile recovery. Glycogen synthase kinase 3β (GSK-3β) is proposed as one of the key molecules that regulate mitochondrial dysfunction and injury during IR. Oxidative stress increases activation of matrix metalloproteinase 2 (MMP2) and subsequent cleavage (amino acids 1-34) of GSK-3ß to form a constitutively-active truncated form. However, the molecular mechanism underlying how GSK-3β activity controls mitochondrial form/function under oxidative stress condition is not fully understood.

Hypothesis:
Oxidative stress induces GSK-3ß to form a constitutively-active truncated form through MMP2-mediated cleavage, which leads to increased susceptibility of mPTP opening and activation of cell death signaling.

Methods:
Using overexpression of full length and truncated GSK-3β fused to GFP in H9C2 cells we determined the effects of GSK-3β on mitochondrial morphology and reactive oxygen species (ROS) generation using confocal microscopy. Activation of cell death signaling was evaluated by cytochrome c release, caspase activation, and GSK-3β activity using Western blotting.

Results:
Non-transfected and full-length GSK-3ß had comparable mitochondrial networking and morphology whereas cells overexpressing truncated GSK-3ß showed distinct mitochondrial fragmentation and swelling. Similarly, the non-transfected and full-length GSK-3ß overexpressing cells had equivalent levels of ROS whereas the truncated-GSK-3ß overexpressing cells had markedly increased levels of basal ROS that was further enhanced with transient H2O2 treatment. Cells overexpressing truncated GSK-3ß had an increased susceptibility to mPTP opening as measured by cytochrome C release to the cytoplasm when challenged with H2O2.

Conclusion:
Truncation of GSK-3ß induces increased mitochondrial ROS formation as well as mitochondrial fragmentation that is indicative of mitochondrial damage and an increased susceptibility to mPTP opening.
Abstract 274: Polarization of Macrophage Confers Regenerative Capacity of Using Adipose Derived Regenerative Cells
Authors: Shintani Satoshi , Shimizu Yuuki , Hao Changning , Kondo Kazuhisa , Hayashida Ryo , Murohara Toyoaki
Published: July 2021

Background:
Recent studies indicate that macrophages (Mφ) have conflicting characteristics, pro-inflammatory or anti-inflammatory phenotypes. We previously demonstrated that implantation of adipose derived regenerative cells (ADRCs) augmented angiogenesis and lymph angiogenesis by modulating Mφ phenotype in animal models. We thus examine whether Mφ polarization to M2 type is important for neovascularization in various models.

Methods and Results:
Culture medium of ADRCs accelerated not only migration of human umbilical vein endothelial cells (HUVECs) but also polarization of M2 type Mφ. Cultured ADRCs released SDF-1, VEGF-C, and prostaglandin E2 (PGE2). PGE2 plays a key role for the polarization of M2 type Mφ via EP2/4 receptors. Matrigel tube formation assay conformed that ADRCs were incorporated into HUVEC network. In vivo, implanted ADRCs participated in the formation of capillary networks in ischemic tissue. In a mice model of tail lymphedema, the number of bone marrow derived Mφ was significantly higher in the ADRCs treated group than in the un-treated group. Most of Mφ differentiated into lymphatic endothelial cell in the edematous tissue and were polarized to M2 phenotype. Moreover, in a mice model of hind limb ischemia, implantation of ADRCs facilitated the polarization of Mφ into M2 type Mφ and up regulated IL-10 expression to suppress inflammation at ischemic tissue.

Conclusion:
Polarization into anti-inflammatory phenotype of Mφ plays an important role for regenerative action of ADRCs.
Abstract 277: Acute Cardiac Insulin Resistance During Cardiopulmonary Bypass in Mini-pigs: Role of Hexosamine Biosynthesis and Protein O-Glycosylation
Authors: Zhao Kun , Li Jia , Zheng Qijun , Fu Feng , Jin Zhenxiao , Chen Wensheng , Wang Xinli , Zhang Yue , Jin Yan , Chen Min , Zhu Hailong , Feng Jianyu , Qin Fuen , Cui Qin , Yi Dinghua , Gao Feng
Published: July 2021

Our previous study as well as others has demonstrated a strong positive correlation between cardiac insulin signaling and myocardial function in ischemic/reperfused hearts. This study was designed to determine whether cardiac insulin signaling is impaired during cardiopulmonary bypass (CPB) and the possible mechanisms involved. Twelve male mini-pigs were anesthetized and subjected to CPB for 30 min. Blood samples and left ventricle biopsies were taken at pre-bypass (control), aortic cross clamp (AXC) 5 min and AXC release 120 min. Compared with sham-operated group, both blood glucose and insulin levels went up at 120 min after AXC release. Glucose uptake in heart dropped significantly as measured by attenuated coronary arterio-venous glucose difference and reduced cardiac
18
F-fluorodeoxyglucose uptake by Positron Emission Tomography imaging. Furthermore, myocardial insulin signaling was blunted as manifested by decreased phosphorylation of IRS-1, Akt and GSK-3beta (P<0.01, n=4). Interestingly, these changes were associated with a substantial increase of GFAT activity (the rate-limiting enzyme for hexosamine biosynthesis) and a significant increase in protein O-GlcNAcylation in cardiac tissues (P<0.05, n=6). Moreover, pretreatment with Alloxan, an inhibitor of O-GlcNAc-transferase, blocked both the impaired GSK-3beta phosphorylation and the increase in O-GlcNAcylation. Enhancement of cardiac insulin signaling with glucose-insulin-potassium before AXC decreased protein O-GlcNAcylation and ameliorated myocardial dysfunction after CPB. These data indicate the existence of acute myocardial insulin resistance during CPB which is attributable to elevated hexosamine biosynthesis and protein O-glycosylation.
Abstract 280: Deficiency of FKBP12 in the Heart Leads to Impaired Contractility and Pregnancy Induced Cardiomyopathy
Authors: Oakes Joshua , Hamilton Susan
Published: July 2021

FK506 Binding Proteins (FKBPs) are a family of cis-trans prolyl isomerases that bind rapamycin and FK506. FKBP12 and 12.6 interact with ryanodine receptors (RyR), homotetrameric transmembrane ion channels that regulate Ca2+ release from the sarcoplasmic reticulum (SR). FKBP12 interacts with RyR1 in skeletal muscle and FKBP12.6 interacts with RyR2 in cardiac muscle to regulate the Ca2+ leak properties of these channels. Recently it has been suggested that FKBP12 also plays a role in regulating RyR2 activity. Using mice with a cardiac specific deficiency in FKBP12, we analyzed the role of FKBP12 in cardiac function.

We found that both male and female mice with a α-MyHC Cre/Lox mediated deficiency in FKBP12 in the heart (FKBP12 KD) developed a mild dilated cardiomyopathy, with enlarged left ventricular diameter both during systole and diastole, decreased ejection fraction and decreased fractional shortening. To elucidate the mechanism for these effects we assessed Ca2+ sparks in isolated cardiomyocytes. We found an increase in both Ca2+ spark frequency and spark amplitude in FKBP12 cardiac deficient mice without a change in spark duration. Despite a mild phenotype in adult mice, we found that approximately 25% of all pregnancies (26/106) in the FKBP12 deficient mice resulted in the mothers dying following the birth. Autopsies show that these cardiac specific FKBP12 deficient mice had increased heart weight and significantly dilated ventricles compared to female Cre mice. Our data suggest that a cardiac specific deficiency in FKBP12 leads to the development of pregnancy induced cardiomyopathy. Echocardiography on FKBP12 deficient mice one day after giving birth found that there was no significant difference in ejection fraction or fractional shortening compared to α-MyHC Cre control mice. FKBP12 deficient females, however, had larger hearts and 50% (2/4) displayed heart failure and died. In conclusion, we show that FKBP12 does indeed alter Ca2+ handling in the heart and that a loss of FKBP12 leads to the development of pregnancy induced cardiomyopathies in females.
Abstract 285: Overexpression of SK3 in a Murine Model Results in Sudden Cardiac Death
Authors: Tucker Nathan R , Mahida Saagar , Mills Robert , Simonson Bridget , Macri Vincenzo , Lemoine Marc , Das Saumya , Milan David J , Ellinor Patrick T
Published: July 2021

Background:
Recently, the gene responsible for encoding SK3, KCNN3, was implicated in a genome-wide association study as a susceptibility locus for atrial fibrillation. SK3 is one of a family of three small conductance, voltage-independent, calcium-activated potassium channels which is abundantly expressed in cardiac tissues.

Therefore, we hypothesized that transcriptional misregulation of the KCNN3 gene could result in arrhythmogenesis.

Methods:
We characterized a mouse line which had constitutive ubiquitous overexpression of
Kcnn3
using ambulatory cardiac rhythm monitoring, optical mapping and
in vivo
electrophysiology studies. Both homozygote (SK3
T/T
) and heterozygote (SK3
T/+
) animals were compared to their wild-type (SK3
+/+
) littermates for assessment of cardiac phenotypes.

Results:
SK3
T/T
mice displayed increased expression of SK3, as assessed by qRT-PCR, in all four chambers of the heart, although highest increase of expression was observed in the ventricles. Examinations of SK3
T/T
mice revealed no gross morphological changes in the myocardium, no observable cardiac fibrosis, and normal echocardiograms when compared to SK3
+/+
mice. However, pups from SK3
T/+
crosses did not produce expected Mendelian ratios (SK3
+/+
= 17%), and 7 out of 19 SK3
+/+
mice died suddenly by 3 months of age, whereas all of the SK3
T/+
or SK3
+/+
survived. To address potential mechanisms, we performed ambulatory monitoring on mice beginning at approximately one month of age. Of the 6 SK3
+/+
mice, 4 died suddenly, whereas neither of the wild-type controls expired. The cardiac rhythm recorded at the time of death in three of the four mice was heart block followed by severe bradycardia. Additionally, during the period of ambulatory monitoring, SK3
T/T
mice had a lower mean heart rate compared to the SK3
+/+
control mice, and SK3
T/T
mice also displayed frequent episodes of atrioventricular dissociation, both at rest and during periods of activity. SK3
T/T
mice also displayed more pronounced variability of the heart rate and the PR interval. Optical mapping revealed slower ventricular conduction velocity.

Conclusion:
These data suggest a mechanism whereby overexpression of SK3 leads to sudden death due to bradyarrhythmias and heart block.
Abstract 294: Hematopoietic Cells Are Required For Proper Development Of Coronary Vasculature
Authors: Lluri Gentian , Liu Xiaoqian , N Atsushi
Published: July 2021

Objective:
We examined whether the hematopoietic cells induce the coronary artery formation using genetically modified mouse models of hematopoietic ablation
in vivo
and
ex vivo
.

Methods:
As a model of for hematopoietic cell deficient animals, we used Runx1 (a transcription factor required for definitive hematopoiesis) knockout embryos and Vav1-cre; R26-DTA embryos, which ablates 2/3 of CD45+ hematopoietic cells. The coronary growth and the hematopoietic cells were evaluated in whole-mount, section and
ex vivo
explant culture.

Results:
The developing coronary endothelial cells form blood-island-like structure at around E12.5 in the subepicardial region. Interestingly, however, the histological analyses suggest that the first Ter119+ and CD45+ blood cells appear in the subendocardial area at E10.5, even before the formation of coronary channels. These initial hematopoietic cells in the heart are not likely derived from the epicardium, as the sorted epicardial cells yielded no hematopoietic cell in colony formation assay. These observations raised a question whether these heart-resident hematopoietic cells rather play an inductive role during coronary formation. To examine this possibility, we analyzed two hematopoietic ablation models. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 knockout embryos and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation
ex vivo.

Conclusion:
Hematopoietic cells are not merely transported via coronary vessels, but substantially involved in the induction of the coronary vessels during cardiogenesis.
Abstract 301: Targeting Mixed Lineage Kinases in pathological cardiac fibroblast activation and intercellular communication
Authors: Schulz Emily M , Martin Melissa L , Goodfellow Val S , Dewhurst Stephen , Gelbard Harris A , Blaxall Bruns C
Published: July 2021

Cardiovascular disease (CVD) and the final clinical iteration, heart failure (HF), affect more than 82 million Americans yearly, affects >5.7 million). Although the cardiac myocyte has long been the focus of cardiac cellular research, our lab and others have demonstrated that pathological activation of cardiac fibroblasts (CF) after onset of disease or cardiac injury is also a key player in HF pathogenesis. Pathologic activation of CFs causes a release of various paracrine and autocrine factors that target cardiomyocytes (CM), local inflammatory cells and the CFs themselves. Elucidating the mechanisms involved in pathological ‘support cell (CF)’- ‘functional cell (CM)’ communication may hold therapeutic promise.

Mixed lineage kinase 3 (MLK3) is a stress activated mitogen-activated protein kinase kinase kinase (MAPKKK) involved in pro-apoptotic pathways. Inhibition of MLK3 in HAND results in the attenuation of microglial cell activation, preservation of neuronal function and synaptic structures. The parity between microglia-neuron communication and CF-CM communication and the effects of pathological activation on the respective support cells for each tissue type suggested a role for MLK3 in aberrant CF-CM cross-talk associated with the development and progression of HF. Preliminary data in mice subjected to a pharmacological model of HF (chronic isoproterenol) that were treated with a small molecule inhibitor (URMC-099) of MLK3 demonstrated a reduction in the development of myocardial fibrosis compared to vehicle treated animals. Additionally, small molecule MLK3 inhibition attenuated the development of myocardial hypertrophy as measured by heart weight: body weight and heart weight: tibia length ratios. Studies in cultured neonatal rat ventricular fibroblasts (NRVF) demonstrated that small molecule MLK3 inhibition attenuated CF activation and transition to a myofibroblast phenotype, including reductions of pathologic CF markers such as α-SMA, IL-6, IL-1β, and others. Our data demonstrate that MLK3 plays an important role in pathologic CF-CM communication and myocardial hypertrophy, and suggest that small molecule inhibition of MLK3 holds therapeutic promise for HF.
Abstract 306: Mitochondrial Aldehyde Dehydrogenase, A Therapeutic Target In Post-myocardial Infarction Heart Failure In Diabetes
Authors: Palaniyandi Suresh , Mali Vishal , Xu Jiang , Yang Xiao-Ping
Published: July 2021

Development of heart failure (HF) and pathological remodeling occurs after myocardial infarction (MI). However, in diabetic patients the prognosis is far worse. Hyperglycemia leads to accumulation of reactive aldehydes like 4-hydroxy-2 nonenal (4-HNE) in the heart. 4-HNE impairs cellular function and causes organ damage by forming adducts with critical proteins. In this study, we hypothesized that 1) 4-HNE-induced impairment of mitochondrial aldehyde dehydrogenase (ALDH) i.e. ALDH2 contributed to myocardial ischemia-induced HF in diabetes mellitus (DM) and 2) pharmacological activation of ALDH2 by Alda-1 prevent post-MI cardiac damage in DM. To test these hypotheses, first 8-week old C57BLC/6J male mice were injected with streptozotocin (STZ) (150 mg/Kg). After 8 weeks of STZ, mice were infused with isoproterenol (Iso) (45 mg/Kg) for 2 weeks by mini pumps to induce chronic myocardial ischemia and HF. Cardiac dysfunction was assessed in conscious mice by echocardiography periodically until sacrifice (16 weeks after STZ). We had normal control (Con), DM control (DM), DM + Iso and non-diabetic Iso control (Iso) with n 6 in each group. Fractional shortening (FS) was reduced in DM + Iso group (47 ± 1.2%) compared to Con (53 ± 0.9 %) and DM (55 ± 1.6 %) P<0.01, but not with Iso (49 ± 1.4%). Decrease in myocardial ALDH2 activity and increase in 4-HNE adduct formation in ALDH2 and mitochondrial damage were observed in DM + Iso at various degrees compared to Con, DM and Iso. Increased cardiac fibrosis was also recorded in DM + Iso (4 ± 0.4%) relative to Con (1 ± 0.2%; P<0.001), DM (2 ± 0.1%; P<0.05) and Iso (2.4 ± 0.3%; P<0.01). In the treatment protocol, DM + Iso group were treated with Alda-1 (10mg/kg/day) from weeks 12 to 16. Alda-1 ameliorated cardiac damage in DM as evident from enhanced FS (55 ± 3.5%; P<0.01) and reduced fibrosis. Besides improving ALDH activity Alda-1 also attenuated 4-HNE adduct formation on ALDH2 and mitochondrial damage. Myocardial levels of ALDH2 activity was lower in diabetic patients with cardiomyopathy (50% reduction; P<0.007). Finally, we conclude that activation of ALDH2 ameliorates HF in diabetic mice and which has a potential to be developed as a therapeutic strategy for HF in diabetic patients.