bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3725: Myocardial Microvascular Obstruction in Sudden Death from Acute Myocardial Infarction Occurs more often in Coronary Plaque Erosion than in Plaque Rupture
Authors: Schwartz Robert S , Burke Allen , Farb Andrew , Kaye David , Lesser John R , Virmani Renu
Published: July 2021

Epicardial coronary artery thrombosis is associated with myocardial microvascular obstruction (MVO) and microemboli. Clinical consequences include acute myocardial infarction and sudden cardiac death from myocardial MVO and myocyte necrosis. We thus characterized myocardial histopathology and morphometry and related these findings to epicardial coronary artery plaque in patients dying from coronary artery occlusion. Hearts from patients with proven sudden coronary death from acute coronary artery occlusion underwent histopathologic characterization for MVO, cardiac microemboli, and epicardial coronary artery plaque. Immunohistochemistry was performed on microvascular thrombus to evaluate for platelet and fibrin content. Forty four cases were studied (mean age at death 51 ± 15 yrs). All emboli and MVO thrombus stained for both fibrin and platelets, and occurred in 54% of all patients, mean 4.5 microemboli/heart. Of all occluded microvessels, 89% were less than 120 um diameter (39% were < 30 um dia), and only 4% were greater than 200 um diameter. Microvascular obstruction and emboli occurred more often in epicardial plaque erosion (74% vs 40% of cases with MVO), and was most common in the LAD territory. Histopathologic epicardial coronary stenosis severity was (mean) 74% in those with emboli and 75% in those without (p=ns). Microemboli and microvascular obstruction are very common in patients dying of acute coronary artery thrombosis. Plaque erosion may be most likely to cause such emboli, with accumulation in sub-200 micron vessels. These emboli and microvessel obstruction have a prominent clinical role due to associated myonecrosis. Interventional strategies for eliminating MVO may be useful to limit myocardial damage and prevent long term sequelae.
Abstract 3732: Long-Term Safety and Efficacy of ABT-335 (Fenofibric Acid) in Combination with Statin Therapy for the Treatment of Patients with Mixed Dyslipidemia
Authors: Bays Harold E. , Jones Peter H. , Mohiuddin Syed M. , Kelly Maureen T. , Sun Hsiaoming , Setze Carolyn M. , Buttler Susan M. , Sleep Darryl J. , Stolzenbach James C.
Published: July 2021

Background:
The efficacy and safety of ABT-335 + statin combination therapy was demonstrated in three 12-week, controlled studies of patients with mixed dyslipidemia randomized to ABT-335 (135mg) + low or moderate dose rosuvastatin (R), simvastatin (S), or atorvastatin (A), or ABT-335 or statin monotherapy. A subsequent 52-week open label extension study evaluated the long-term safety and efficacy of ABT-335 combined with statins.

Methods:
Patients who completed 12 weeks of treatment in the 3 controlled studies were eligible to enroll in the 52-week extension study to receive ABT-335 + the moderate dose statin that was used in their initial study: R 20mg, S 40mg, or A 40mg. This was a pre-specified, integrated analysis of patients receiving ABT-335 + statin in the 12- and 52-week studies.

Results:
Of the 2715 randomized patients, 2316 completed the 12-week studies and 1911 enrolled in the extension study. A total of 2201 patients received at least 1 dose of ABT-335 + statin for a median duration of 364 days; 1139 patients were treated for ≥52 weeks. The incidence of adverse events (AEs) was similar across all 3 combination therapy groups (Table

). The most common AEs were headache, upper respiratory tract infection, nasopharyngitis, and back pain. Rhabdomyolysis was not reported in any group. Patients who were initially randomized to ABT-335 + statin and continued in the extension study (N=979) had sustained improvements in multiple lipids. After 12 and 52 weeks of treatment, combination therapy resulted in mean percent decreases in TG (−45.7% and −47.5%, respectively) and LDL-C (−32.2% and −37.8%), and increases in HDL-C (17.6% and 23.8%). Mean values at 12 and 52 weeks were: TG 141.2 and 136.3 mg/dL; LDL-C 101.6 and 93.5 mg/dL; and HDL-C 45.0 and 47.1 mg/dL, respectively.

Conclusions:
Long-term ABT-335 + statin combination therapy was generally well tolerated and resulted in comprehensive and sustained improvements in TG, HDL-C, and LDL-C in adults with mixed dyslipidemia.

Summary of safety, n/N (%)
Abstract 3734: Differential Platelet Proteome Analysis Following Specific Activation Of Glycoprotein VI
Authors: Schulz Christian , Leuschen Nina V , Fröhlich Thomas , Lorenz Michael , Arnold Georg J , Massberg Steffen
Published: July 2021

Background: Platelets play a key role in haemostasis and in the pathophysiology of various diseases including arterial thrombosis. Glycoprotein (GP)VI, the major platelet collagen receptor, mediates platelet activation and firm adhesion to collagen structures exposed at sites of vascular injury. Here, we determined the effects of specific activation of GPVI on the human platelet proteome by two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry.

Methods and Results: Platelets from healthy human donors were isolated and purified. Platelets were stimulated with an activating monoclonal antibody specific for GPVI, or control IgG. Platelet proteins were subjected to 2D-DIGE with extensive software-assisted image analysis. Subsequent identification of the differentially abundant proteins was carried out by MALDI TOF/TOF MS and LC ESI-MS/MS. We identified 8 differentially regulated proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included pleckstrin, beta-centractin, src substrate cortactin, charged multivesicular body protein 3, aldose reductase and protein disulfide isomerase-associated 3 precursor.

Conclusion: Specific platelet activation via GPVI results in the differential regulation of several proteins. The results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor, which may help to identify novel pharmacological targets.
Abstract 3736: The HDL Receptor SR-BI Modulates Platelet Function and Susceptibility to Thrombosis
in vivo
Authors: Korporaal Suzanne J , Meurs Illiana , Vergeer Menno , Hauer Arnaud , Hildebrand Reeni B , Praticò Domenico , Akkerman Jan-Willem N , Van Berkel Theo J , Kuiper Johan , Kuivenhoven Jan Albert , Van Eck Miranda
Published: July 2021

Objective:
Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from HDL by the liver. In mice, SR-BI deficiency leads to increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. Recently, a family was identified in which heterozygous carriers of a mutation (P297S) in SR-BI showed elevated HDL cholesterol levels, suggesting that SR-BI is equally important in controlling HDL cholesterol levels in humans. Interestingly, SR-BI is also expressed on platelets, which play a crucial role in atherothrombosis. The aim of this study was to investigate the role of SR-BI in platelet function in both SR-BI knockout (KO) mice and human P297S carriers.

Methods & Results:
Hematological analysis showed that SR-BI deficiency in mice caused thrombocytopenia (381±69x10
9
SR-BI KO platelets/L vs 878±162x10
9
wildtype (WT) platelets/L, p=0.001). Correspondingly, SR-BI deficiency increased tail bleeding times from 85±8 to 134±16 sec (p=0.02). SR-BI KO platelets were abnormally large (7.1±0.3 fl vs 5.8±0.0 fl for WT platelets, p=0.0004), probably caused by an increased cholesterol content. Platelet count and size of the human P297S carriers were, however, not significantly different from matched controls. The FeCl
3
acute injury model was used to study the effect of SR-BI deficiency in mice on the susceptibility to arterial thrombosis. WT mice developed total arterial occlusion after 24±2 min. In SR-BI KO mice, however, the time to total arterial occlusion was reduced 2-fold to 13±1 min (p=0.02). In line with this finding, SR-BI KO and P297S platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. The activated state of the platelets
in vivo
led to desensitization of the receptors for ADP and thromboxane that are essential for positive feedback mechanisms to enhance the platelet response. As a result,
ex vivo
aggregation of platelets from SR-BI KO mice and P297S carriers was inhibited in response to agonist stimulation as compared to control platelets.

Conclusion:
SR-BI is not only essential for HDL cholesterol homeostasis and atherosclerosis susceptibility, but also for maintaining normal platelet aggregation and prevention of thrombosis.
Abstract 3751: Selective Improvement of Renal Function in Renovascular Disease by Intra-Renal Infusion of Endothelial Progenitor Cells Restores Myocardial Microvascular Integrity
Authors: Zhu Xiangyang , Lin Jing , Textor Kyle , Chade Alejandro R , Krier James D , Lerman Amir , Lerman Lilach O
Published: July 2021

Background
: Renovascular disease (RVD) and impaired renal function are important risk factors for cardiac disease, but it remained unclear whether selective recovery of renal function can reverse cardiac dysfunction.

Aims:
This study tested the hypothesis that improvement in renal function using selective intra-renal administration of endothelial progenitor cells (EPC) in swine renovascular hypertension would improve myocardial microvascular (MV) integrity.

Methods:
The functions of the myocardial MV (response to IV adenosine) and the stenotic kidney of 7 pigs were assessed in-vivo using multi-detector CT after 6 weeks of RVD and hypertension. Autologous EPC expanded in culture were then infused (10x10
6
cells) intra-renally, and in vivo studies repeated 4 wks later. Six normal pigs served as controls.

Results
: Glomerular filtration rate was lower in RVD compared to normal (47.9±10.1 vs. 70.8±4.3 mL/min, p<0.05) and improved after EPC (to 62.7±1.5 mL/min, p=NS vs. normal), as did renal blood flow. Mean arterial pressure remained elevated after EPC (120.7±11 and 129.4±6 vs. 99.9±4.7 mmHg in normal, p<0.05), and plasma renin activity was unchanged. Myocardial MV permeability-surface area increased in RVD in response to adenosine (0.009±0.001 to 0.011±0.002 AU, p=0.01), while MV volume fraction decreased (4.3±0.8 to 2.7±0.6%, p=0.01), indicating impaired MV barrier function and integrity. Contrarily, both remained unchanged in response to adenosine 4 wks after EPC (0.01±0.002 to 0.01±0.002 AU, p=0.24, and 3.5±0.8 to 3.4±0.5%, p=0.46, respectively), as did normal MV.

Conclusion
: A single intra-renal infusion of EPC that improved renal function in experimental RVD also preserved remote myocardial MV function, despite sustained systemic hypertension. These findings underscore the cross talk between renal and cardiac function, and the potential of selective renal EPC intervention to preserve both the kidney and heart in RVD.

Renal and myocardial microvascular function change in normal and RVD before and after EPC treatment
Abstract 3810: Antiviral RNA Interference Mediated by a Recombinant Cardiotropic AAV9 Vector Improves Cardiac Function in Coxsackievirus B3 Cardiomyopathy
Authors: Poller Wolfgang , Sipo Isaac , Westermann Dirk , Kurreck Jens , Vetter Roland , Wang Xiaomin , Suckau Lennart , Zeichhardt Heinz , Pinkert Sandra , Erdmann Volker , Tschope Carsten , Fechner Henry
Published: July 2021

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. We report here on targeted RNAi for the treatment of a viral cardiomyopathy which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of Coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs), and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated intravenous treatment of mice with an AAV2.9-shRdRp vector since AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax 3006±287 vs. 7482±487 mmHg/s, p<0.01) and diastolic relaxation (dP/dtmin -2224±195 vs. -6456±356 mmHg/s, p<0.01 and Tau 16.2±1.1 vs. 10.7±0.6 ms, p<0.01) as compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax 3865±354 vs. 3006±287 mmHg/s (p<0.05) and dP/dtmin -3245±231 vs. −2224±195, mmHg/s (p<0.05), and Tau 11.9±0.5 vs. 16.2±1.1 ms (p<0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.
Abstract 3818: Notch1 Receptor Blockade Results in Dilated Cardiomyopathy in the Neonatal Mouse Heart
Authors: Urbanek Konrad , Maestroni Silvia , Sohn Regina Lee , Delucchi Francesca , Monte Federica Del , Liao Ronglih , Signoretti Sabina , Rizzi Robero , Quaini Federico , Rota Marcello , Anversa Piero , Leri Annarosa
Published: July 2021

The objective of this study was to determine whether Notch1 receptor plays a crucial role in the commitment of c-kit-positive cardiac progenitor cells (CPCs) to the myocyte lineage. This possibility was tested in the neonatal mouse heart because of the dramatic increase in the myocyte compartment early postnatally. CPCs are located within the primitive heart and their number increases progressively with gestational age and after birth. c-kit colocalizes with transcription factors and contractile proteins specific of myocytes, pointing to a lineage relationship between CPCs and myocytes in the prenatal and postnatal heart. Notch1 intracellular domain (N1ICD) was consistently found together with Nkx2.5 suggesting that Notch1 receptor represents an early determinant of myocyte differentiation. The critical function of Notch1 in the generation of myocytes was established by interfering with this pathway through the administration of a γ-secretase inhibitor to newborn mice for 3–7 days; γ-secretase inhibition opposes the cleavage of the active fragment of Notch and initiation of transcription. Treated mice showed a dilated myopathy characterized by decreased muscle mass, wall thinning and impaired fractional shortening and ejection fraction. There was also a marked reduction in the fraction of CPCs and myocytes expressing the Notch1 active fragment N1ICD. Myocyte number decreased 28% while myocyte volume remained constant, suggesting that Notch1 blockade affects myocyte formation and conditions the development of a dilated myopathy. This hypothesis is supported further by a 57% decrease in replicating myocytes positive for Ki67. These anatomical and functional changes were accompanied by downregulation of Nkx2.5 transcripts in the pathologic heart. A perfect consensus site for the target gene of Notch, RBP-Jk, was recognized in the promoter of Nkx2.5. The formation of a functional complex between Nkx2.5 promoter and RBP-Jk protein was demonstrated by electrophoretic mobility assay, chromatin immunoprecipitation and luciferase reporter assay. In conclusion, Notch1 promotes the commitment of CPCs to the myocyte lineage and inhibition of Notch1 interferes with cardiomyogenesis leading to a dilated cardiomyopathy in the postnatal heart.
Abstract 3822: Different microRNA In Mouse Heart, Cardiac Stem Cells And Cardiac Stem Cells With GATA4, Indicate Different Sets Of microRNA Involved In Stem Cell Differentiation By Gene Repression
Authors: de Oliveira Edilamar M , Tang Yao Liang , Qian Keping , Shen leping , Ong Luvena , Phillips M.Ian
Published: July 2021

Several studies suggest that miRNA have important roles in the development of the heart and cardiac function. We hypothesized that specific miRNA are involved in the differentiation steps of cardiac stem cells to heart cells. We studied 569 unique miRNAs probes in mouse heart cells (MHtC), cardiac stem cells (CSC) and CSC with GATA4 (CSCG) . Based on Sanger miRMouse -10.1, we compared miRNAs in the following groups 1) MHtC vs. CSC, 2) CSC vs.CSCG , 3) MHtCs v CSCG. We also compared CSCs under hypoxia and normoxia. miRNAs reported in the results were confirmed by PCR As previously reported miRNA 1, 133a and 133b were highly expressed in MHt. However, we also found previously unreported, relatively high expression of miRNAs 126 –3p, 145, , 451and 499 in MHtC. MicroRNAs have not previously been reported in CSCs or CSCG. We found unique expression miRNAs 10a,10b, 31, 214 and 762 in CSCs and increased expression of miRNAs 705,709,762, and 1224 in CSCG. In MHt, miR 21 was lower compared to miR21 in CSCs . Expression of miRNA-762 was expressed significantly more in CSC-G than in CSC. Therefore, miR-762 could be the direct transcriptional target of GATA-4 in CSC and a part of the differentiation control pathway from CSC to MHtC. In addition we studied miRNA from CSCs exposed to hypoxia. Hypoxia suppressed all miRs found except miR-574 –5p, which was increased in hypoxia. The results led us to identify which genes are targeted by these miRNAs. miRNA 762 is located into the BCl-7c gene and by miRNA target predictions it was found to target activin A receptor (type IB and II-like-1) gene, bone morphogenic gene and transforming growth factor, beta receptor III (TGFβIII). Embryonic stem cells differentiate into cardiomyocytes in the presence of activin A and BMP4, therefore we suggest that the miRNA 700 family ( miR762 in particluar) in cardiac stem cells inhibits activin A and BMP4 genes, possibly through TGFβIII signaling, to maintain stem cells in the undifferentiated state. The results reveal new miRNAs in cardiac stem cells differentiation to adult heart cells and suggest their role of specific gene targeting.
Abstract 3878: Glucagon-Like Peptide-1 Metabolite Protects the Myocardium Against Ischemia-Reperfusion Injury in Diabetes Mellitus
Authors: Gundewar Susheel , Elrod John W , Calvert John W , Du Xuelaing , Edelstein Diane , Lefer David J , Brownlee Michael
Published: July 2021

Background:
Glucagon-Like Peptide-1 (GLP-1) is an incretin hormone with potent plasma glucose lowering actions that is rapidly degraded by the enzyme Dipeptidyl Peptidase-IV to GLP-1(9 –36)
amide
. GLP-1(9 –36)
amide
(GLP1dp) has previously been viewed to be biologically inactive. However, our laboratory has demonstrated that GLP1dp inhibits hyperglycemia-induced production of oxidant species and prevents the inactivation of both eNOS and prostacyclin synthase in cells and diabetic animals. We investigated the effects of GLP1dp in two
in vivo
diabetic murine models of myocardial ischemia-reperfusion (MI-R) injury.

Methods:
Diabetic (db/db and STZ-diabetic) mice were treated with 2.4 μg/day of GLP1dp via Alzet pump for 7 days and subjected to 45 min of left coronary artery occlusion and 2 hr of R. At 2 hr of R, hearts were excised and evaluated for infarct (INF) size.

Results:
Diabetic (db/db) and STZ-diabetic mice treated with GLP1dp exhibited a 37% and 33% reduction in myocardial infarct size following MI-R respectively. Additionally, GLP1dp significantly reduced oxidative stress in the myocardium of these mice. Both models of diabetic mice (db/db and STZ-diabetic) exhibited elevated baseline blood glucose (BG) values of 386 ± 25 and 435 ± 15 mg/dl respectively. After 7 days of GLP1dp therapy, db/db mice exhibited a 48% reduction in BG values. In contrast, no reduction in BG was observed in the STZ-diabetic mice.

Conclusion
: Administration of GLP1dp peptide confers cardioprotection in diabetic mice by attenuating the extent of oxidant-mediated injury following MI-R. The cardioprotective actions of GLP1dp appear to be independent of any effects on blood glucose.
Abstract 3887: Wnt11 Overexpression in MSCs Protects Myocardium Against Ischemic Reperfusion Injury via Upregulation of GATA-4
Authors: Dai Ying , Zuo Shi , Ning Xu , Wang Yigang , Ashraf Muhammad , Xu Meifeng
Published: July 2021

We have studied the augmented effect of Wnt11 transfected mesenchymal stem cells (MSCs
Wnt11
) which show upregulation of GATA-4 on cardioprotection. We hyothesize that GATA-4 overexpression mediates cardioprotection. Cardioprotection by MSCs was investigated by 1) co-culture CM with MSCs and 2) transplantation of MSCs (1.5×10
6
/50μl) into the border area of ischemic rat hearts after ligation of the left anterior descending coronary artery. GATA-4 was overexpressed by transfection of GATA-4 gene via adenoviral vectors to MSCs (MSCs
GATA-4
). Loss of GATA-4 in MSCs
Wnt11
was carried out by transfecting GATA-4-siRNA into MSCs
Wnt11
using oligofectamine. Expression of GATA-4 in MSCs
Wnt11
was assayed by quantitative PCR. GATA-4 was significantly upregulated in MSCs
Wnt11
compared to MSCs
GFP
, especially, when cells were exposed to low oxygen microenvironment

(Fig. 1

).

Hearts transplanted with MSCs
Wnt11
showed a significant improvement in cardiac function compared to those transplanted with MSCs
GFP
. TUNEL positive cells in hearts treated with MSCs
Wnt11
were significantly reduced (0.57±0.17%) compared to MSCs
GFP
(1.84±0.38% VS 6.51±1.00%, p<0.05). Moreover, annexin-V
+
CM were significantly less when co-cultured with MSCs
Wnt11
and MSCs
GATA-4
than those co-cultured with MSCs
GFP
(

Fig. 2

). The effect of MSCs
Wnt11
was abolished by GATA-4-siRNA-MSCs
Wnt11
(
Tab. 1
). These results suggest that MSCs
Wnt11
exert a powerful cardioprotective effect via overexpression of GATA-4.

Figure 1.

Figure 2.

Table 1.

Cardiacfunction aftertransplantation of various MSCS
Abstract 5247: MiR-132 Regulates Angiotensin II Type 1 Receptor Expression Through a Protein Coding Region Binding Site
Authors: Elton Terry S , Kuhn Donald E , Malana Geraldine E , Martin Mickey M , Nuovo Gerard J , Pleister Adam P , Feldman David S
Published: July 2021

Most of the known physiological and pathophysiological effects of angiotensin II (Ang II) are mediated via the angiotensin II type 1 receptor (AT1R). A number of studies suggest that AT1R expression levels are predominantly regulated by post-transcriptional mechanisms. Recently, it has been suggested that 30% of human genes may be regulated, by a novel post-transcriptional mechanism involving microRNAs (miRNAs). MiRNAs are natural, single-stranded, small RNA molecules that regulate gene expression by binding to target mRNAs and suppressing its translation or initiating its degradation. Our previous studies demonstrated that miR-155 can bind to the 3′-untranslated region (3′-UTR) of human AT1R mRNAs and translationally repress the expression of this protein. In this study, we demonstrate, for the first time, that miRNAs can also bind to sequence recognition sites harbored in the coding region of the human AT1R mRNA and suppress its translation. Specifically, i
n situ
hybridization experiments demonstrate that, like the AT1R, miR-132 is abundantly expressed in vascular smooth muscle cells (VSMCs)
in vivo
. Further, we show that over-expression of miR-132 (“gain-of function”) represses human AT1R expression in VSMCs. In contrast, “loss-of-function” experiments demonstrate that transfection of the miR-132 antisense inhibitor results in augmented AT1R protein levels. Importantly, miR-132 regulation of AT1R expression was abolished by mutating the putative miR-132 target site harbored in the AT1R mRNA open reading frame. Taken together, our study demonstrates that the AT1R and miR-132 are co-expressed and that miR-132 translationally represses the expression of AT1R
in vivo
. Our data support the hypothesis that Ang II responsiveness is “fine-tuned” by exquisitely regulating the expression levels of the AT1R by a number of distinct mechanisms including miRNA regulation.
Abstract 5270: Crystal Structure of Nkx2.5 Homeodomain in Complex with Anf-242 Motif
Authors: Nam Hyun-Joo , Genis Caroli , Scone Peyton , Kasahara Hideko
Published: July 2021

NKX2.5 is a homeodomain (HD) containing transcription factor preferentially expressed in cardiomyocytes. Mutations in the NKX2.5 gene have been identified in patients with congenital heart disease, suggesting its critical roles in cardiac development and maturation. NKX2.5 is reported to regulate transcription of atrial natriuretic factor (ANF), potentially through direct binding to -242 bp proximal promoter region (ANF-242). ANF-242 contains two palidromic NKX2.5 binding sites, and occupation of both sites by NKX2.5 has been reported. In addition, physical interaction between NKX2.5 proteins has been suggested to facilitate DNA binding through domains outside of the HD. However, detailed crystal structure of NKX2.5 protein is currently not available. The HD of human NKX2.5 (aa 138–197; C193S) with the oxidizable Cys193 substituted with Ser was co-crystallized with 19bp double stranded DNA including ANF-242 region. This mutant was reported to have the same DNA binding characteristics as the wild type. The crystal structure was determined at 1.8 Å resolution with final R-working and R-free values of 0.234 and 0.278, respectively. Consistent with previous studies, two molecules of NKX2.5 HD are bound to ANF-242 region without physical interaction between the two proteins. Column purification demonstrated that NKX2.5 behaves as dimers in solution in a concentration dependent manner. These results suggest that dimeric NKX2.5 HD in solution may change conformation upon DNA binding. Among various NK class proteins, the three-dimensional structure has only been determined in the HD of Drosophila vnd/NK2 (ventral nervous system defective proteins) using NMR. When the two HDs are compared, NKX2.5 HD shows extended DNA contacts to a total of 8 base pairs. Most of the residues of HD implicated in pathology of mutants are involved in DNA binding and structural stabilization. The structure of NKX2.5 HD with ANF-242 shows that the HD occupies two binding sites of ANF-242 without physical interaction between themselves. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.

This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
Abstract 5283: Perfluorocarbon Microcapsules for X-ray Visualization of Mesenchymal Stem Cell Delivery and Engraftment
Authors: Fu Yingli , Kedziorek Dorota , Crisostomo Veronica , Gilson Wesley , Azene Nicole , Arepally Aravind , Lorenz Christine , Bulte Jeff , Kraitchman Dara
Published: July 2021

Mesenchymal stem cell (MSC) transplantation offers an alternative strategy for therapeutic angiogenesis for the management of ischemic cardiovascular disease. However, the poor survival of transplanted cells and lack of sustained engraftment severely limits therapeutic efficacy. We propose a novel cell microencapsulation method utilizing perfluoro-octyl-bromide (PFOB) impregnated alginate-poly-L-lysine-alginate microcapsules (PFOB Caps) that enable noninvasive cell delivery and tracking using conventional clinical X-ray fluoroscopy. Microen-capsulation of rabbit MSCs (1x10
6
cells/ml) combined with PFOB-loaded alginate was performed with an electrostatic droplet generator. Control capsules lacked PFOB. MSC viability post-encapsulation was determined using a microfluorometric assay. X-ray fluoroscopy was performed during direct intramuscular/intramyocardial injection of 2300 capsules/injection in a rabbit peripheral arterial disease (PAD) model (n=2) and a swine reperfused myocardial infarction (MI) model (n=3). Fluoroscopy and angiographic computed tomography (DynaCT) were performed to assess the ability to monitor microcapsule delivery and tracking with X-ray. As compared to control capsules, PFOB Caps showed similar swelling, when subjected to 55 mM sodium citrate incubation (mean diameter increased 5.4% for PFOB vs. 4.5% for control, P=NS), and mechanical strength (broken capsules from osmotic pressure tests: 2/591 for PFOB and 2/603 for control). The PFOB Caps showed a small viability decrease immediately after encapsulation (90±3%) with no significant further viability loss up to 4 weeks
in vitro
(79±5%). In both PAD and MI animals, successful injection of PFOB Caps could be demonstrated with DynaCT but not with real-time fluoroscopic images. Control capsules were never detected. CT tracking of PFOB Caps was possible up to 5 weeks post-injection. PFOB microcapsules provide an ideal microenvironment for maintaining MSC viability
in vitro
. In addition, PFOB proves to be a non-toxic, radiopaque agent suitable for cell tracking using conventional fluoroscopy
in vivo
. PFOB microencapsulation of MSCs may be a novel approach for X-ray tracking therapeutic angiogenesis.
Abstract 5323: Expression of Calsequestrin D307H Mutant Protein Partially Rescues the Abnormal Calcium Handling and Ultra Structural Defects of CASQ2 Null Hearts
Authors: Kalyanasundaram Anuradha , Viatchenko-Karpinski Serge , Lacombe Veronique , Franzini-Armstrong Clara , Györke Sandor , Knollmann Bjorn C , Periasamy Muthu
Published: July 2021

Calsequestrin (CASQ2) is the major calcium binding protein located in the cardiac junctional sarcoplasmic reticulum (jSR). Several mutations have been reported in human
CASQ2
and have been linked to Catecholamine induced Polymorphic Ventricular Tachycardia (CPVT). To understand how a point mutation
CASQ2
D307H
that changes the negative aspartate (D) to a positive histidine (H), causes ventricular tachycardia, we generated a mouse model that expresses the D307H mutant protein in a CASQ2 null background (CASQ2
−/−
mice). Our results demonstrate that the mutant protein can be stably expressed, properly targeted and localized to the jSR. This refutes a recently reported study that suggested a highly unstable, easily degraded CASQ2
D307H
mutant protein (J Clin Invest. 2007, 117(7):1814–23). Expression of the mutant CASQ2
D307H
protein did not affect RyR2, DHPR, SERCA2a and calreticulin levels, but significantly restored both triadin and junctin from the very low levels in the CASQ2
−/−
mouse. Expression and appropriate targeting of CASQ2
D307H
was directly confirmed by electron microscopy: the “tightly” packed arrangement of CASQ2 within the jSR, which was completely missing in CASQ2
−/−
mouse myocardium, was restored. This requires both the presence of CASQ2 and its interaction with triadin/junctin. Calcium transients and calcium load in ventricular myocytes isolated from CASQ2
D307H
hearts were not significantly different from those of WT cells. Standard beta-adrenergic stimulation protocols did not induce spontaneous calcium releases and triggered beats in isolated CASQ2
D307H
myocytes. Studies are in progress using telemetry to examine if the D307H mice in the null background develop VT upon exercise, a hallmark of CPVT linked mutations. Our studies thus far suggest that mutant CASQ2
D307H
protein is capable of interacting with either triadin or junctin, or both, allowing it to be localized within the jSR. In conclusion, our data demonstrate that altered protein stability of CASQ2 D307H is not the causative mechanism for the CPVT phenotype. Other mechanisms such as altered interaction between jSR proteins, decreased SR buffering and/or altered regulation of the RyR2 channels by mutant CASQ2 D307H should be explored.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 5331: Gene Transfer Of The Peripheral Type Benzodiazepine Receptor Alters The Spatio-temporal Dynamics Of The Mitochondrial Membrane Potential (ΔΨ
M
) Across The Intact Heart
Authors: Lyon Alexander R , Park Jaeho , Liang Lifan , Hajjar Roger J , Akar Fadi G
Published: July 2021

The peripheral type benzodiazepine receptor (mBZR) is a key mediator of mitochondrial dysfunction during oxidative stress. In isolated cardiomyocytes agonists of the mBZR cause ΔΨ
m
depolarization, KATP channel activation, and subsequent loss of cellular excitability. However, the spatiotemporal dynamics of ΔΨ
m
in the intact heart and their relationship to arrhythmias are unknown. Here, we investigate the mechanistic link between mBZR and ΔΨ
m
directly using adenoviral mediated gene transfer of the mBZR encoding gene (PKBS) in rat myocardium.

Methods
: Optical imaging was performed at a subcellular resolution in ex vivo perfused hearts from AdPKBS (n=5), AdGFP (n=2), and normal (n=4) rats. Normalized ΔΨ
m
was measured using a novel quantitative technique of TMRM imaging during global ischemia (7.5 min) followed by reperfusion. Mean and SD of baseline corrected & normalized ΔΨ
m
across 6400 sites were compared between groups.

Results
: In sharp contrast to normal and GFP groups, 4 of 5 PKBS hearts exhibited a paradoxical increase in ΔΨ
m
during ischemia (Fig A

) as ΔΨ
m
was significantly (p<0.05) greater in PKBS (+0.18±0.07) compared to GFP (−0.62±0.21) and normal hearts (−0.17±0.16) following 7.5min of ischemia. A transient overshoot in ΔΨ
m
observed in all groups during early (<30sec) reperfusion was greatest in PKBS hearts (Fig A

). Finally, ΔΨ
m
heterogeneity indexed by SD was markedly greater in PKBS compared to GFP and normal hearts during ischemia (p<0.01) and reperfusion (p<0.01) (Fig B

).

Conclusion
: PKBS alters spatiotemporal dynamics of ΔΨ
m
and increases ΔΨ
m
heterogeneity across the intact heart. Modulating PKBS expression may be a novel therapeutic strategy for arrhythmias.

Figure 1.

Influence of PKBS gene transfer on myocardial ΔΨm during ischemia and reperfusion. Mean myocardial ΔΨm (
A
) and spatial Δψm heteroogeneity (
B
) during ischemia and reperfusion.
Abstract 5347: Alpha-Adrenergic-Regulation of I
K1
and Acetylcholine-gated I
K,ACh
is Impaired in Patients with Atrial Fibrillation
Authors: Voigt Niels , Bollmann Birgit , Wettwer Erich , Matschke Klaus , Ravens Ursula , Dobrev Dobromir
Published: July 2021

In chronic atrial fibrillation (cAF) the major effector of vagal excitation I
K,ACh
develops arrhythmogenic ACh-independent constitutive activity with limited additional activation via muscarinic receptors. Since the sympathetic nervous system prevents excessive vagal nerve activation we tested whether modulation of I
K,ACh
by adrenoceptors (ARs) is affected in cAF. I
K,ACh
was measured with whole-cell voltage-clamp in isolated right atrial myocytes from 29 sinus rhythm (SR) and 17 cAF patients. Compared to SR basal current was higher in cAF whereas carbachol (CCh, 2 μM)-activated I
K,ACh
was smaller (Figure

). Activation of beta-ARs with isoproterenol (ISO, 1 μM) increased CCh-activated I
K,ACh
by ~25% and ~20% in SR and cAF, respectively, whereas activation of adenylyl cyclase with forskolin (FSK, 1 μM) was without effect suggesting channel activation by G-beta/gamma-binding rather than PKA. Stimulation of alpha-1-ARs with phenylephrine (PE, 100 μM) reduced CCh-activated I
K,ACh
in SR by ~80%, but was ineffective in cAF. Neomycin (NEO, 500 μM), a blocker of phospholipase-C (PLC), prevented the PE effect suggesting that PE blunts I
K,ACh
activation via PLC-induced depletion of membrane PIP2 content required for I
K,ACh
stimulation. PE, but not ISO or FSK, reduced basal current in SR by ~50%, suggesting inhibition of I
K1
, but was ineffective on basal current in cAF which consists of I
K1
and constitutively active I
K,ACh
. Inhibition of CCh-activated I
K,ACh
and suppression of I
K1
by alpha-1-ARs are impaired in cAF. These changes may shift the balance to stronger beta-AR-mediated I
K,ACh
activation promoting the maintenance of AF.
Abstract 5382: Accelerated Triacylglyceride Dynamics Induced by Cardiac PPARα Overexpression are Potentiated by Adrenergic Stress to Reveal Increased Reliance on Endogenous Lipid Stores
Authors: Banke Natasha H , Leone Teresa C , Kelly Daniel P , Lewandowski E. D
Published: July 2021

Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor involved in regulating fatty acid utilization and is implicated in the pathogenesis of diabetic cardiomyopathy. However, the involvement of PPARα in the specific utilization of endogenous lipid stores and the consequences for triacylglyceride (TAG) formation in the cardiomyocyte remain unknown. Thus, we examined TAG dynamics in isolated perfused hearts of transgenic mice exhibiting cardiac-specific over-expression of PPARα under normal and stressed conditions. Hearts from MHC-PPARα overexpressing mice (PPAR) and non-transgenic (NTG) littermates were isolated and retrogradely perfused in a 14.1 T NMR magnet with buffer containing 0.4 mM [2,4,6,8,10,12,14,16]-13C palmitate, as a source of long chain fatty acids (LCFA) + 10 mM glucose. Dynamic 13C NMR observations of the beating heart and endpoint mass spectrometry provided TAG turnover at baseline work (PPAR, n=12; NTG, n=7) and adrenergic stress (10–7 M isoproterenol, ISO) (PPAR ISO, n=5; NTG ISO, n=6). ISO elevated rate pressure product by 27% and 25% in NTG and PPAR, respectively (P<0.05). At baseline workloads, PPAR contained 45% more TAG than NTG (P <0.05) and TAG turnover was elevated in PPARα mice by 191% over NTG. ISO did not affect TAG content in either NTG or PPAR. However, ISO induced large increases in TAG turnover in PPAR from 1454 ± 232 to 2532 ±334 nanomoles/mg prot/min (P<0.05), without affecting TAG turnover in NTG. The near doubling of TAG turnover with elevated content in MHC-PPARα hearts indicates upregulated baseline lipid metabolism via both utilization and storage pathways. Surprisingly, the adrenergic stress response potentiates the already elevated TAG dynamics in MHC-PPARα hearts, indicating persistence in matching LCFA storage rates to maintain this elevated endogenous lipid pool. Elevated TAG content in PPAR was maintained during ISO, despite increased reliance on endogenous TAG to meet the increased energy demand as compared to NTG. Therefore, activation of LCFA oxidation enzymes by PPARα is coupled in complementary fashion to the activation of LCFA storage kinetics, suggesting an expanded role for PPARα in the induction of TAG turnover.
Abstract 5383: The Circadian Clock within the Cardiomyocyte is a Novel Mechanism Regulating Myocardial Triglyceride Metabolism
Authors: Tsai Ju-Yun , Nannegari Vijayalakshmi , Heird William C , Young Martin E
Published: July 2021

Virtually every mammalian cell, including the cardiomyocyte, has an intrinsic molecular circadian clock. The cardiomyocyte circadian clock has recently been shown to directly mediate diurnal variations in myocardial gene expression, metabolism, and contractile function. This includes modulation of the heart’s acute transcriptional response to increased circulating fatty acids, in a time-of-day-dependent manner. We therefore hypothesized that disruption of the cardiomyocyte circadian clock would impair metabolic adaptation of the heart to high fat feeding. Wild type (WT) and cardiomyocyte-specific circadian clock mutant (CCM) mice were fed either a control or high fat diet (10% and 45% calories from fat, respectively) for 16 weeks, after which myocardial adaptation was assessed at transcriptional, metabolic, and functional levels.
Ex vivo
mouse heart perfusions in the working mode revealed that high fat feeding induced alterations in myocardial metabolism (
e.g.
reduced carbohydrate oxidation; p<0.01) and contractile function (
e.g.
depressed cardiac power; p<0.01) in WT, but not CCM, hearts. Furthermore, the ability to decrease reliance on endogenous substrate utilization during an acute increase in fatty acid availability
ex vivo
was abolished in hearts from CCM mice fed the high fat diet (p<0.05), suggesting potential alterations in triglyceride metabolism. Consistent with the latter, microarray analysis revealed that the cardiomyocyte circadian clock regulates expression of several genes involved in triglyceride turnover. For example, diurnal variations in expression of diacylglycerol acyltransferase 2, which promotes lipogenesis, and rates of myocardial triglyceride synthesis both peak in the middle of the active phase (2- and 3- fold, trough-to-peak, respectively; p<0.05), which are abolished in CCM hearts. These data provide direct evidence that the cardiomyocyte circadian clock regulates myocardial triglyceride metabolism at multiple levels. We postulate that disruption of this intramyocellular mechanism may contribute to altered myocardial metabolism and contractile dysfunction observed during cardiac hypertrophy, diabetes mellitus, and/or shift work.
Abstract 5385: Transport of Free Fatty Acids in Cardiac Myocytes is Mediated by a Plasma Membrane Pump as Revealed by Monitoring Cytoplasmic Unbound Free Fatty Acids
Authors: Carley Andrew N , Kampf J P , Kleinfeld Alan M
Published: July 2021

The transport of FFA across the plasma membrane represents one of the earliest points at which FFA metabolism can be controlled by cardiac myocytes. Using novel methods to measure the intracellular unbound concentration of FFA ([FFA
i
]), the first direct measurements of FFA transport across cardiac plasma membranes have been performed in freshly isolated cardiac myoctyes. Measurements of the unbound concentrations of FFA (FFA
u
) in the aqueous phase were performed using the fluorescent ratio probe ADIFAB. Cardiac myocytes were microinjected with ADIFAB, and the transport of oleate and palmitate was determined by monitoring [FFA
i
] using fluorescence ratio microscopy. FFA influx was initiated by rapidly increasing the extracellular concentration of FFA
u
([FFA
o
]) using FFA-BSA complexes, which clamped [FFA
o
] at fixed values. The time course of influx was monitored from the change in [FFA
i
], which rose exponentially to a steady state level (k
influx
~ 0.01 s
−1
). Once steady state was achieved, efflux was initiated by changing the extracellular media back to zero [FFA
o
]. Efflux was monitored by the decrease in [FFA
i
] which, like influx, revealed exponential behavior (k
efflux
~ 0.02 s
−1
). At steady state [FFA
i
] was greater than [FFA
o
] by a factor of ~3.5, indicating that during influx FFA are pumped up a concentration gradient. Both the initial rate of transport and the gradient ([FFA
i
] > [FFA
o
]) revealed saturation with increasing [FFA
o
]. The initial rate of influx saturated at [FFA
o
] > 200 nM, while the [FFA
i
] > [FFA
o
] gradient was relatively constant (~ 3.5) but began to decrease and approached 1 at [FFA
o
] > 200 nM. The efflux rate constant decreased for [FFA
o
] > zero, suggesting that efflux may be regulated by a mechanism that senses the level of circulating FFA
u
. Our results indicate that the mechanism of FFA transport across cardiac myocytes is regulated by the plasma membrane and allows for the efficient storage and release of FFA from cardiac myocytes. We suggest that this mechanism involves an as yet unknown membrane protein pump which enables the cells to accumulate surprisingly high concentrations of FFA. The ability to measure [FFA
i
] and the demonstration of efflux are significant steps in understanding cardiac FFA metabolism.

This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 5399: Myocardial Contractile Dysfunction is Associated with Fetal Cardiac Bypass in a Sheep Model
Authors: Duffy Jodie Y , Baker R. Scott , Basu Mitali , Reed Casey , Heeb Emily A , Eghtesady Pirooz
Published: July 2021

Declining cardiac output and placental dysfunction are the key barriers to successful fetal cardiac bypass for
in utero
repair of congenital heart defects. Identifying myocardial dysfunction, especially in the RV that is considered the systemic ventricle in fetal life, may be important for success. We hypothesized that fetal cardiac bypass results in myocardial contractile dysfunction that reduces cardiac output and placental perfusion, leading to placental dysfunction and fetal demise. Three fetuses from mid-term pregnant ewes were subjected to 30 min of cardiac bypass and followed for 120 min after bypass. Piezoelectric crystals (Sonometrics, Ontario, CA) were placed on three axes of the fetal heart and pressure catheters (Millar Instruments, Houston, TX) were inserted in the left and right ventricle (RV). Data were analyzed by repeated measures ANOVA with Fisher’s PLSD post hoc analyses. Representative RV pressure-volume loops in the figure showed that preload increased after bypass, indicated by increased end-diastolic volume (mean 4.1±.7 vs. 4.4±1 mL) and pressure (mean 14.3 ±7 vs. 31.3± 3 mmHg, P<.05). RV contractility decreased after bypass: pre-bypass dPdt
max
(mmHg/sec) - 880±93 vs. 731±102 at 120 min after bypass (P≤.1) and dP/dt
min
- 798±4 vs. 689±11 (P≤.1). Preload recruitable stroke work (slope of regression line) also decreased from 28.5±7 pre-bypass to 22.1±5 at 120 min post-bypass. Increased RV preload and RV contractile dysfunction are evident after fetal cardiac bypass. Minimizing the associated myocardial dysfunction is important for the success of fetal surgery to repair complex congenital heart defects.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5402: Two Photon Microscopy Measurements Of Sub-epicardial Sarcomere Length In Perfused Rat Hearts
Authors: Camelliti Patrizia , Bub Gil , Stuckey Daniel J , Bollensdorff Christian , Tyler Damian J , Helmes Michiel , Clarke Kieran , Kohl Peter
Published: July 2021

Sarcomere length (SL) is a fundamental parameter underlying the Frank Starling relation in the heart, as it offers an absolute representation of myocardial stretch. Previous studies addressed the Frank Starling relation by measuring SL in isolated myocytes or muscle strips. Here, we report first data obtained using a novel technique to measure sub-epicardial SL in perfused hearts. Rat hearts were Langendorff perfused (normal Tyrode solution) at a constant pressure of 90mmHg, labeled with the fluorescent membrane marker di-4-ANEPPS, and then arrested with high-K
+
Tyrode for either 2-photon microscopy (n=4) or MRI (n=4). Image analysis software was developed to extract SL at the cell level from >1,400 2-photon images (Fig 1

) and correct for cell angle. SL increased by 10±2 % between 30 and 80 min of perfusion (1.98±0.04 to 2.17±0.03 μm; p<0.05; Fig 1

). Measurements of left ventricular myocardial volume (LVMV) were made
in vivo
and in perfused hearts using 3D MRI. LVMV increased by 24±7% from
in vivo
to 30 min of perfusion, and by 11±3 % between 30 and 90 min (539±35; 664±44; 737±49 mm
3
, respectively; p<0.05; Fig 1

). We show that SL can be measured in isolated perfused hearts. The method allowed monitoring of changes in SL over time, and showed that SL and LVMV increase to a similar extent during 30–80 min perfusion with crystalloid solution, probably due to tissue oedema. This result, together with the increase in LVMV during the first 30 min, highlights the pronounced differences between
in vivo
,
in situ
, and
in vitro
model systems for studies of cardiac physiology and mechanics. Future research will compare changes in SL in healthy hearts and disease models involving contractile dysfunction.

Figure 1:

Left: 2-photon microscopy image of di-4-ANEPPS labeled myocardium. Right: SL and LVMV changes over time.